Proteomics in studies of Staphylococcus aureus virulence.

Staphylococcus aureus is a widespread, opportunistic pathogen that causes community and hospital acquired infections. Its high pathogenicity is driven by multifactorial and complex mechanisms determined by the ability of the bacterium to express a wide variety of virulence factors. The proteome secreted into extracellular milieu is a rich reservoir of such factors which include mainly nonenzymatic toxins and enzymes. Simultaneously, membrane proteins, membrane-cell wall interface proteins and cell wall-associated proteins also strongly influence staphylococcal virulence. Proteomics shows a great potential in exploring the role of the extracellular proteome in cell physiology, including the pathogenic potential of particular strains of staphylococci. In turn, understanding the bacterial physiology including the interconnections of particular factors within the extracellular proteomes is a key to the development of the ever needed, novel antibacterial strategies. Here, we briefly overview the latest applications of gel-based and gel-free proteomic techniques in the identification of the virulence factors within S. aureus secretome and surfacome. Such studies are of utmost importance in understanding the host-pathogen interactions, analysis of the role of staphylococcal regulatory systems and also the detection of posttranslational modifications emerging as important modifiers of the infection process.

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title: The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections.
abstract: Staphylococcus aureus remains a leading cause of hospital-acquired and community-associated infection worldwide. The burden of disease is exacerbated by the emergence of virulent strains with reduced susceptibility to commonly used antibiotics and their dissemination in healthcare settings and in the community. Whole genome sequencing (WGS) has the potential to revolutionize our understanding and management of S. aureus infection. As a research tool, WGS has provided insights into the origins of antibiotic-resistant strains, the genetic basis of virulence, the emergence and spread of lineages, and the population structure of S. aureus. As a frontline tool, WGS offers the prospect of a method that could be used to predict resistance, assess virulence, and type isolates at the highest possible resolution. The results generated could be used to guide clinical management and infection control practice. Studies using bench-top sequencing machines have already demonstrated the feasibility of such approaches. Infection control management is compromised by our incomplete understanding of transmission, which in turn reflects the suboptimal resolution offered by conventional typing methods. As the costs of sequencing begin to approach those of conventional methods, high-resolution typing with WGS could realistically be implemented for hospital infection control, as well as for local and national surveillance practice. Translation into routine practice will require the development of a knowledge base, reliable automated bioinformatic tools, the capacity to store, exchange and interrogate large volumes of genomic data, and an acceptance of WGS by clinicians, infection control specialists, and laboratory staff.

title: Proteomic analysis of exoproteins expressed by enterotoxigenic Staphylococcus aureus strains
abstract: Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel‐based bottom‐up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one‐ and 2‐DE based proteomics. Detailed analysis of enterotoxin region of the 2‐D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin‐like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al..,  2004, 42, 2134–2143). Exoprotein patterns at the late‐exponential (7 h) and stationary (24 h) phases of cellular growth show a high‐level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB‐8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP  was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified.

title: A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome
abstract: The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen.

title: Comprehensive Characterization of Methicillin-resistant Staphylococcus aureus subsp. aureus COL Secretome by Two-dimensional Liquid Chromatography and Mass Spectrometry*
abstract: Two-dimensional LC combined with whole protein and peptide mass spectrometry is used to characterize proteins secreted by methicillin-resistant Staphylococcus aureus COL. Protein identifications were accomplished via off-line protein fractionation followed by digestion and subsequent peptide analysis by reverse phase LC-ESI-LTQ-FT-MS/MS. Peptide MS/MS analysis identified 127 proteins comprising 59 secreted proteins, seven cell wall-anchored proteins, four lipoproteins, four membrane proteins, and 53 cytoplasmic proteins. The identified secreted proteins included various virulence factors of known functions (cytotoxins, enterotoxins, proteases, lipolytic enzymes, peptidoglycan hydrolases, etc.). Accurate whole protein mass measurement (±1.5 Da) of the secreted proteins combined with peptide analysis enabled identification of signal peptide cleavage sites and various post-translational modifications. In addition, new observations were possible using the present approach. Although signal peptide cleavage is highly specific, signal peptide processing can occur at more than one site. Surprisingly, cleaved signal peptides and their fragments can be observed in the extracellular medium. The prediction accuracies of several signal peptide prediction programs were also evaluated.

title: Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation
abstract: Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2‐DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post‐transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

title: Staphylococcus aureus Produces Membrane-Derived Vesicles That Induce Host Cell Death
abstract: Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by Gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

title: Global proteome analysis of vancomycin stress in Staphylococcus aureus.
abstract: Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureus infections. Several transcriptomics and proteomics studies have investigated the bacterium's cellular response to vancomycin, but quantitative proteomic studies have been limited in the number of proteins and restricted to certain sub-cellular compartments so far. Here, we combined the enrichment of different proteomic sub-fractions with in vivo metabolic labeling and shotgun proteomics to analyze the vancomycin induced stress response. Quantitative data for approximately 1400 proteins could be obtained, covering the majority of cytosolic as well as membrane localized proteins, cell surface associated and extracellular proteins. Besides major adaptive processes induced by limited growth of the cells due to the sublethal vancomycin exposure, specific cellular responses are seen on proteome level, e.g. the specific increase of proteins synthesizing amino acids which are essential for the peptidoglycan synthesis or the decrease of most proteins with a virulence related function. Most important, the influence on regulatory targets of the two-component system VraSR as the main regulatory system known for cell wall stress as well as for global regulons like SigB and SaeR was detected.

title: Extracellular proteases are key mediators of Staphylococcus aureus virulence via the global modulation of virulence-determinant stability
abstract: Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community‐associated methicillin‐resistant S. aureus (CA‐MRSA). The reason for enhanced CA‐MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr‐regulated toxins, including secreted proteases. In this study, we explore the contribution of exo‐proteases to CA‐MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide‐rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. We also reveal that extracellular proteases are important for resisting phagocytosis by human leukocytes. Using murine infection models, we reveal contrasting roles for the proteases in morbidity and mortality. Upon exo‐protease deletion, we observed decreases in abscess formation, and impairment during organ invasion. In contrast, we observed hypervirulence of the protease‐null strain in the context of mortality. This dichotomy is explained by proteomic analyses, which demonstrates exo‐proteases to be key mediators of virulence‐determinant stability. Specifically, increased abundance of both secreted (e.g. α‐toxin, Psms, LukAB, LukE, PVL, Sbi, γ‐hemolysin) and surface‐associated (e.g. ClfA+B, FnbA+B, IsdA, Spa) proteins was observed upon protease deletion. Collectively, our findings provide a unique insight into the progression of CA‐MRSA infections, and the role of secreted proteolytic enzymes.

title: Proteomic analysis of the proteins released from Staphylococcus aureus following exposure to Ag(I).
abstract: The silver ion (Ag(I)) has well established antimicrobial properties and is widely used in a variety of anti-bacterial ointments and plasters for the control of wound infections. Wounds are frequently colonised by the bacterium Staphylococcus aureus and the aim of the work presented here was to establish how S. aureus responded following exposure to Ag(I). Exposure of S. aureus to Ag(I) resulted in the release of a range of proteins from cells. Analysis of proteins released revealed a number of proteins associated with the stress response (e.g. alkaline shock protein, methionine sulfoxide reductase), virulence (e.g. signal transduction protein) and metabolism (e.g. lipase, acetate kinase, phosphoglycerate mutase). The release of toxins (e.g. α-hemolysin, bifunctional autolysin, leucocidin F) was decreased. These results indicated that, while silver is a potent antimicrobial agent, exposure of S. aureus to this metal results in the release of a variety of proteins from the cell. Many of the proteins showing increased release were antigenic and would have the potential to induce an inflammatory response at the site of infection and thus delay healing.

title: The phosphoproteome and its physiological dynamics in Staphylococcus aureus.
abstract: Phosphorylation events on proteins during growth and stress/starvation can represent crucial regulation processes inside the bacterial cell. Therefore, serine, threonine and tyrosine phosphorylation patterns were analyzed by two powerful complementary proteomic methods for the human pathogen Staphylococcus aureus. Using 2D-gel analysis with a phosphosensitive stain (Pro-Q Diamond) and gel-free titanium dioxide based phosphopeptide enrichment, 103 putative phosphorylated proteins with successfully mapped 68 different phosphorylation sites were found in the soluble proteome of S. aureus. Additionally, in a proof of concept study, 8 proteins phosphorylated on arginine residues have been identified. Most important for functional analyses of S. aureus, proteins related to pathogenicity and virulence were found to be phosphorylated: the virulence regulator SarA, the potential antimicrobial target FbaA and the elastin-binding protein EbpS. Besides newly identified phosphorylation sites we compared our dataset with existing data from literature and subsequent experiments revealed additional phosphorylation events on highly conserved localizations in FbaA. Differential analysis of phosphorylation signals on the 2D-gels showed significant changes in phosphorylation under different physiological conditions for 10 proteins. Among these, we were able to detect newly appearing signals for phosphorylated isoforms of FdaB and HchA under nitrosative stress conditions.

title: Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions
abstract: BackgroundThe biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm.ResultsGlobal protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins.ConclusionsThe reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections.

title: Secretome of Staphylococcus aureus
abstract: Presence of secreted proteins is the key for sustenance of Staphylococcus aureus strains in the host system. General secretory (Sec) system is a well known and major secretion system that is responsible for the transportation of exoproteins across the bacterial cytoplasmic membrane and cell wall. In order to define the secretome of S. aureus, the availability of combination of two-dimensional gel electrophoresis (2-DE) with the latest advanced techniques (MALDI-TOF and ESI-MS/MS) has provided a powerful tool for the investigation of a large number of protein spots with the aim of identifying new drug candidates and discovering new diagnostics tools. In this paper, we present a short review regarding S. aureus general secretory system and role of secreted protein in host cells, and discuss the proteomic, together with genomics of S. aureus.

title: Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface
abstract: The human commensal bacterium Staphylococcus aureus is renowned as a causative agent of severe invasive diseases. Upon entering the bloodstream, S. aureus can infect almost every tissue and organ system in the human body. To withstand insults from the immune system upon invasion, several immune‐evasive mechanisms have evolved in S. aureus, such as complement inhibition by secreted proteins and IgG‐binding by surface‐exposed protein A. While it is generally accepted that S. aureus cells bind a range of host factors for various purposes, no global analyses to profile staphylococcal host factor binding have so far been performed. Therefore, we explored the possibility to profile the binding of human serum proteins to S. aureus cells by “surface shaving” with trypsin and subsequent MS analysis of liberated peptides. This resulted in the identification of several components of the complement system, the platelet factor 4 and the isoform 1 of the inter‐α‐trypsin inhibitor heavy chain H4 on the staphylococcal cell surface. We conclude that surface shaving is a versatile tool to profile global interactions between human serum proteins and the S. aureus cell surface.

title: Staphylococcus aureus virulence factors in evasion from innate immune defenses in human and animal diseases.
abstract: In the last decades, Staphylococcus aureus acquired a dramatic relevance in human and veterinary medicine for different reasons, one of them represented by the increasing prevalence of antibiotic resistant strains. However, antibiotic resistance is not the only weapon in the arsenal of S. aureus. Indeed, these bacteria have plenty of virulence factors, including a vast ability to evade host immune defenses. The innate immune system represents the first line of defense against invading pathogens. This system consists of three major effector mechanisms: antimicrobial peptides and enzymes, the complement system and phagocytes. In this review, we focused on S. aureus virulence factors involved in the immune evasion in the first phases of infection: TLR recognition avoidance, adhesins affecting immune response and resistance to host defenses peptides and polypeptides. Studies of innate immune defenses and their role against S. aureus are important in human and veterinary medicine given the problems related to S. aureus antimicrobial resistance. Moreover, due to the pathogen ability to manipulate the immune response, these data are needed to develop efficacious vaccines or molecules against S. aureus.

title: The current state of microbial proteomics: Where we are and where we want to go
abstract: Proteomics allows the assessment of cellular processes in an unprecedented scale by providing a comprehensive quantitative and qualitative overview of the protein content of a cell. Consequently, proteomics has been employed to investigate a multitude of bacterial processes ranging from the analysis of environmental communities, identification of virulence factors to the proteome‐guided optimization of production strains. Proteomics has, in short, become an indispensable tool for the global analysis of bacterial physiology. Nonetheless, challenges exist, especially in the accurate prediction of phenotypic consequences based on any given proteome composition. In this review, we will give an overview of current highlights in the area of microbial proteomics, discuss some current challenges and present new developments that may help in overcoming them.

title: Proteomic and bioinformatics tools to understand virulence mechanisms in Staphylococcus aureus
abstract: Staphylococcus aureus, one of the major pathogenic bacteria, is associated with substantial morbidity and mortality. The disease burden of staphylococcal infections is significant, which is primarily attributed to its adaptability and resistance to environmental stresses. S. aureus has the ability to develop multiple resistances to antimicrobial agents. These high resistances make pathogenicity of S. aureus one of the most complex mechanisms to understand and manage. Proteomic and bioinformatics approaches show great potential in exploring microbial adaptation strategies, ability to cause disease by pathogenic bacteria and the development of diagnostic tools. A summary of the latest developments in the application of ‘omics’ technologies to understand resistance mechanisms in S. aureus and their future role in antistaphylococcal vaccine and/or drug discovery is given here.

title: Epidemiology of methicillin-resistant Staphylococcus aureus.
abstract: The frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections continues to grow in hospital-associated settings and, more recently, in community settings in the United States and globally. The increase in the incidence of infections due to S. aureus is partially a consequence of advances in patient care and also of the pathogen's ability to adapt to a changing environment. Infection due to S. aureus imposes a high and increasing burden on health care resources. A growing concern is the emergence of MRSA infections in patients with no apparent risk factors. MRSA infection in community settings involves considerable morbidity and mortality, as does nosocomial MRSA infection. For community-associated MRSA, person-to-person transmission has been reported, and several factors have been shown to predict disease. We examine the trends in both nosocomial and community-associated MRSA infections and explore recent studies of the mechanisms that allow S. aureus to become resistant to currently available drugs.

title: The broad landscape of immune interactions with Staphylococcus aureus: from commensalism to lethal infections.
abstract: Staphylococcus aureus is a gram-positive bacterium that is present in the nostrils of a quarter of the general population without causing any apparent disease. However, S. aureus can also act as a pathogen to cause severe infections. The factors determining the balance between its commensal and pathogenic states are not understood. Emerging evidence suggests that S. aureus, in addition to inducing a pro-inflammatory response, may have the capacity to modulate the host immune system. The latter is in part the result of recognition of specific molecules embedded in the peptidoglycan layer of the staphylococcal cell wall that bind to TLR2 on host antigen-presenting cells and induce a strong IL-10 response that down regulates the adaptive T cell response. This mechanism can partially explain the duality of interactions between S. aureus and the human immune system by favoring nasal colonization instead of staphylococcal diseases. In this review, we discuss the molecular and cellular basis of this mechanism and explore its clinical implications.

title: Host-pathogen interactions between the skin and Staphylococcus aureus.
abstract: Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.

title: Colonization and infection of the human host by staphylococci: adhesion, survival and immune evasion.
abstract: The natural habitat of Staphylococcus aureus in humans is the moist squamous epithelium of the anterior nares. Several bacterial surface proteins are implicated in promoting adhesion to desquamated epithelial cells. Clumping factor B (ClfB) and iron-regulated surface determinant A both promote nasal colonization in rodent models, and in the case of ClfB, humans. One of the ligands involved in adhesion is cytokeratin 10. Reduction in nasal colonization can be achieved by active and passive immunization. S. aureus is well endowed with secreted and surface components that compromise innate immune responses, particularly the function of neutrophils. S. aureus secretes proteins that reduce migration of neutrophils from the bloodstream to the site of infection by impeding diapedesis and receptors for chemotactic molecules. Several secreted proteins interfere with complement C3 and C5 convertases, thus reducing the level of C3b opsonin and the chemotactic peptide C5a. Host proteases are recruited to the cell surface to enhance destruction of opsonic C3b and IgG. Surface components ClfA, protein A and polysaccharide capsule compromise the recognition of opsonins on the bacterial cell surface. If engulfed by neutrophils the intracellular bacterium can resist reactive oxygen intermediates, nitric oxide radicals, defensin peptides and bactericidal proteins. A prior infection by S. aureus does not induce complete protective immunity. This could be due to immunosuppression caused by expression of superantigen proteins that disrupt normal activation of T cells and B cells during antigen presentation. By studying the molecular pathogenesis of S. aureus infections markers might be found for investigating S. pseudintermedius infections of dogs.

title: Staphylococcus aureus Secretes Coagulase and von Willebrand Factor Binding Protein to Modify the Coagulation Cascade and Establish Host Infections
abstract: Clinical isolates of Staphylococcus aureus secrete coagulases, polypeptides that bind to and activate prothrombin, thereby converting fibrinogen to fibrin and promoting the clotting of plasma or blood. Two staphylococcal products, the canonical coagulase (Coa) as well as the recently identified von Willebrand factor binding protein (vWbp), promote similar modifications of the coagulation cascade during host infection. Staphylococcal binding to fibrinogen or fibrin is an important attribute of disease pathogenesis, which leads to the formation of abscesses and bacterial persistence in host tissues and also enables the pathogen to cause lethal sepsis. Circumstantial evidence suggests that the product of coagulase activity, staphylococci captured within a fibrin meshwork, enable this pathogen to disseminate as thromboembolic lesions and to resist opsonophagocytic clearance by host immune cells. In addition, the coagulation products of staphylococci appear to display discrete differences when compared to those of thrombin-mediated coagulation, the latter representing a key innate defense mechanism against many invading pathogens. Preclinical evidence suggests that inactivation or neutralization of coagulases may prevent the pathogenesis of staphylococcal infections, a strategy that could be used to combat the current epidemic of hospital-acquired infections with drug-resistant S. aureus isolates.

title: Bacterial proteases in disease - role in intracellular survival, evasion of coagulation/ fibrinolysis innate defenses, toxicoses and viral infections.
abstract: Pathogenic bacteria have evolved multiple mechanisms aimed to evade host defenses. This review summarizes selected examples of how bacteria utilize proteolytic enzymes to efficiently establish and spread infection systemically. First, the role of proteases in intracellular survival and persistence - the primary means used by bacteria to endure phagocytosis and/or avoid the vigilance of the immune system - is discussed. Second, it is demonstrated how some bacteria escape entanglement in fibrin(ogen) meshes, by inducing their proteolytic dissolution while other species modify the proteolytic cascade of mesh formation to divert this important innate immune defense for their own benefit. Third, bacterial proteolytic toxins are introduced, which allow microorganisms to exert and take advantage of systemic effects already during primary, localized infection. Finally, it is discussed how viruses utilize bacterial proteases by taking advantage of concurrent infection, and how pathogens may even mutually benefit from the joint presence of other pathogens. The reviewed adaptations are often essential for pathogen survival in the hostile environment of a host organism. As such, the potential benefits of pharmacological interference in relevant pathways for the struggle against bacterial pathogens are also discussed.

title: Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis
abstract: Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.  We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×105 and 4×108 CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.  PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.

title: Current concepts on the virulence mechanisms of meticillin-resistant Staphylococcus aureus.
abstract: Meticillin-resistant Staphylococcus aureus (MRSA) strains are prevalent bacterial pathogens that cause both health care and community-associated infections. Increasing resistance to commonly prescribed antibiotics has made MRSA a serious threat to public health throughout the world. The USA300 strain of MRSA has been responsible for an epidemic of community-associated infections in the US, mostly involving skin and soft tissue but also more serious invasive syndromes such as pneumonia, severe sepsis and endocarditis. MRSA strains are particularly serious and potentially lethal pathogens that possess virulence mechanisms including toxins, adhesins, enzymes and immunomodulators. One of these is Panton-Valentine leukocidin (PVL), a toxin associated with abscess formation and severe necrotizing pneumonia. Earlier studies suggested that PVL was a major virulence factor in community-associated MRSA infections. However, some recent data have not supported this association while others have, leading to controversy. Therefore, investigators continue to search for additional mechanisms of pathogenesis. In this review, we summarize the current understanding of the biological basis of MRSA virulence and explore future directions for research, including potential vaccines and antivirulence therapies under development that might allow clinicians to more successfully treat and prevent MRSA infections.

title: Staphylococcus aureus leukotoxin GH promotes inflammation.
abstract: Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo.



We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models.



Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain.



Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.

title: Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins.
abstract: The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

title: Exfoliative Toxins of Staphylococcus aureus
abstract: Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1), enterotoxins, and exfoliative toxins (ETs). The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS), a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.

title: Exotoxins of Staphylococcus aureus.
abstract: This article reviews the literature regarding the structure and function of two types of exotoxins expressed by Staphylococcus aureus, pyrogenic toxin superantigens (PTSAgs) and hemolysins. The molecular basis of PTSAg toxicity is presented in the context of two diseases known to be caused by these exotoxins: toxic shock syndrome and staphylococcal food poisoning. The family of staphylococcal PTSAgs presently includes toxic shock syndrome toxin-1 (TSST-1) and most of the staphylococcal enterotoxins (SEs) (SEA, SEB, SEC, SED, SEE, SEG, and SEH). As the name implies, the PTSAgs are multifunctional proteins that invariably exhibit lethal activity, pyrogenicity, superantigenicity, and the capacity to induce lethal hypersensitivity to endotoxin. Other properties exhibited by one or more staphylococcal PTSAgs include emetic activity (SEs) and penetration across mucosal barriers (TSST-1). A detailed review of the molecular mechanisms underlying the toxicity of the staphylococcal hemolysins is also presented.

title: Discovery of virulence factors of pathogenic bacteria.
abstract: Discovering virulence factors of pathogenic bacteria is a key in understanding pathogenesis and for identification of targets for novel drugs and design of new vaccines. Comparative genomics, transcriptomics, and proteomics have become the popular tools in discovering the virulence factors in bacterial pathogens, such as Neisseria meningitidis, Yersinia pestis, Mycobacterium tuberculosis, and Staphylococcus aureus. In addition, proteomics has been employed successfully in the study of the mechanism of post-translationally modified proteins of bacterial pathogens. Once the putative virulence factors are identified by genomics and/or proteomics, their functions and mechanisms can be further investigated by phenotypic analyses including mutagenesis and biochemical methods and/or structural biology. Combination of these techniques will accelerate the developments of therapeutic drugs and vaccines in combating bacterial diseases.

title: Whole genome sequencing of meticillin-resistant Staphylococcus aureus

abstract: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism.



Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction.



The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors.



The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

title: Genome and virulence determinants of high virulence community-acquired MRSA
abstract: A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non b-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium.



We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50.



Meticillin resistance gene (mecA) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCCmec), by contrast with type II in N315 and Mu50. Type IVa SCCmec did not carry any of the multiple antibiotic resistance genes reported in type II SCCmec. By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2.



MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus, including those of community-acquired MRSA strains.

title: Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100.
abstract: Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain. Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by sigma(B) (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown to be present at significantly altered levels. SarA production was induced in TX-100-treated cultures. A protein of the sigma(B) operon, RsbV, was only detected in COL and its production was down-regulated in COL when the strain was treated with TX-100, whereas RsbW was present at reduced levels in both strains. Upon growth of both strains in the presence of TX-100, no effects on the production of the essential methicillin-resistance factor FemA were detected, whereas phosphoglucosamine mutase (GlmM) production was reduced in COL alone. This study suggests that proteins of the sigma(B) and sarA regulons, as well as other factors, are involved in methicillin resistance in S. aureus.

title: VFDB: a reference database for bacterial virulence factors
abstract: Bacterial pathogens continue to impose a major threat to public health worldwide in the 21st century. Intensified studies on bacterial pathogenesis have greatly expanded our knowledge about the mechanisms of the disease processes at the molecular level over the last decades. To facilitate future research, it becomes necessary to form a database collectively presenting the virulence factors (VFs) of various medical significant bacterial pathogens. The aim of virulence factor database (VFDB) (http://www.mgc.ac.cn/VFs/) is to provide such a source for scientists to rapidly access to current knowledge about VFs from various bacterial pathogens. VFDB is comprehensive and user-friendly. One can search VFDB by browsing each genus or by typing keywords. Furthermore, a BLAST search tool against all known VF-related genes is also available. VFDB provides a unified gateway to store, search, retrieve and update information about VFs from various bacterial pathogens.

title: Secrets of the secretome in Staphylococcus aureus.
abstract: Secreted proteins constitute a reservoir of virulence factors. Here, an in silico analysis of the secretome of 15 S. aureus reference strains is presented revealing that about 30% of the encoded proteome of this bacterium could be secreted. In total 1354 proteins were predicted to be localized outside the cytoplasm representing the pan-secretome of S. aureus. 41% of these proteins have been identified to be expressed using different approaches. The function of at least 50% of the secreted proteins encoded by S. aureus is not yet clear and a possible role in virulence has to be elucidated.

title: High-resolution two-dimensional electrophoresis of plant proteins.
abstract: A technique for the analysis of plant proteins from seed, leaf, root, and coleoptile tissues by high resolution two-dimensional electrophoresis is described. This technique is based primarily on the procedure of P. O'Farrell (1975, ); however, a number of improvements and simplifications have been introduced. We have found that resolution of polypeptides from a range of plant tissues is improved if the concentrations of nonionic detergent and ampholytes used in the isoelectric focusing (IEF) step are increased to 4 and 5% (w/v), respectively. Further increase in the concentrations of these two components results in gels of decreased resolution and low mechanical strength. We have also found that substitution of n-octyl beta-D-glucopyranoside or 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate for Triton X-100 or Nonidet-P40 in the IEF dimension significantly increases the resolution of polypeptides in these gels. This technique also allows minor polypeptide differences between closely related cultivars of plants to be identified.

title: Proteomic approaches to study Staphylococcus aureus pathogenesis.
abstract: Staphylococcus aureus is an important human and veterinary pathogen that causes a wide variety of infections ranging from benign skin infections to life threatening diseases. Recently, important changes in the epidemiology have been reported demonstrating that S. aureus, and particularly its methicillin-resistant variant, is now recognized as a ubiquitous pathogen responsible for both, hospital- and community-acquired infections. In these settings, the bacterium is responsible for various acute or chronic diseases and shows particular ability to adapt its metabolism to major environmental changes. Despite the fact that S. aureus infections are common worldwide, the factors triggering the different steps of infection (colonization, expression of virulence factors, invasion of host tissues, and persistence in hostile environments) are not fully characterized. Over the last decade highly parallel methods of analysis led to the release of whole genome sequences of numerous pathogenic bacteria. These efforts coupled to the parallel improvements of proteomic procedures permitted to study S. aureus transcriptome or proteome at the organism level. This now provides a sound basis for a comprehensive understanding of bacterial metabolism, adaptability to environment and pathogenicity mechanisms. This review summarizes the impact of proteomic on our comprehension of S. aureus virulence and pathogenesis.

title: A proteomic view of cell physiology and virulence of Staphylococcus aureus.
abstract: Staphylococcus aureus is a human pathogen that has advanced to a main problem in hospital settings since effective treatment options for infections caused by this pathogen are limited. Thus, new strategies to prevent and treat S. aureus infections and rapid diagnostic tools are urgently needed. The course of an S. aureus infection largely depends on successful adaptation to the host environment and a very complex and poorly understood interplay of bacterial virulence factors with each other and with host components. Over the last years, genome sequences of different S. aureus strains have been published permitting a high-throughput proteomic analysis of this pathogen. This review summarizes the impact of 2D gel- and mass spectrometry-based proteomic approaches on a more comprehensive understanding of S. aureus pathophysiology and virulence. We show that only a combination of both techniques allows a proteomic view which adequately considers all subproteomic fractions of a bacterium, i.e. cytosolic, membrane, cell surface-associated, and extracellular proteins. By this means, the majority of proteins expressed in S. aureus can be identified and even quantified. In addition, posttranslational processes such as protein secretion, modification, processing, damages, and degradation can be adequately studied.

title: The Staphylococcus aureus proteome.
abstract: Staphylococcus aureus is a Gram-positive commensal bacterium that is regarded as a major threat for modern health care systems. This relates both to the ability of S. aureus to overcome antibiotic therapy by developing high-level resistance against multiple antibiotics and this bacterium's extensive arsenal of virulence factors. Understanding the mechanisms of resistance and functional studies on stress and starvation responses are the main goals of proteomics in staphylococcal research. This review high-lights recent advances in gel-based and gel-free proteomics analyses of S. aureus and pinpoints the importance of location-specific proteomics studies targeting the cytosol, the membrane, the cell surface and the extracellular milieu in combination with integrated global proteome studies. Emerging hot topics in staphylococcal proteomics are discussed with special focus on in vivo proteomics, membrane vesicles, biofilm formation and the acquisition of absolute proteome data for systems biological modeling approaches.

title: Proteome analyses of Staphylococcus aureus in growing and non-growing cells: a physiological approach.
abstract: Staphylococcus aureus is a versatile human pathogen causing a wide variety of diseases ranging from wound infection to endocarditis, osteomyelitis, and sepsis. In order to investigate this pathogen, we sought to analyze the cytoplasmic proteome of S. aureus COL by using two different approaches: two-dimensional (2D) gel analyses combined with matrix-assisted laser ionization-time of flight mass spectrometry and a gel-free system using multidimensional liquid chromatography followed by mass spectrometry. By combining both analyses we identified 1123 cytoplasmic proteins that represent two-thirds of the cytoplasmic proteome of the organism. With our standard 2D gel setup (pI 4-7) we identified 473 proteins that cover about 40% of the cytoplasmic proteome predicted for this proteomic window. The identified proteins belong to a variety of cellular functions ranging from the transcriptional and translational machinery, tricarboxylic acid cycle (TCC), glycolysis, and fermentation pathways to biosynthetic pathways of nucleotides, fatty acids, and cell wall components. While most of the metabolic pathways predicted for S. aureus were covered by this gel-based proteomics 650 additional proteins were identified by the gel-free approach, among them alkaline or hydrophobic proteins. In our work, we established a master 2D gel that enabled us to study the regulation of core carbon metabolism in S. aureus cells grown in a complex medium. Our comparison of the protein pattern of exponentially growing cells with that of stationary-phase cells revealed a higher amount of enzymes involved in protein synthesis, transcription, and glycolysis in exponentially growing cells. In contrast, enzymes of the TCC and gluconeogenesis are increased at the stationary phase. With this comprehensive proteome map we have an essential tool for a better understanding of cell physiology of the human pathogen, S. aureus.

title: Profiling the surfacome of Staphylococcus aureus
abstract: Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life‐threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface‐exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface‐exposed protein domains – the surfacome – of S. aureus that are accessible to extracellular bio‐macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface‐exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.

title: Proteomic profiling of cell envelope‐associated proteins from Staphylococcus aureus
abstract: The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin‐digested cell envelope from cultured vancomycin‐intermediate S. aureus (VISA) cells. Combining approaches of 2‐DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan‐anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2‐DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall‐anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall‐anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2‐DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.

title: Extensive proteomic profiling of the secretome of European community acquired methicillin resistant Staphylococcus aureus clone
abstract: European community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) clone remains a striking pathogenic clone spreading in European and Mediterranean countries. Since analysis of the secretome produced from this clone by proteomics could provide a comprehensive picture of both core exoproteins as well as virulence factors, we applied two proteomic approaches, pre-fractionation of proteins on SDS-PAGE followed by in-gel trypsin digestion, and in-solution trypsin-digestion followed by off-line SCX fractionation, both of which were coupled with LC-MS/MS analyses. A total of 174 distinct proteins were identified with a high-confidence. Functional classification of these identified proteins resulted in16.09% of protein synthesis, 13.79% of virulence, 6.89% of toxin, and 17.24% of unknown function. Prediction of their cellular localizations revealed 18.39% in extracellular space, 36.20% in cytoplasm, 5.17% in cytoplasmic membranes, 6.89% in cell wall, 1.14% in multiple localizations, and 32.18% in unknown localization. Among them, 52% proteins were predicted to be secreted through signal peptide-independent pathways. Most notably, the expression of some proteins such as enterotoxins U and B were identified for the first time in this clone.

title: Proteomic tracking and analysis of a bacterial mixed culture
abstract: To improve the understanding of microbial behaviors in communities, proteomic tracking, an approach for relative quantification of species‐specific population dynamics of mixed cultures, was developed. Therefore, a bacterial mixed culture was analyzed during batch cultivations with and without addition of the antibiotic Ceftazidime. The community was composed of Burkholderia cepacia, Pseudomonas aeruginosa, and Staphylococcus aureus, pathogens causing infections in cystic fibrosis patients. Gel‐based proteomics and mass spectrometry were used to obtain qualitative and quantitative proteomic data. During cultivation, P. aeruginosa became dominant within the mixed culture while S. aureus was inhibited in growth. Analysis of samples – taken along cultivation – revealed about 270 differentially expressed proteins. Some of those proteins are related to bacterial interactions, response to antibiotic treatment or metabolic shifts. For instance, the enzymes PhzS(flavin‐containing monooxygenase), PhzD (phenazine biosynthesis protein), and PhzG2 (pyridoxamine 5′‐phosphate oxidase) indicated the production of the antibiotic pigment pyocyanine by P. aeruginosa that is related to oxidative stress and therefore, might inhibit growth of S. aureus. Overall, the strategy applied not only allows species‐specific tracking of the community composition but also provides valuable insights into the behavior of mixed cultures.

title: Difference gel electrophoresis. A single gel method for detecting changes in protein extracts
abstract: We describe a modification of two‐dimensional (2‐D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2‐D gel, post‐run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2‐D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.

title: The development of the DIGE system: 2D fluorescence difference gel analysis technology
abstract: Two-dimensional (2D) gel electrophoresis is a powerful technique enabling simultaneous visualization of relatively large portions of the proteome. However, the well documented issues of variation and lack of sensitivity and quantitative capabilities of existing labeling reagents, has limited the use of this technique as a quantitative tool. Two-dimensional difference gel electrophoresis (2D DIGE) builds on this technique by adding a highly accurate quantitative dimension. 2D DIGE enables multiple protein extracts to be separated on the same 2D gel. This is made possible by labeling of each extract using spectrally resolvable, size and charge-matched fluorescent dyes known as CyDye DIGE fluors. 2D DIGE involves use of a reference sample, known as an internal standard, which comprises equal amounts of all biological samples in the experiment. Including the internal standard on each gel in the experiment with the individual biological samples means that the abundance of each protein spot on a gel can be measured relative (i.e. as a ratio) to its corresponding spot in the internal standard present on the same gel. Ettan DIGE is the system of technologies that has been optimized to fully benefit from the advantages provided by 2D DIGE.

title: New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564.
abstract: In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

title: Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus.
abstract: In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.

title: Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus.
abstract: Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.

title: Correlation of proteomic and transcriptomic profiles of Staphylococcus aureus during the post-exponential phase of growth.
abstract: A combined proteomic and transcriptomic analysis of Staphylococcus aureus strain N315 was performed to study a sequenced strain at the system level. Total protein and membrane protein extracts were prepared and analyzed using various proteomic workflows including: 2-DE, SDS-PAGE combined with microcapillary LC-MALDI-MS/MS, and multidimensional liquid chromatography. The presence of a protein was then correlated with its respective transcript level from S. aureus cells grown under the same conditions. Gene-expression data revealed that 97% of the 2'596 ORFs were detected during the post-exponential phase. At the protein level, 23% of these ORFs (591 proteins) were identified. Correlation of the two datasets revealed that 42% of the identified proteins (248 proteins) were amongst the top 25% of genes with highest mRNA signal intensities, and 69% of the identified proteins (406 proteins) were amongst the top 50% with the highest mRNA signal intensities. The fact that the remaining 31% of proteins were not strongly expressed at the RNA level indicates either that some low-abundance proteins were identified or that some transcripts or proteins showed extended half-lives. The most abundant classes identified with the combined proteomic and transcriptomic approach involved energy production, translational activities and nucleotide transport, reflecting an active metabolism. The simultaneous large-scale analysis of transcriptomes and proteomes enables a global and holistic view of the S. aureus biology, allowing the parallel study of multiple active events in an organism.

title: Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis
abstract: Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.

title: Microbiology and proteomics, getting the best of both worlds!
abstract: High-throughput identification of proteins with the latest generation of hybrid high-resolution mass spectrometers is opening new perspectives in microbiology. I present, here, an overview of tandem mass spectrometry technology and bioinformatics for shotgun proteomics that make 2D-PAGE approaches obsolete. Non-labelling quantitative approaches have become more popular than labelling techniques on most proteomic platforms because they are easier to carry out while their quantitative outcome is rather robust. Parameters for recording mass spectrometry data, however, need to be chosen carefully and statistics to assess the confidence of the results should not be neglected. Interestingly, next-generation sequencing methodologies make any microbial model quickly amenable to proteomics, leading to the documentation of a wide range of organisms from diverse environments. Some recent discoveries made using microbial proteomics have challenged some biological dogma, such as: (i) initiation of the translation does not occur predominantly from ATG codons in some microorganisms, (ii) non-canonical initiation codons are used to regulate the production of specific but important proteins and (iii) a gene may code for multiple polypeptide species, heterogeneous in terms of sequences. Microbial diversity and microbial physiology can now be revisited by means of exhaustive comparative proteomic surveys where thousands of proteins are detected and quantified. Proteogenomics, consisting of better annotating of genomes with the help of proteomic evidence, is paving the way for integrated multi-omic approaches in microbiology. Finally, meta-proteomic tools and approaches are emerging for tackling the high complexity of the microbial world as a whole, opening new perspectives for assessing how microbial communities function.

title: Proteomics by mass spectrometry: approaches, advances, and applications.
abstract: Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.

title: A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.
abstract: The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10⁶ cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

title: Comparative phosphoproteomics studies of macrophage response to bacterial virulence effectors.
abstract: Lipopolysaccharide (LPS) from Gram-negative bacteria and cell wall components from Gram-positive bacteria are pathogenic inducers of host cell innate immune systems. In this study, we adapted stable isotope labeling with amino acid in cell culture (SILAC) and Fe(3+)-IMAC phosphopeptide enrichment method to study phosphoproteomic changes in bacterial virulence factors induced macrophage RAW 264.7 cells. In total, we quantified 2657 phosphopeptides and 1990 phosphopeptides in LPS treated and heat-killed Staphylococcus aureus (HKSA) treated macrophage samples respectively. Functional bioinformatics analysis was followed to show differences between LPS and HKSA stimulated macrophage signaling pathways. We identified differences in immune-related signaling networks, including Erk1/2 signaling pathway, Jak/Stat signaling pathway, Jnk signaling pathway, and revealed differences in cytoskeleton reorganization, GTPase regulators and in phosphorylation motifs.

title: The impact of CodY on virulence determinant production in community‐associated methicillin‐resistant Staphylococcus aureus
abstract: Staphylococcus aureus is a leading human pathogen of both hospital and community‐associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ‐based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA‐MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post‐exponential and stationary growth. Specifically, we observe hyper‐production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry‐based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

title: An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry
abstract: A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography‐based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser‐induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side‐reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5‐iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1–105) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.

title: An exclusion list based label-free proteome quantification approach using an LTQ Orbitrap.
abstract: Label-based mass spectrometry is a powerful tool for large-scale protein identification and quantification. However, it requires the chemical or metabolic incorporation of the labeled compound(s) which can be difficult to attain, e.g. for non-cultivable organisms or scarce sample, such as biopsies. Therefore, we set out to develop and validate an efficient label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) workflow based on optimized instrument settings and incremental exclusion lists.



To increase the number of quantified peptides an incremental exclusion list was incorporated along with optimized instrument settings for the used LTQ Orbitrap. As a proof of concept, label-free quantification data from this optimized approach were compared to the results of control measurements without exclusion lists and of an in vivo metabolic labeling GeLC/MS/MS experiment. The data were drawn from Staphylococcus aureus whole cell lysates of non-stressed and nitric oxide (NO)-stressed cells.



Compared to MS analysis without exclusion lists the new approach resulted in an increased number of identified peptides, enabling label-free quantification of more than 990 S. aureus proteins. With respect to the number of quantified proteins and differences in protein levels between the control and NO-treated samples the results of the new method were consistent with those of the GeLC/MS/MS experiment.



The application of exclusion lists and optimized instrument settings in LC/MS/MS analysis significantly enhances the sensitivity and resolution of label-free protein identification and quantification. Therefore, the new workflow is a powerful alternative to label-based quantification methods.

title: Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging.
abstract: As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its β-galactosidase activity found 8.3 and 9.8 ng/μg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/μg are obtained for top-down analysis, while 14.5 and 15.9 ng/μg were found in the bottom-up analysis.

title: Protein analysis by shotgun/bottom-up proteomics.
abstract: According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics. 
 
Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge. 
 
The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis. 
 
“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins . The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future. 
 
 
 
Figure 1 
 
Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ... 
 
 
 
In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.

title: Top-Down Proteomic Identification of Furin-Cleaved α-Subunit of Shiga Toxin 2 from Escherichia coli O157:H7 Using MALDI-TOF-TOF-MS/MS
abstract: A method has been developed to identify the α-subunit of Shiga toxin 2 (α-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. Expression of Stx2 was induced by culturing E. coli O157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleaves α-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at m/z 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment of α-Stx2.

title: Comparative proteomics analyses reveal a potential biomarker for the detection of vancomycin-intermediate Staphylococcus aureus strains.
abstract: Vancomycin-intermediate Staphylococcus aureus (VISA) strains tend to develop during glycopeptide treatment of infections caused by methicillin-resistant S. aureus (MRSA). Rapid and effective detection methods for VISA strains are lacking, and mechanisms of resistance are unclear. Here, global comparative proteomic approaches have been used to identify potential biomarkers of intermediate vancomycin resistance. With the use of high-resolution two-dimensional gels and iTRAQ mass tagging, numerous proteins were found to be differentially expressed between clinical MRSA and VISA isolates of the same multilocus sequence type. One of these, the predicted lytic transglycosylase SAV2095 (SceD-like protein), was selected for further study based on both its high level of induction in Mu50 and its predicted role in modeling the cell wall, which is the target of vancomycin. Relative SAV2095 mRNA expression levels were compared between 25 MRSA and VISA/heterogeneous VISA clinical isolates by real-time RT-PCR. The SAV2095 mRNA was significantly induced in all VISA isolates relative to all MRSA strains ( p < 0.001), and significant induction of SAV2095 was also seen for several potential heterogeneous VISA strains that appear vancomycin-sensitive by standard minimum inhibitory concentration-determining methods. Furthermore, strains selected in vitro for increasing levels of resistance from four unrelated clinical MRSA isolates displayed concomitant increases in levels of SAV2095 expression. Together, these results suggest that SAV2095 expression level could serve as a molecular diagnostic marker for the rapid detection of VISA.

title: Selection of bacterial virulence genes that are specifically induced in host tissues
abstract: A genetic system was devised that positively selects for bacterial genes that are specifically induced when bacteria infect their host. With the pathogen Salmonella typhimurium, the genes identified by this selection show a marked induction in bacteria recovered from mouse spleen. Mutations in all ivi (in vivo-induced) genes that were tested conferred a defect in virulence. This genetic system was designed to be of general use in a wide variety of bacterial-host systems and has several applications in both vaccine and antimicrobial drug development.

title: Simultaneous identification of bacterial virulence genes by negative selection.
abstract: An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

title: Application of subproteomics in the characterization of Gram-positive bacteria.
abstract: Gram-positive bacteria cause a series of diseases in human, animals and plants. There has been increasing interest in efforts to investigate pathogenesis of bacteria using multiple "omic" strategies including proteomics. Proteins in different cell fractions of bacteria may play different vital roles in various physiological processes, such as adhesion, invasion, internalization, sensing, respiration, oxidative stress protection and pathogenicity. Subproteomics specifically focuses on the pre-fractionated cellular proteins and thus may be able to characterize more low-abundance molecules that are usually overlooked by the traditional whole-cell proteomics, providing comprehensive information for further investigations. This review intends to outline the current progress, challenges and future development of subproteomics in the characterization of Gram-positive bacteria. This article is part of a Special Issue entitled: Proteomics: The clinical link.

title: Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection
abstract: Community‐associated methicillin‐resistant Staphylococcus aureus (CA‐MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA‐MRSA pathogenesis are poorly understood and a comprehensive analysis of CA‐MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2‐dimensional gel electrophoresis coupled with automated direct infusion‐tandem mass spectrometry (ADI‐MS/MS) analysis. Eleven known virulence‐related exoproteins differed in abundance between the strains, including alpha‐haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1‐phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA‐MRSA as a human pathogen.

title: Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.
abstract: Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

title: Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles
abstract: Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.

title: Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease
abstract: Men may not be able to multitask, but it is emerging that proteins can. This capacity of proteins to exhibit more than one function is termed protein moonlighting, and, surprisingly, many highly conserved proteins involved in metabolic regulation or the cell stress response have a range of additional biological actions which are involved in bacterial virulence. This review highlights the multiple roles exhibited by a range of bacterial proteins, such as glycolytic and other metabolic enzymes and molecular chaperones, and the role that such moonlighting activity plays in the virulence characteristics of a number of important human pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Helicobacter pylori, and Mycobacterium tuberculosis.

title: Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus.
abstract: We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.

title: The staphylococcal transferrin receptor: a glycolytic enzyme with novel functions.
abstract: To obtain iron from the host for growth, staphylococci have evolved sophisticated iron-scavenging systems including siderophores and a cell surface receptor for transferrin, the mammalian iron-transporting glycoprotein. The staphylococcal transferrin receptor has been identified as a member of a newly emerging family of multifunctional, cell-surface-associated glyceraldehyde-3-phosphate dehydrogenases, which not only retain their glycolytic enzyme activities but also bind diverse human serum proteins and possess NAD-ribosylating activity. These multiple functions suggest a potential contribution to virulence far beyond iron acquisition.

title: Evaluation of P1' substrate specificity of staphylococcal SplB protease.
abstract: Staphylococcus aureus is a dangerous human pathogen characterized by growing antibiotic resistance. Virulence of S. aureus relies on a variety of secreted and cell surface associated virulence factors among which certain proteolytic enzymes play an important role. Amid staphylococcal extracellular proteases, those encoded by the spl operon remain poorly characterized, both in terms of enzymology and their physiological role. Initial data demonstrated that Spl proteases exhibit restricted substrate specificity. This study describes development of convenient protein FRET substrates for SplB protease and characterization of the substrate preference of the protease at the P1' position. Kinetic data on hydrolysis of a panel of substrates substituted at the said position is provided.

title: Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage.
abstract: Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.

title: Quantitative NMR Metabolite Profiling of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Discriminates between Biofilm and Planktonic Phenotypes
abstract: Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.

title: Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics
abstract: Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

title: Proteomics-Based Identification of Anchorless Cell Wall Proteins as Vaccine Candidates against Staphylococcus aureus
abstract: Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization—time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.

title: Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false‐positive control
abstract: Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface‐exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital‐acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface “shaving” technique with either trypsin or proteinase‐K combined with LC‐MS/MS. Trypsin‐derived data were controlled using a “false‐positive” strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin‐shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface‐exposed peptides. Trypsin and proteinase‐K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase‐K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false‐positive strategy improved enrichment of surface‐exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance–surface protein SACOL0050 (pls) and penicillin‐binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix‐binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface‐exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.

title: Exploring extra-cellular proteins in methicillin susceptible and methicillin resistant Staphylococcus aureus by liquid chromatography–tandem mass spectrometry
abstract: Staphylococcus aureus (S. aureus) strains cause several diseases in humans from minor skin infections to severe lethal infections. To explore the virulence determinants of this important microorganism, two clinical isolates of methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) were subjected to proteomic analysis of their extracellular products using liquid chromatography–tandem mass spectrometry. The numbers of proteins identified in MSSA and MRSA extracellular products were 168 and 261; respectively, from them 117 were shared, while 144 proteins were unique to MRSA. The shared proteins, having a higher protein score with increased number of peptide matches in MRSA over MSSA, reflect the relatively active secretory state of MRSA rather than biased analytical variances. Characteristic determinants for MRSA were identified; mostly found to play a role in the virulence. We conclude that MRSA produces distinct proteins considered as its virulence determinants and we found that the shared extracellular products are more abundant in MRSA than MSSA that supporting the high invasiveness of MRSA over MSSA in pathogenesis.

title: Oxidative stress triggers thiol oxidation in the glyceraldehyde‐3‐phosphate dehydrogenase of Staphylococcus aureus
abstract: The high‐resolution two‐dimensional protein gel electrophoresis technique combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was used to analyse the oxidative stress response in Staphylococcus aureus COL. Exponentially growing cells were supplemented with 100 mM H2O2 leading to a growth arrest lasting 30 min. The comparison of the two‐dimensional pattern of cytoplasmic protein extracts of stressed and unstressed cells revealed only a few changes in the protein synthesis profile. However, the isoelectric points of Gap (glyceraldehyde‐3‐phosphate dehydrogenase), AhpC (alkylhydroperoxide reductase) and MvaS (HMG‐CoA‐synthase) changed strikingly. For analysis of the modification of Gap, tandem hybrid mass spectrometry (Q‐Star) was used. The observed pI shift resulted from the oxidation to sulphonic acid of cysteine 151, which is crucial for catalytic activity. A drop in ATP and a complete inactivation of Gap was accompanied by the growth arrest. About 30 min after the addition of H2O2, the damaged Gap was still present, but a new protein spot at the original location became visible, representing the newly synthesized enzyme that is active again. This is accompanied by the restoration of Gap enzyme activity, ATP levels and recovery of growth. There is a strong correlation between growth, ATP level and Gap activity under oxidative stress conditions, indicating that the H2O2‐triggered Gap inactivation might be one reason for growth arrest under these conditions. Our data indicate that the damaged Gap protein was not repaired.

title: Nitric oxide dioxygenase: an enzymic function for flavohemoglobin.
abstract: Nitric oxide (NO•) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO•-resistant mutants of Escherichia coli were isolated that rapidly consumed NO•. An NO•-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3−. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO•. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO•. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO• detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage.

title: Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme.
abstract: Cell-wall sorted proteins of the Staphylococcus aureus iron-regulated surface determinant system bind human hemoproteins, remove the heme molecule, and transport heme through the cell wall and plasma membrane for accumulation in the bacterial cytoplasm. Once inside the cell, the porphyrin ring of heme is degraded by heme degrading monooxygenases, leading to the formation of free iron for use by the bacterium as a nutrient source.

title: Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo.
abstract: Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.

title: Proteome analysis of host–pathogen interactions: Investigation of pathogen responses to the host cell environment
abstract: Infectious diseases are still a major health risk, and thus a better understanding of the interaction between human host cells and pathogenic microbes is urgently required. Since the interplay between both partners is highly complex, genome‐wide analysis by OMICs approaches will likely make a major contribution to the elucidation of the pathophysiology of infection processes. In the concert of OMICs technologies, proteomics is particularly important because it reveals changes in the active players of the cell and has thus a close relationship to the phenotypic changes observed. While proteomic studies of in vitro‐grown microbial pathogens are routinely established in many labs, in vivo proteomic approaches are still rare. Here, we will review the challenges and recent developments of proteomic analysis of microbial pathogens derived from cell culture or in vivo infection settings and summarize some lessons that have been learned from these studies.

title: Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells
abstract: Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

title: Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus
abstract: Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.

title: Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release
abstract: Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

title: Bacterial outer membrane vesicles and the host-pathogen interaction.
abstract: Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.

title: Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis-like skin inflammation
abstract: To cite this article: Hong S‐W, Kim M‐R, Lee E‐Y, Kim JH, Kim Y‐S, Jeon SG, Yang J‐M, Lee B‐J, Pyun B‐Y, Gho YS, Kim Y‐K. Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis‐like skin inflammation. Allergy 2011; 66: 351–359.

title: Bacterial biofilms: a common cause of persistent infections.
abstract: Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

title: Antibiotic resistance of bacteria in biofilms
abstract: Bacteria that adhere to implanted medical devices or damaged tissue can encase themselves in a hydrated matrix of polysaccharide and protein, and form a slimy layer known as a biofilm. Antibiotic resistance of bacteria in the biofilm mode of growth contributes to the chronicity of infections such as those associated with implanted medical devices. The mechanisms of resistance in biofilms are different from the now familiar plasmids, transposons, and mutations that confer innate resistance to individual bacterial cells. In biofilms, resistance seems to depend on multicellular strategies. We summarise the features of biofilm infections, review emerging mechanisms of resistance, and discuss potential therapies.

title: Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection
abstract: The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.

title: Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction
abstract: Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

title: Comparative Exoproteomics and Host Inflammatory Response in Staphylococcus aureus Skin and Soft Tissue Infections, Bacteremia, and Subclinical Colonization
abstract: The exoproteome of Staphylococcus aureus contains enzymes and virulence factors that are important for host adaptation. We investigated the exoprotein profiles and cytokine/chemokine responses obtained in three different S. aureus-host interaction scenarios by using two-dimensional gel electrophoresis (2-DGE) and two-dimensional immunoblotting (2D-IB) combined with tandem mass spectrometry (MS/MS) and cytometric bead array techniques. The scenarios included S. aureus bacteremia, skin and soft tissue infections (SSTIs), and healthy carriage. By the 2-DGE approach, 12 exoproteins (the chaperone protein DnaK, a phosphoglycerate kinase [Pgk], the chaperone GroEL, a multisensor hybrid histidine kinase, a 3-methyl-2-oxobutanoate hydroxymethyltransferase [PanB], cysteine synthase A, an N-acetyltransferase, four isoforms of elongation factor Tu [EF-Tu], and one signature protein spot that could not be reliably identified by MS/MS) were found to be consistently present in more than 50% of the bacteremia isolates, while none of the SSTI or healthy-carrier isolates showed any of these proteins. By the 2D-IB approach, we also identified five antigens (methionine aminopeptidase [MetAPs], exotoxin 15 [Set15], a peptidoglycan hydrolase [LytM], an alkyl hydroperoxide reductase [AhpC], and a haptoglobin-binding heme uptake protein [HarA]) specific for SSTI cases. Cytokine and chemokine production varied during the course of different infection types and carriage. Monokine induced by gamma interferon (MIG) was more highly stimulated in bacteremia patients than in SSTI patients and healthy carriers, especially during the acute phase of infection. MIG could therefore be further explored as a potential biomarker of bacteremia. In conclusion, 12 exoproteins from bacteremia isolates, MIG production, and five antigenic proteins identified during SSTIs should be further investigated for potential use as diagnostic markers.

title: Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.
abstract: A single insertion of transposon Tn917LTV1 into the chromosome of a Staphylococcus aureus clinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extracellular and cell-wall-associated proteins. Detailed comparison of phenotypes associated with the mutant, 11D2, and the parent, DB, indicated that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of alpha-hemolysin, which is not detectable in Agr- mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agr- phenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.

title: Regulation of virulence determinants in Staphylococcus aureus: complexity and applications.
abstract: The virulence of Staphylococcus aureus is essentially determined by cell wall associated proteins and secreted toxins that are regulated and expressed according to growth phases and/or growth conditions. Gene expression is regulated by specific and sensitive mechanisms, most of which act at the transcriptional level. Regulatory factors constitute numerous complex networks, driving specific interactions with target gene promoters. These factors are largely regulated by two-component regulatory systems, such as the agr, saeRS, srrAB, arlSR and lytRS systems. These systems are sensitive to environmental signals and consist of a sensor histidine kinase and a response regulator protein. DNA-binding proteins, such as SarA and the recently identified SarA homologues (SarR, Rot, SarS, SarT, SarU), also regulate virulence factor expression. These homologues might be intermediates in the regulatory networks. The multiple pathways generated by these factors allow the bacterium to adapt to environmental conditions rapidly and specifically, and to develop infection. Precise knowledge of these regulatory mechanisms and how they control virulence factor expression would open up new perspectives for antimicrobial chemotherapy using key inhibitors of these systems.

title: Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus
abstract: We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system.

title: Regulation of virulence determinants in Staphylococcus aureus.
abstract: The pathogenicity of Staphylococcus aureus depends on the combined action of more than 40 different extracellular toxins, enzymes and cell surface proteins. A global regulator agr controls the production of many of these virulence factors by a regulating RNA molecule, RNAIII. Most of the virulence genes regulated by RNAIII are also regulated by SarA and a family of homologous proteins. The Sar proteins appear to repress transcription of individual virulence genes or sets of genes. As some Sar proteins also repress one or more sar homologous genes an increased production of a single Sar protein can result in decreased expression of some virulence genes, and an increased expression of others. Results are presented suggesting that RNAIII might function as an antirepressor, binding one or more of the Sar proteins.

title: The role of environmental factors in the regulation of virulence-determinant expression in Staphylococcus aureus 8325-4.
abstract: Staphylococcus aureus is a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulence-determinant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator (sarA), alpha-haemolysin (hla), surface protein A (spa) and toxic-shock syndrome toxin-1 (tst) genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression of hla, tst and spa was strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), but sarA was relatively unaffected. The global regulator of expression of virulence-determinant genes, agr (accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression of tst in wild-type or agr backgrounds and failed to relieve the salt suppression. Expression of tst was strongly stimulated in several low-metal environments, independently of agr, whilst spa levels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.

title: Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry
abstract: One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.

title: Staphylococcus aureus CodY Negatively Regulates Virulence Gene Expression
abstract: CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.

title: Role of Staphylococcus aureus Global Regulators sae and σB in Virulence Gene Expression during Device-Related Infection
abstract: The ability of Staphylococcus aureus to adapt to different environments is due to a regulatory network comprising several loci. Here we present a detailed study of the interaction between the two global regulators sae and σB of S. aureus and their influence on virulence gene expression in vitro, as well as during device-related infection. The expression of sae, asp23, hla, clfA, coa, and fnbA was determined in strain Newman and its isogenic saeS/R and sigB mutants by Northern analysis and LightCycler reverse transcription-PCR. There was no indication of direct cross talk between the two regulators. sae had a dominant effect on target gene expression during device-related infection. σB seemed to be less active throughout the infection than under induced conditions in vitro.

title: Influence of the Two-Component System SaeRS on Global Gene Expression in Two Different Staphylococcus aureus Strains
abstract: The two-component system SaeRS consisting of the histidin kinase SaeS and the response regulator SaeR is known to act on virulence gene expression in Staphylococcus aureus. In order to get a more comprehensive picture on SaeR-regulated genes, we studied the contribution of the two-component system on global gene expression by using both the proteomic and transcriptomic approach. Altogether, a loss of SaeRS resulted in a decreased amount of at least 17 extracellular proteins and two cell surface-associated proteins, among them several important virulence factors such as HlgA, HlgB, HlgC, LukF, and LukM. SaeRS activates the expression of these genes at the transcriptional level. The amount of the five proteins Aur, SspA, SsaA, Plc, and GlpQ was negatively influenced by SaeRS. However, the transcription of the corresponding genes was not affected by the two-component system. SaeRS had also no measurable influence on the transcription of the regulatory genes agr, sarA, arlRS, and sigB that contribute to the regulation of SaeRS-dependent virulence factors identified in this investigation. Our results clearly show that SaeRS is strongly involved in the tight temporal control of virulence factor expression in S. aureus. Its precise role within the regulatory network remains to be determined.

title: Phosphorylation of Isocitrate dehydrogenase of Escherichia coli.
abstract: Addition of acetate to a stationary phase culture of Escherichia coli in glycerol mineral salts medium containing phosphorus-32-labeled orthophosphate results in rapid loss of isocitrate dehydrogenase activity and concomitant incorporation of phosphorus-32 into the enzyme. This is the first example of protein phosphorylation in a bacterium in which the endogenous substrate for the protein kinase has been identified.

title: A Eukaryotic Type Serine/Threonine Kinase and Phosphatase inStreptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and Virulence*
abstract: Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.

title: A Eukaryotic-like Ser/Thr Kinase Signals Bacteria to Exit Dormancy in Response to Peptidoglycan Fragments
abstract: Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.

title: Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes
abstract: The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [32P]‐orthophosphate, one‐unit pH 2‐DE, combined with MS. Highly reproducible 2‐D profiles of phosphoproteins were obtained with early stationary‐phase cells. The 2‐D profiles contained at least 80 clearly labelled spots in the pH range 4–7. Forty‐six spots were analysed by MS (confirmed in most cases by LC‐MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary‐phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [32P]‐labelled spots were also stained by Pro‐Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro‐Q.

title: Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis
abstract: The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2‐D gel‐based approach combining Pro‐Q Diamond staining and [33P]‐labeling. In exponentially growing B. subtilis cells 27 proteins could be identified after staining with Pro‐Q Diamond and/or [33P]‐labeling and one additional protein was labeled solely by [33P] resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor σB. We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C‐termini were verified by MS (T171, T186, T181, respectively).

title: The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins
abstract: Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over‐representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species–specific, and therefore have probably co‐evolved with the adaptation of the bacterial species to their present‐day ecological niches.

title: Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.
abstract: Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

title: Hydrophilic interaction chromatography for fractionation and enrichment of the phosphoproteome.
abstract: Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formidable challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. It is recognized that reduction of sample complexity or enrichment of the phosphopeptide pool is a necessary prerequisite for global phospho-proteomics. Immobilized metal affinity chromatography (IMAC) and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic-based enrichment strategies. However, each is not without shortcomings. Both techniques provide little fractionation of phosphorylated species and are compromised by competition and co-elution of highly acidic peptides. Here, we describe a phosphopeptide prefractionation scheme using hydrophilic interaction chromatography, which both enriches the phosphopeptide pool and efficiently fractionates the remaining peptides. When used in front of IMAC, the selectivity of the metal affinity resin is improved to greater than 95%. The lack of significant numbers of nonphosphorylated peptides also allows for more efficient use of the mass spectrometer duty cycle in that the instrument spends nearly all of its time in sequencing the phosphopeptides.

title: Bacterial Phosphoproteomic Analysis Reveals the Correlation Between Protein Phosphorylation and Bacterial Pathogenicity
abstract: Increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. Recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaustive site-specific bacterial phosphoproteomes. The high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen–host interactions; the high abundance of multi-phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions.

title: A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.
abstract: Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

title: Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species
abstract: Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.

title: The impact of serine/threonine phosphorylation in Staphylococcus aureus.
abstract: In prokaryotes and eukaryotes, phosphotransfer represents a common mechanism to regulate cellular functions. Recent work revealed that modulation of cellular processes by eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) are widespread in bacteria. During the last two years, first evidence on the role of Ser/Thr phosphorylation/dephosphorylation in Staphylococcus aureus has emerged leading to the identification of a functional STK and corresponding STP. Due to homology to known STKs/STPs in other bacterial species the kinase was designated PknB or alternatively Stk/Stk1, and the phosphatase Stp. The role of these enzymes in S. aureus has been examined by use of knock-out mutants and a kinase-overexpressing strain. These studies uncovered PknB/Stk and Stp as modulators of cell wall structure and susceptibility to cell wall-acting antibiotics such as certain beta-lactams and tunicamycin. By utilizing transcriptional profile analysis a strong regulatory impact of PknB/Stk on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis could be identified. Moreover, PknB/Stk is able to phosphorylate MgrA, thereby regulating activity of the efflux pump NorA. In a mouse pyelonephritis model PknB/Stk has been shown to play a role in virulence. Overall, Ser/Thr phosphorylation/dephosphorylation is a common theme in regulation of cellular functions determining metabolic activity and virulence also in the major human pathogen S. aureus.

title: Insights from site-specific phosphoproteomics in bacteria.
abstract: Recent advances in mass spectrometry allowed the charting of bacterial serine/threonine/tyrosine phosphoproteomes with unprecedented accuracy, including the acquisition of a large number of phosphorylation sites. Phosphorylated bacterial proteins are involved in some key housekeeping processes, and their phosphorylation is expected to play an important regulatory role. When coupled to stable isotope labeling by amino acids in cell culture (SILAC), high-resolution mass spectrometry allows the detection of changes in the occupancy of phosphorylation sites in response to various stimuli. This and similar approaches promise to lead bacterial phosphoproteomics into the era of systems biology, where the entire phosphorylation-based regulatory networks will be charted, modelled, and ultimately engineered to obtain desired properties.

title: Characterization of a Serine/Threonine Kinase Involved in Virulence of Staphylococcus aureus
abstract: Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Δstk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

title: Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase
abstract: Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

title: Evidence for a new post-translational modification in Staphylococcus aureus: hydroxymethylation of asparagine and glutamine.
abstract: Staphylococcus aureus is an opportunistic pathogen whose infectious capacity depends on surface proteins, which enable bacteria to colonize and invade host tissues and cells. We analyzed "trypsin-shaved" surface proteins of S. aureus cultures by high resolution LC-MS/MS at different growth stages and culture conditions. Some modified peptides were identified, with a mass shift corresponding to the addition of a CH₂O group (+30.0106 u). We present evidence that this shift corresponds to a hyxdroxymethylation of asparagine and glutamine residues. This known but poorly documented post-translational modification was only found in a few proteins of S. aureus grown under specific conditions. This specificity seemed to exclude the hypothesis of an artifact due to sample preparation. Altogether hydroxymethylation was observed in 35 peptides from 15 proteins in our dataset, which corresponded to 41 modified sites, 35 of them being univocally localized. While no function can currently be assigned to this post-translational modification, we hypothesize that it could be linked to modulation of virulence factors, since it was mostly found on some surface proteins of S. aureus.

title: A role for glycosylated serine-rich repeat proteins in gram-positive bacterial pathogenesis.
abstract: Bacterial attachment to host surfaces is a pivotal event in the biological and infectious processes of both commensal and pathogenic bacteria, respectively. Serine-rich repeat proteins (SRRPs) are a family of adhesins in Gram-positive bacteria that mediate attachment to a variety of host and bacterial surfaces. As such, they contribute towards a wide-range of diseases including sub-acute bacterial endocarditis, community-acquired pneumonia, and meningitis. SRRPs are unique in that they are glycosylated, require a non-canonical Sec-translocase for transport, and are largely composed of a domain containing hundreds of alternating serine residues. These serine-rich repeats are thought to extend a unique non-repeat (NR) domain outward away from the bacterial surface to mediate adhesion. So far, NR domains have been determined to bind to sialic acid moieties, keratins, or other NR domains of a similar SRRP. This review summarizes how this important family of bacterial adhesins mediates bacterial attachment to host and bacterial cells, contributes to disease pathogenesis, and might be targeted for pharmacological intervention or used as novel protective vaccine antigens. This review also highlights recent structural findings on the NR domains of these proteins.

title: Both GtfA and GtfB are Required for SraP Glycosylation in Staphylococcus aureus
abstract: Staphylococcus aureus has been shown to bind to human platelets through a variety of surface molecules, including serine-rich adhesin for platelets (SraP). The SraP mutant strain of S. aureus is significantly impaired in its ability to initiate infection compared with the wild strain. SraP is a cell wall-anchored, glycosylated protein. A previous study revealed that SecY2, Asp1, Asp2, Asp3, and SecA2 in the SraP operon were required for the efficient transport of glycosylated SraP from the cytoplasm to the bacterial cell surface. However, no glycosyltransferase (Gtf) was found to be involved in the glycosylation of SraP. In this study, SraP was found in all of the 55 clinical isolates of S. aureus using a real-time polymerase chain reaction assay. Sequence and phylogenetic analysis showed that GtfA and GtfB in the SraP operon were highly conserved in most of these clinical isolates. Conserved domains analysis revealed that both GtfA and GtfB contained a GT1_GtfA-like domain. Structural homology analysis inferred that they are both Gtfs. We then constructed an in vivo glycosylation system in Escherichia coli using SraP1–743 as the substrate and GtfA and GtfB as the Gtfs. Using this system, we found that GtfA and GtfB were the Gtfs that transferred the N-acetylglucosamine-containing oligosaccharides to the recombinant SraP1–743. Deletion of either one or both of the Gtfs abolished the glycosylation of SraP. In summary, GtfA and GtfB in the SraP operon are highly conserved in most clinical isolates of S. aureus, and both GtfA and GtfB are required for SraP glycosylation.

title: Bacterial glycoproteins: Functions, biosynthesis and applications
abstract: Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S‐layer glycoproteins of Archeabacteria. Apart from these, membrane‐associated, surface‐associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.

title: Glycosylation and biogenesis of a family of serine-rich bacterial adhesins.
abstract: Glycosylation of bacterial proteins is an important process for bacterial physiology and pathophysiology. Both O- and N-linked glycan moieties have been identified in bacterial glycoproteins. The N-linked glycosylation pathways are well established in Gram-negative bacteria. However, the O-linked glycosylation pathways are not well defined due to the complex nature of known O-linked glycoproteins in bacteria. In this review, we examine a new family of serine-rich O-linked glycoproteins which are represented by fimbriae-associated adhesin Fap1 of Streptococcus parasanguinis and human platelet-binding protein GspB of Streptococcus gordonii. This family of glycoproteins is conserved in streptococcal and staphylococcal species. A gene cluster coding for glycosyltransferases and accessory Sec proteins has been implicated in the protein glycosylation. A two-step glycosylation model is proposed. Two glycosyltransferases interact with each other and catalyse the first step of the protein glycosylation in the cytoplasm; the cross-talk between glycosylation-associated proteins and accessory Sec components mediates the second step of the protein glycosylation, an emerging mechanism for bacterial O-linked protein glycosylation. Dissecting the molecular mechanism of this conserved biosynthetic pathway offers opportunities to develop new therapeutic strategies targeting this previously unrecognized pathway, as serine-rich glycoproteins have been shown to play a role in bacterial pathogenesis.