bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents
Authors: Vaara Martti , Fox John , Loidl Günther , Siikanen Osmo , Apajalahti Juha , Hansen Frank , Frimodt-Møller Niels , Nagai Junya , Takano Mikihisa , Vaara Timo 
Published: July 2008

ABSTRACT

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-
d
-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of
Escherichia coli
were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of
Enterobacteriaceae
and against
Acinetobacter baumannii
. At subinhibitory concentrations, it dramatically sensitized
A. baumannii
to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against
Pseudomonas aeruginosa
. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.
Antifungal Resistance of
Candida glabrata
Vaginal Isolates and Development of a Quantitative Reverse Transcription-PCR-Based Azole Susceptibility Assay
Authors: Gygax Scott E. , Vermitsky John-Paul , Chadwick Sean G. , Self Matthew J. , Zimmerman Jessica A. , Mordechai Eli , Adelson Martin E. , Trama Jason P. 
Published: July 2008

ABSTRACT

A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in
Candida glabrata
, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a ≥3-fold increase in
CDR1
expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.
Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from
Neisseria gonorrhoeae
and YdhE from
Escherichia coli
Authors: Long Feng , Rouquette-Loughlin Corinne , Shafer William M. , Yu Edward W. 
Published: July 2008

ABSTRACT

Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of
Neisseria gonorrhoeae
and the YdhE protein of
Escherichia coli
have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro.
E. coli
strains expressing
norM
or
ydhE
showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in
E. coli
, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na
+
-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.
Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of
Yersinia pseudotuberculosis
by Macrophages
Authors: Zhang Yue , Murtha James , Roberts Margaret A. , Siegel Richard M. , Bliska James B. 
Published: July 2008

ABSTRACT

Yersinia pseudotuberculosis
uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and
Y. pseudotuberculosis
can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV
+
wild-type, and
yop
mutant
Y. pseudotuberculosis
strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV
+
Y. pseudotuberculosis
. Interestingly, macrophages restricted survival of pYV
+
but not pYV-cured or
ΔyopB Y. pseudotuberculosis
within phagosomes: only a small fraction of the pYV
+
bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV
+
Y. pseudotuberculosis
died of apoptosis after 20 h. Analysis of
yop
mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV
+
Y. pseudotuberculosis
with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of
Y. pseudotuberculosis
suggests new roles for the T3SS in
Yersinia
pathogenesis.
Stages of Meningococcal Sepsis Simulated In Vitro, with Emphasis on Complement and Toll-Like Receptor Activation
Authors: Hellerud Bernt Christian , Stenvik Jørgen , Espevik Terje , Lambris John D. , Mollnes Tom Eirik , Brandtzaeg Petter 
Published: July 2008

ABSTRACTThe clinical presentation of meningococcal disease is closely related to the number of meningococci in the circulation. This study aimed to examine the activation of the innate immune system after being exposed to increasing and clinically relevant concentrations of meningococci. We incubated representativeNeisseria meningitidisserogroup B (ST-32) and serogroup C (ST-11) strains and a lipopolysaccharide (LPS)-deficient mutant (the 44/76lpxAmutant) in human serum and whole blood and measured complement activation and cytokine secretion and the effect of blocking these systems. HEK293 cells transfected with Toll-like receptors (TLRs) were examined for activation of NF-κB. The threshold for cytokine secretion and activation of NF-κB was 103to 104meningococci/ml. LPS was the sole inflammation-inducing molecule at concentrations up to 105to 106meningococci/ml. The activation was dependent on TLR4-MD2-CD14. Complement contributed to the inflammatory response at ≥105to 106meningococci/ml, and complement activation increased exponentially at ≥107bacteria/ml. Non-LPS components initiated TLR2-mediated activation at ≥107bacteria/ml. As the bacterial concentration exceeded 107/ml, TLR4 and TLR2 were increasingly activated, independent of CD14. In this model mimicking human disease, the inflammatory response toN. meningitidiswas closely associated with the bacterial concentration. Therapeutically, CD14 inhibition alone was most efficient at a low bacterial concentration, whereas addition of a complement inhibitor may be beneficial when the bacterial load increases.
Bacterial Protein Secretion Is Required for Priming of CD8+T Cells Specific for theMycobacterium tuberculosisAntigen CFP10
Authors: Woodworth Joshua S. , Fortune Sarah M. , Behar Samuel M. 
Published: July 2008

ABSTRACTMycobacterium tuberculosisinfection elicits antigen-specific CD8+T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted byM. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion byM. tuberculosisis required for the priming of CD8+T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8+T cells in vivo. By using well-definedM. tuberculosismutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8+T cells specific for CFP10. CD4+and CD8+T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8+T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8+T-cell response duringM. tuberculosisinfection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation duringM. tuberculosisinfection and in vaccine development.
Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation
Authors: Overhage Joerg , Campisano Andrea , Bains Manjeet , Torfs Ellen C. W. , Rehm Bernd H. A. , Hancock Robert E. W. 
Published: July 2008

ABSTRACT

The ability to form biofilms is a critical factor in chronic infections by
Pseudomonas aeruginosa
and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 μg/ml, far below that required to kill or inhibit growth (MIC = 64 μg/ml). LL-37 also affected existing, pregrown
P. aeruginosa
biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.
A Functional Collagen Adhesin Gene,
acm
, in Clinical Isolates of
Enterococcus faecium
Correlates with the Recent Success of This Emerging Nosocomial Pathogen
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Okhuysen Pablo C. , Murray Barbara E. 
Published: July 2008

ABSTRACT

Enterococcus faecium
recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some
E. faecium
strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90
E. faecium
isolates (99%
acm
+
) and found that the Acm protein was detected predominantly in clinically derived isolates, while the
acm
gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (
P
≤ 0.0003) was also demonstrated, and levels of adherence were highly correlated (
r
= 0.879) with the amount of cell surface Acm detected by whole-cell enzyme-linked immunosorbent assay and flow cytometry. Thirty-seven of 41 sera from patients with
E. faecium
infections showed reactivity with recombinant Acm, while only 4 of 30 community and hospitalized patient control group sera reacted (
P
≤ 0.0003); importantly, antibodies to Acm were present in all 14
E. faecium
endocarditis patient sera. Although pulsed-field gel electrophoresis indicated that multiple strains expressed collagen adherence, multilocus sequence typing demonstrated that the majority of collagen-adhering isolates, as well as 16 of 17 endocarditis isolates, are part of the hospital-associated
E. faecium
genogroup referred to as clonal complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of
E. faecium
and CC17 in nosocomial infections.
Contribution of the Collagen Adhesin Acm to Pathogenesis of
Enterococcus faecium
in Experimental Endocarditis
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Murray Barbara E. 
Published: July 2008

ABSTRACT

Enterococcus faecium
is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating
E. faecium
virulence determinants. Our previous studies showed that some clinical
E. faecium
isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic
acm
deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Δ
acm
::
cat
is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (
P
< 0.0001) and for valve colonization 1 and 3 hours after infection (
P
≤ 0.0002), making Acm the first factor shown to be important for
E. faecium
pathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conserved
acm
locus. Highly reduced
acm
mRNA levels (≥50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that
acm
transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in
E. faecium
collagen adherence by affinity-purified anti-Acm antibodies from
E. faecium
endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.
The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative
Escherichia coli
Mediates Binding to Extracellular Matrix Proteins
Authors: Farfan Mauricio J. , Inman Keith G. , Nataro James P. 
Published: July 2008

ABSTRACT

Enteroaggregative
Escherichia coli
(EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene,
aafA
. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of
aafA
. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.
Proteomic Analysis of
Vibrio cholerae
in Human Stool
Authors: LaRocque Regina C. , Krastins Bryan , Harris Jason B. , Lebrun Lauren M. , Parker Kenneth C. , Chase Michael , Ryan Edward T. , Qadri Firdausi , Sarracino David , Calderwood Stephen B. 
Published: July 2008

ABSTRACT

An effective vaccine for
Vibrio cholerae
is not yet available for use in the developing world, where the burden of cholera disease is highest. Characterizing the proteins that are expressed by
V. cholerae
in the human host environment may provide insight into the pathogenesis of cholera and assist with the development of an improved vaccine. We analyzed the
V. cholerae
proteins present in the stools of 32 patients with clinical cholera. The
V. cholerae
outer membrane porin, OmpU, was identified in all of the human stool samples, and many
V. cholerae
proteins were repeatedly identified in separate patient samples. The majority of
V. cholerae
proteins identified in human stool are involved in protein synthesis and energy metabolism. A number of proteins involved in the pathogenesis of cholera, including the A and B subunits of cholera toxin and the toxin-coregulated pilus, were identified in human stool. In a subset of stool specimens, we also assessed which in vivo expressed
V. cholerae
proteins were recognized uniquely by convalescent-phase as opposed to acute-phase serum from cholera patients. We identified a number of these in vivo expressed proteins as immunogenic during human infection. To our knowledge, this is the first characterization of the proteome of a pathogenic bacteria recovered from a natural host.
ACandida albicansMannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production
Authors: Pietrella Donatella , Lupo Patrizia , Rachini Anna , Sandini Silvia , Ciervo Alessandra , Perito Stefano , Bistoni Francesco , Vecchiarelli Anna 
Published: July 2008

ABSTRACTMannoproteins are cell wall components of pathogenic fungi and play major virulence and immunogenic roles with both their mannan and protein moieties. The 65-kDa mannoprotein (MP65) ofCandida albicansis a β-glucanase adhesin recognized as a major target of the human immune response against this fungus, and its recombinant product (rMP65; devoid of the mannan moiety) is presently under consideration as a vaccine candidate. Here we investigated cellular and molecular aspects of the interaction of rMP65 with human antigen-presenting cells. We also assessed the ability of rMP65 to initiate a T-cell response. Both the native mannosylated MP65 (nMP65) and the recombinant product were efficiently bound and taken up by macrophages and dendritic cells. However, contrarily to nMP65, rMP65 did not induce tumor necrosis factor alpha and interleukin-6 release from these cells. On the other hand, rMP65 was rapidly endocytosed by both macrophages and dendritic cells, in a process involving both clathrin-dependent and clathrin-independent mechanisms. Moreover, the RGD sequence inhibited rMP65 uptake to some extent. After internalization, rMP65 partially colocalized with lysosomal membrane-associated glycoproteins 1 and 2. This possibly resulted in efficient protein degradation and presentation to CD4+T cells, which proliferated and produced gamma interferon. Collectively, these results demonstrate that the absence of the mannan moiety does not deprive MP65 of the capacity to initiate the pattern of cellular and molecular events leading to antigen presentation and T-cell activation, which are essential features for further consideration of MP65 as a potential vaccine candidate.
The Interleukin-17 Receptor Plays a Gender-Dependent Role in Host Protection againstPorphyromonas gingivalis-Induced Periodontal Bone Loss
Authors: Yu Jeffrey J. , Ruddy Matthew J. , Conti Heather R. , Boonanantanasarn Kanitsak , Gaffen Sarah L. 
Published: July 2008

ABSTRACTInterleukin-17 (IL-17) is a proinflammatory cytokine secreted by the newly described CD4+Th17 subset, which is distinct from classic Th1 and Th2 lineages. IL-17 contributes to bone destruction in rheumatoid arthritis but is essential in host defense against pathogens that are susceptible to neutrophils. Periodontal disease (PD) is a chronic inflammatory condition initiated by anaerobic oral pathogens such asPorphyromonas gingivalis, and it is characterized by host-mediated alveolar bone destruction due primarily to the immune response. The role of IL-17 in PD is controversial. Whereas elevated IL-17 levels have been found in humans with severe PD, we recently reported that female C57BL/6J mice lacking the IL-17 receptor (IL-17RAKO) are significantly more susceptible to PD bone loss due to defects in the chemokine-neutrophil axis (J. J. Yu, M. J. Ruddy, G. C. Wong, C. Sfintescu, P. J. Baker, J. B. Smith, R. T. Evans, and S. L. Gaffen, Blood109:3794-3802, 2007). Since different mouse strains exhibit differences in susceptibility to PD as well as Th1/Th2 cell skewing, we crossed the IL-17RA gene knockout onto the BALB/c background and observed a similar enhancement in alveolar bone loss followingP. gingivalisinfection. Unexpectedly, in both strains IL-17RAKOfemale mice were much more susceptible to PD bone loss than males. Moreover, female BALB/c-IL-17RAKOmice were defective in producing anti-P. gingivalisimmunoglobulin G and the chemokines KC/Groα and MIP-2. In contrast, male mice produced normal levels of chemokines and anti-P. gingivalisantibodies, but they were defective in granulocyte colony-stimulating factor upregulation. This study demonstrates a gender-dependent effect of IL-17 signaling and indicates that gender differences should be taken into account in the preclinical and clinical safety testing of anti-IL-17 biologic therapies.