bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens
Authors: Ariel N. , Zvi A. , Makarova K. S. , Chitlaru T. , Elhanany E. , Velan B. , Cohen S. , Friedlander A. M. , Shafferman A. 
Published: July 2003

ABSTRACTBacillus anthracis(Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected asB. anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of aB. anthracismembrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera fromB. anthracisimmunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.
Interleukin-4 and Transforming Growth Factor β Have Opposing Regulatory Effects on Gamma Interferon-Mediated Inhibition ofCryptosporidium parvumReproduction
Authors: Lean I.-Sarah , McDonald Stuart A. C. , Bajaj-Elliott Mona , Pollok Richard C. G. , Farthing Michael J. G. , McDonald Vincent 
Published: July 2003

ABSTRACTIt was shown previously that enterocytes activated by gamma interferon (IFN-γ) are efficient effector cells in the killing ofCryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor β (TGF-β) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-β might modulate the effect of IFN-γ in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-β can abolish the inhibitory effect onC. parvumdevelopment and that neither IL-13 nor IL-10 influenced the action of IFN-γ. In contrast, IL-4 cooperated with low concentrations of IFN-γ (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-γ and IL-4 was intracellular Fe2+deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-γ signaling, were similar when cells were stimulated with IFN-γ alone and with IFN-γ and IL-4γ, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-β and IL-4 on IFN-γ function did not appear to involve any alteration in the level of expression of IFN-γ receptors.
Diminished Adhesion ofAnaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin
Authors: Choi Kyoung-Seong , Garyu Justin , Park Jinho , Dumler J. Stephen 
Published: July 2003

ABSTRACTAnaplasma phagocytophilumpropagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection withA. phagocytophilummodifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of β2integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h withA. phagocytophiluminfection, whereasEscherichia coliandStaphylococcus aureusrapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur withA. phagocytophiluminfections.
Requirement of the
Yersinia pseudotuberculosis
Effectors YopH and YopE in Colonization and Persistence in Intestinal and Lymph Tissues
Authors: Logsdon Lauren K. , Mecsas Joan 
Published: July 2003

ABSTRACT

The gram-negative enteric pathogen
Yersinia pseudotuberculosis
employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic
yop
mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a
yopHEMOJ
mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A
yopH
mutant strain specifically fails to colonize the mesenteric lymph nodes, but
yopE
and
yopO
mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that
Y. pseudotuberculosis
employs multiple strategies for colonization. In competition infections with wild-type
Y. pseudotuberculosis
, the presence of wild-type bacteria severely hindered the ability of the
yopH
,
yopE
, and
yopO
mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the
yop
mutants are unable to withstand.
The Absence of Linear Plasmid 25 or 28-1 ofBorrelia burgdorferiDramatically Alters the Kinetics of Experimental Infection via Distinct Mechanisms
Authors: Labandeira-Rey Maria , Seshu J. , Skare Jonathan T. 
Published: July 2003

ABSTRACTThe 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection inBorrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected withB. burgdorferilacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1−strains were able to disseminate throughout the host, whereas the lp25−strain was cleared within 48 h of inoculation. While the wild-typeB. burgdorferipersisted in tissues for the duration of the study, the lp28-1−mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1−mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1−mutant was able to disseminate and persist in C3H-scidmice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.
Pseudomonas aeruginosaSlime Glycolipoprotein Is a Potent Stimulant of Tumor Necrosis Factor Alpha Gene Expression and Activation of Transcription Activators Nuclear Factor κB and Activator Protein 1 in Human Monocytes
Authors: Lagoumintzis George , Christofidou Myrto , Dimitracopoulos George , Paliogianni Fotini 
Published: July 2003

ABSTRACTPseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts. Production of tumor necrosis factor alpha (TNF-α), primarily by cells of monocytic lineage, is a crucial event in the course of these infections. During in vivo infections withP. aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released. In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-α production by human monocytes. To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated withP. aeruginosaLPS or GLP. GLP was found to be a more potent stimulus for TNF-α production (threefold higher) by human monocytes than LPS. Moreover, its effect was comparable to that of viable bacteria. Quantitative mRNA analysis revealed predominantly transcriptional regulation. Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-κB and activator protein 1 (AP-1). NF-κB activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-κB through GLP. MoreoverP. aeruginosaGLP induced the formation of AP-1 complex with delayed kinetics compared with NF-κB but much more efficiently than the homologous LPS. These results identify GLP as the most important stimulant for TNF-α production by human monocytes. Activation of NF-κB and AP-1 byP. aeruginosaGLP may be involved not only in TNF-α induction but also in many of the inflammatory responses triggered in the course of infection withP. aeruginosa.
The C Terminus of YopT Is Crucial for Activity and the N Terminus Is Crucial for Substrate Binding
Authors: Sorg Isabel , Hoffmann Claudia , Dumbach Juergen , Aktories Klaus , Schmidt Gudula 
Published: July 2003

ABSTRACTRecently, it was shown thatYersiniaouter protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathioneS-transferase fusion proteins inEscherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.
Functional Selection of Vaccine Candidate Peptides from
Staphylococcus aureus
Whole-Genome Expression Libraries In Vitro
Authors: Weichhart Thomas , Horky Markus , Söllner Johannes , Gangl Susanne , Henics Tamàs , Nagy Eszter , Meinke Andreas , von Gabain Alexander , Fraser Claire M. , Gill Steve R. , Hafner Martin , von Ahsen Uwe 
Published: July 2003

ABSTRACT

An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen
Staphylococcus aureus
. A library built up from genomic fragments of the virulent
S. aureus
COL strain (methicillin-resistant
S. aureus
) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against
S. aureus
were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.
Delineation of the Molecular Mechanisms ofFrancisella tularensis-Induced Apoptosis in Murine Macrophages
Authors: Lai Xin-He , Sjöstedt Anders 
Published: July 2003

ABSTRACTFrancisella tularensisis a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection withF. tularensisstrain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochromecinto the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, theF. tularensisinfection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.
Stimulation of Neutrophil Granulocytes withMycobacterium bovisBacillus Calmette-Guérin Induces Changes in Phenotype and Gene Expression and Inhibits Spontaneous Apoptosis
Authors: Suttmann Henrik , Lehan Nadine , Böhle Andreas , Brandau Sven 
Published: July 2003

ABSTRACTPolymorphonuclear neutrophil granulocytes (PMN) have been implicated in the early inflammatory response against mycobacteria besides monocytes/macrophages. Yet, little is known about the interaction of mycobacteria with PMN. We investigated the potential ofMycobacterium bovisbacillus Calmette-Guérin (BCG) to stimulate and influence PMN phenotype, gene expression profile and spontaneous apoptosis. Flow cytometric analyses revealed an upregulation of the function-associated molecules Fcγ receptor III (FcγR III) and II (CD16 and CD32) as well as MAC-1 (CD11b and CD18) on BCG-stimulated PMN. As determined by cDNA microarrays and multiplex reverse transcriptase PCR, stimulation with BCG alters the expression of various genes for proinflammatory cytokines/chemokines or receptors in PMN. We detected an upregulation or de novo synthesis of interleukin 1α (IL-1α), IL-1β, IL-8, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, GRO-α, transforming growth factor β, MCP-1, IL-2 receptor γ (IL-2Rγ), IL-10Rα, and IL-6R. Genes for IL-9, IL-12α, IL-15, IL-5Rα, and IL-13Rα1were found to be downregulated or switched off. Furthermore, Giemsa and annexin V-propidium iodide double staining demonstrated an inhibition of spontaneous PMN apoptosis following BCG stimulation. Changes in phenotype and inhibition of apoptosis did not depend on direct mycobacterial stimulation alone, but were a result of an autocrine-paracrine stimulation mechanism. Our findings support the hypothesis that PMN become activated at the site of mycobacterial infections and that this activation might set the stage for a subsequent antimycobacterial immune response.
Binding of
Actinobacillus pleuropneumoniae
to Phosphatidylethanolamine
Authors: Jeannotte Marie-Eve , Abul-Milh Maan , Dubreuil J. Daniel , Jacques Mario 
Published: July 2003

ABSTRACT

The gram-negative bacterium
Actinobacillus pleuropneumoniae
is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of
A. pleuropneumoniae
serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that
A. pleuropneumoniae
serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of
A. pleuropneumoniae
in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against
A. pleuropneumoniae
serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of
A. pleuropneumoniae
serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for
A. pleuropneumoniae
serotype 1.
A. pleuropneumoniae
was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of
A. pleuropneumoniae
binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.
Construction, Characterization, and Immunogenicity of an AttenuatedSalmonella entericaSerovar TyphimuriumpgtEVaccine Expressing Fimbriae with Integrated Viral Epitopes from thespiCPromoter
Authors: Chen Huaiqing , Schifferli Dieter M. 
Published: July 2003

ABSTRACTTransmissible gastroenteritis virus (TGEV) is a porcine coronavirus that causes diarrhea, leading to near 100% mortality in neonatal piglets with corresponding devastating economic consequences. For the protection of neonatal and older animals, oral live vaccines present the attractive property of inducing desired mucosal immune responses, including colostral antibodies in sows—an effective means to passively protect suckling piglets. Newly attenuatedSalmonellavaccine constructs expressing TGEV S protein epitopes were studied and evaluated for improved humoral immune response to TGEV. The macrophage-inducibleSalmonella ssaHandspiC/ssaBpromoters were compared for their ability to express the TGEV C and A epitopes in the context of the heterologous 987P fimbriae onSalmonellavaccines. Compared to thessaHpromoter, theSalmonella cya crpvector elicited significantly higher levels of mucosal and systemic antibodies in orally immunized mice when the chimeric fimbriae were expressed from thespiCpromoter. TheSalmonella spiCpromoter construct induced the highest level of chimeric fimbriae after being taken up by the J774A.1 macrophagelike cells. TheSalmonella cya crpvaccine vector was shown to incorporate into 987P partially degraded chimeric subunits lacking the TGEV epitopes. In contrast, its isogenicpgtEmutant produced fimbriae consisting exclusively of intact chimeric subunits. Mice immunized orally with theSalmonella pgtEvaccine expressing chimeric fimbriae from thespiCpromoter elicited significantly higher systemic and mucosal antibody titers against the TGEV epitopes compared to the parental vaccine. This study indicates that theSalmonella cya crp pgtEvector and thespiCpromoter can be used successfully to improve immune responses toward heterologous antigens.
Comparative Genomic Indexing Reveals the Phylogenomics of
Escherichia coli
Pathogens
Authors: Anjum Muna F. , Lucchini Sacha , Thompson Arthur , Hinton Jay C. D. , Woodward Martin J. 
Published: July 2003

ABSTRACT

The
Escherichia coli
O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing
E. coli
O26 strains represent the same clonal lineage and that VT-expressing
E. coli
O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the
E. coli
species and identified regions of variation from the
E. coli
K-12 chromosomal backbone.