bluuebunny/crossref_metadata_2025 huggingface dataset:
Aspergillus
Cyclooxygenase-Like Enzymes Are Associated with Prostaglandin Production and Virulence
Authors: Tsitsigiannis Dimitrios I. , Bok Jin-Woo , Andes David , Nielsen Kristian Fog , Frisvad Jens C. , Keller Nancy P.
Published: July 2005

Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological activities in animals, plants, and fungi. Mammalian oxylipins, including the prostaglandins (PGs), mediate many immune and inflammation responses in animals. PG production by pathogenic microbes is theorized to play a role in pathogenesis. We have genetically characterized three
Aspergillus
genes,
ppoA
,
ppoB
, and
ppoC
, encoding fatty acid oxygenases similar in sequence to specific mammalian prostaglandin synthases, the cyclooxygenases. Enzyme-linked immunosorbent assay analysis showed that production of PG species is decreased in both
Aspergillus nidulans
and
A. fumigatus ppo
mutants, implicating Ppo activity in generating PGs. The
A. fumigatus
triple-
ppo
-silenced mutant was hypervirulent in the invasive pulmonary aspergillosis murine model system and showed increased tolerance to H
2
O
2
stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.
Degradation ofChlamydia pneumoniaeby Peripheral Blood Monocytic Cells
Authors: Wolf Katerina , Fischer Elizabeth , Hackstadt Ted
Published: July 2005

Chlamydia pneumoniaeis a common human respiratory pathogen that has been associated with a variety of chronic diseases, including atherosclerosis. The role of this organism in the pathogenesis of atherosclerosis remains unknown. A key question is howC. pneumoniaeis transferred from the site of primary infection to a developing atherosclerotic plaque. It has been suggested that circulating monocytes could be vehicles for dissemination ofC. pneumoniaesince the organism has been detected in peripheral blood monocytic cells (PBMCs). In this study we focused on survival ofC. pneumoniaewithin PBMCs isolated from the blood of healthy human donors. We found thatC. pneumoniaedoes not grow and multiply in cultured primary monocytes. InC. pneumoniae-infected monocyte-derived macrophages, growth of the organism was very limited, and the majority of the bacteria were eradicated. We also found that the destruction ofC. pneumoniaewithin infected macrophages resulted in a gradual diminution of chlamydial antigens, although some of these antigens could be detected for days after the initial infection. The detected antigens present in infected monocytes and monocyte-derived macrophages represented neither chlamydial inclusions nor intact organisms. The use of {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]}-6-aminocaproyl-d-erythro-sphingosine as a vital stain for chlamydiae proved to be a sensitive method for identifying rareC. pneumoniaeinclusions and was useful in the detection of even aberrant developmental forms.
Virulence of the Fungal Pathogen
Candida albicans
Requires the Five Isoforms of Protein Mannosyltransferases
Authors: Rouabhia Mahmoud , Schaller Martin , Corbucci Cristina , Vecchiarelli Anna , Prill Stephan K.-H. , Giasson Luc , Ernst Joachim F.
Published: July 2005

The
PMT
gene family in
Candida albicans
encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of
pmt
mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All
pmt
mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The
pmt5
mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the
pmt4
and
pmt6
mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of
C. albicans
and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.
Role for Nucleotide Excision Repair in Virulence of
Mycobacterium tuberculosis
Authors: Darwin K. Heran , Nathan Carl F.
Published: July 2005

Mutations in
Mycobacterium tuberculosis uvrB
result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a
uvrB
mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a
uvrB
mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of
M. tuberculosis
to both reactive nitrogen and reactive oxygen species in mice.
Invasive Phenotype ofCandida albicansAffects the Host Proinflammatory Response to Infection
Authors: Villar C. C. , Kashleva H. , Mitchell A. P. , Dongari-Bagtzoglou A.
Published: July 2005

Candida albicansis a major opportunistic pathogen in immunocompromised patients. Production of proinflammatory cytokines by host cells in response toC. albicansplays a critical role in the activation of immune cells and final clearance of the organism. Invasion of host cells and tissues is considered one of the virulence attributes of this organism. The purpose of this study was to investigate whether the ability ofC. albicansto invade host cells and tissues affects the proinflammatory cytokine responses by epithelial and endothelial cells. In this study we used the invasion-deficientRIM101gene knockout strain DAY25, the highly invasive strain SC5314, and highly invasiveRIM101-complemented strain DAY44 to compare the proinflammatory cytokine responses by oral epithelial or endothelial cells. Using a high-throughput approach, we found both qualitative and quantitative differences in the overall inflammatory responses toC. albicansstrains with different invasive potentials. Overall, the highly invasive strains triggered higher levels of proinflammatory cytokines in host cells than the invasion-deficient mutant triggered. Significant differences compared to the attenuated mutant were noted in interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha in epithelial cells and in IL-6, growth-related oncogene, IL-8, monocyte chemoattractant protein 1 (MCP-1), MCP-2, and granulocyte colony-stimulating factor in endothelial cells. Our results indicate that invasion of host cells and tissues byC. albicansenhances the host proinflammatory response to infection.
Heparin Stimulates
Staphylococcus aureus
Biofilm Formation
Authors: Shanks Robert M. Q. , Donegan Niles P. , Graber Martha L. , Buckingham Sarah E. , Zegans Michael E. , Cheung Ambrose L. , O'Toole George A.
Published: July 2005

Heparin, known for its anticoagulant activity, is commonly used in catheter locks.
Staphylococcus aureus
, a versatile human and animal pathogen, is commonly associated with catheter-related bloodstream infections and has evolved a number of mechanisms through which it adheres to biotic and abiotic surfaces. We demonstrate that heparin increased biofilm formation by several
S. aureus
strains. Surface coverage and the kinetics of biofilm formation were stimulated, but primary attachment to the surface was not affected. Heparin increased
S. aureus
cell-cell interactions in a protein synthesis-dependent manner. The addition of heparin rescued biofilm formation of
hla
,
ica
, and
sarA
mutants. Our data further suggest that heparin stimulation of biofilm formation occurs neither through an increase in
sigB
activity nor through an increase in polysaccharide intracellular adhesin levels. These finding suggests that heparin stimulates
S. aureus
biofilm formation via a novel pathway.
Human Antibody against Shiga Toxin 2 Administered to Piglets after the Onset of Diarrhea Due to
Escherichia coli
O157:H7 Prevents Fatal Systemic Complications
Authors: Sheoran Abhineet S. , Chapman-Bonofiglio Susan , Harvey Barrett R. , Mukherjee Jean , Georgiou George , Donohue-Rolfe Arthur , Tzipori Saul
Published: July 2005

Infection of children with Shiga toxin (Stx)-producing
Escherichia coli
(STEC) can lead to hemolytic-uremic syndrome (HUS) in 5 to 10% of patients. Stx2, one of two toxins liberated by the bacterium, is directly linked with HUS. We have previously shown that Stx-specific human monoclonal antibodies protect STEC-infected animals from fatal systemic complications. The present study defines the protective antibody dose in relation to the time of treatment after the onset of diarrhea in infected gnotobiotic piglets. Using the mouse toxicity model, we selected 5C12, an antibody specific for the A subunit, as the most effective Stx2 antibody for further characterization in the piglet model in which piglets developed diarrhea 16 to 40 h after bacterial challenge, followed by fatal neurological symptoms at 48 to 96 h. Seven groups of piglets received doses of 5C12 ranging from 6.0 mg/kg to 0.05 mg/kg of body weight, administered parenterally 48 h after bacterial challenge. The minimum fully protective antibody dose was 0.4 mg/kg, and the corresponding serum antibody concentration in these piglets was 0.7 μg (±0.5)/ml, measured 7 to 14 days after administration. Of 40 infected animals which received Stx2 antibody treatment of ≥0.4 mg/kg, 34 (85%) survived, while only 1 (2.5%) of 39 placebo-treated animals survived. We conclude that the administration of the Stx2-specific antibody was protective against fatal systemic complications even when it was administered well after the onset of diarrhea. These findings suggest that children treated with this antibody, even after the onset of bloody diarrhea, may be equally protected against the risk of developing HUS.
Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence ofActinobacillus pleuropneumoniaein the Porcine Respiratory Tract
Authors: Baltes Nina , N′diaye Mohamed , Jacobsen Ilse D. , Maas Alexander , Buettner Falk F. R. , Gerlach Gerald-F.
Published: July 2005

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue ofA. pleuropneumoniae. By constructing an isogenicA. pleuropneumoniae hlyXmutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment theA. pleuropneumoniae hlyXmutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using anA. pleuropneumoniaestrain carrying theluxABgenes as transcriptional fusion toaspAon the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression ofA. pleuropneumoniaein epithelial lining fluid.
Chlamydia pneumoniae
Uses the Mannose 6-Phosphate/Insulin-Like Growth Factor 2 Receptor for Infection of Endothelial Cells
Authors: Puolakkainen Mirja , Kuo Cho-Chou , Campbell Lee Ann
Published: July 2005

Several mechanisms for attachment and entry of
Chlamydia
have been proposed. We previously determined that the major outer membrane protein of
Chlamydia trachomatis
is glycosylated with a high-mannose oligosaccharide, and a similar structure inhibited the attachment and infectivity of
C. trachomatis
in epithelial cells. Because insulin-like growth factor 2 (IGF2) was shown to enhance the infectivity of
Chlamydia pneumoniae
but not
C. trachomatis
in endothelial cells, a hapten inhibition assay was used to analyze whether the mannose 6-phosphate (M6P)/IGF2 receptor that also binds M6P could be involved in infection of endothelial cells (HMEC-1) by
Chlamydia
. M6P and mannose 6-phosphate-poly[
N
-(2-hydroxyethyl)-acrylamide] (M6P-PAA) inhibited the infectivity of
C. pneumonia
e AR-39, but not
C. trachomatis
serovar UW5 or L2, while mannan inhibited the growth of
C. trachomatis
, but not
C. pneumoniae
. Using metabolically labeled organisms incubated with cells at 4°C (organisms attach but do not enter) or at 37°C (organisms attach and are internalized), M6P-PAA was shown to inhibit attachment and internalization of
C. pneumoniae
in endothelial cells but did not inhibit attachment or internalization of
C. trachomatis
serovar E or L2. These findings indicate that
C. pneumoniae
can utilize the M6P/IGF2 receptor and that the use of this receptor for attachment and entry differs between
C. pneumoniae
and
C. trachomatis
.
Toll-Like Receptor 4 Mediates Tolerance in Macrophages Stimulated withToxoplasma gondii-Derived Heat Shock Protein 70
Authors: Mun Hye-Seong , Aosai Fumie , Norose Kazumi , Piao Lian-Xun , Fang Hao , Akira Shizuo , Yano Akihiko
Published: July 2005

Peritoneal macrophages (PMs) from toll-like receptor 4 (TLR4)-deficient and wild-type (WT) mice were responsive to recombinantToxoplasma gondii-derived heat shock protein 70 (rTgHSP70) and natural TgHSP70 (nTgHSP70) in NO release, but those from TLR2-, myeloid differentiation factor 88 (MyD88)-, and interleukin-1R-associated kinase 4 (IRAK4)-deficient mice were not. Polymyxin B did not inhibit PM activation by TgHSP70 and nTgHSP70 from WT and TLR4-deficient mice, while it inhibited PM activation by lipopolysaccharide. Pretreatment of PMs from WT but not from TLR4-deficient mice with rTgHSP70 resulted in suppression of NO release on restimulation with rTgHSP70. Similarly, pretreatment of PMs from WT but not TLR4-deficient mice with nTgHSP70 resulted in suppression of NO release on restimulation with nTgHSP70. Polymyxin B did not inhibit rTgHSP70- and nTgHSP70-induced tolerance of PMs from TLR4-deficient mice. Furthermore, PMs from WT mice increased suppressor of cytokine-signaling-1 (SOCS-1) expression after restimulation with rTgHSP70, while those from TLR4-deficient mice did not. Phosphorylation of JNK and I-κBα occurred in rTgHSP70-induced tolerance of PMs from TLR4-deficient mice, but not in that from WT mice. These data indicated that TgHSP70 signaling mechanisms were mediated by TLR2, MyD88, and IRAK4, but not by TLR4. On the other hand, signaling of TgHSP70-induced tolerance was mediated by TLR4, and the expression of SOCS-1 suppressed the TLR2 signaling pathway.
Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of
Helicobacter pylori
Authors: Bauer Bianca , Moese Stefan , Bartfeld Sina , Meyer Thomas F. , Selbach Matthias
Published: July 2005

Helicobacter pylori
persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent
H. pylori
strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the
H. pylori
genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with
H. pylori
and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.
Illustration of Pneumococcal Polysaccharide Capsule during Adherence and Invasion of Epithelial Cells
Authors: Hammerschmidt Sven , Wolff Sonja , Hocke Andreas , Rosseau Simone , Müller Ellruth , Rohde Manfred
Published: July 2005

The capsular polysaccharide of
Streptococcus pneumoniae
represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation procedure did not require the use of capsule-specific antibodies. Visualization of pneumococci in intimate contact and invading cells demonstrated that pneumococci were devoid of capsular polysaccharide. Pneumococci not in contact with the cells did not show alterations in capsular polysaccharide. After infection of the cells, invasive pneumococci of different strains and serotypes were recovered. Single colonies of these recovered pneumococci exhibited an up-to-10
5
-fold-enhanced capacity to adhere and an up-to-10
4
-fold-enhanced capacity to invade epithelial cells. Electron microscopic studies using a lysine-ruthenium red (LRR) fixation procedure or cryo-field emission scanning electron microscopy revealed a reduction in capsular material, as determined in detail for a serotype 3 pneumococcal strain. The amount of polysaccharide in the serotype 3 capsule was also determined after intranasal infection of mice. This study illustrates for the first time the phenotypic variation of the polysaccharide capsule in the initial phase of pneumococcal infections. The modified LRR fixation allowed monitoring of the state of capsule expression of pathogens during the infectious process.
Exposure to Rumen Protozoa Leads to Enhancement of Pathogenicity of and Invasion by Multiple-Antibiotic-ResistantSalmonella entericaBearing SGI1
Authors: Rasmussen Mark A. , Carlson Steve A. , Franklin Sharon K. , McCuddin Zoe P. , Wu Max T. , Sharma Vijay K.
Published: July 2005

Multiple-antibiotic-resistantSalmonella entericaserotype Typhimurium is a food-borne pathogen that has been purported to be more virulent than antibiotic-sensitive counterparts. The paradigm for this multiresistant/hyperpathogenic phenotype isSalmonella entericaserotype Typhimurium phage type DT104 (DT104). The basis for the multiresistance in DT104 is related to an integron structure designated SGI1, but factors underlying hyperpathogenicity have not been completely identified. Since protozoa have been implicated in the alteration of virulence inLegionellaandMycobacteriumspp., we attempted to assess the possibility that protozoa may contribute to the putative hypervirulence of DT104. Our study reveals that DT104 can be more invasive, as determined by a tissue culture invasion assay, after surviving within protozoa originating from the bovine rumen. The enhancement of invasion was correlated with hypervirulence in a bovine infection model in which we observed a more rapid progression of disease and a greater recovery rate for the pathogen. Fewer DT104 cells were recovered from tissues of infected animals when protozoa were lysed by preinfection chemical defaunation of the bovine or ovine rumen. The protozoan-mediated hypervirulence phenotype was observed only in DT104 and otherSalmonellastrains, including serovars Agona and Infantis, possessing SGI1.
T-Cell-Stimulating Protein A Elicits Immune Responses during Meningococcal Carriage and Human Disease
Authors: Robinson Karen , Wooldridge Karl G. , Wells Damien B. , Hasan Amal , Todd Ian , Robins Adrian , James Richard , Ala'Aldeen Dlawer A. A.
Published: July 2005

In recognition of the need for immunological memory-inducing components for futureNeisseria meningitidisgroup B vaccines, we previously searched the proteome ofN. meningitidisand identified T-cell-stimulating protein A (TspA). This study was designed to confirm the immunogencity of TspA and to examine the subset of T-helper cell responses to the protein in patients and nasopharyngeal carriers. ThetspAgene was reconstructed, cloned, and expressed inEscherichia coli, and the recombinant TspA (rTspA) protein was affinity purified. T-cell proliferative responses to rTspA were detected in the peripheral blood mononuclear cells (PBMCs) of convalescent patients and carriers, confirming that TspA-specific T-cell responses were stimulated by invasive disease and nasopharyngeal colonization. Following stimulation of PBMCs with meningococcal lysate, increased frequencies of both Th1 and Th2 cells were observed, indicating that, as during carriage, invasive meningococcal disease induced an unbiased T-helper subset response. A similar unbiased T-helper response was also detected against rTspA in the PBMCs of convalescent patients. The response of PBMCs from the carriers to TspA stimulation, however, was very weak, and the frequencies of cytokine-positive CD4 cells were not significantly greater than the frequencies in unstimulated control cultures. All of the patients and carriers responded with serum antimeningococcal immunoglobulin G (IgG) antibodies, while four of six samples from patients and 5 of 14 samples from carriers contained detectable anti-rTspA IgG antibodies. Taken together, the results of this study confirmed the immunogenicity of TspA in humans during natural meningococcal infection, and therefore, TspA is worthy of further investigation as a possible T-cell stimulating component of future vaccines.
Protection by Natural Human Immunoglobulin M Antibody to Meningococcal Serogroup B Capsular Polysaccharide in the Infant Rat Protection Assay Is Independent of Complement-Mediated Bacterial Lysis
Authors: Toropainen Maija , Saarinen Leena , Wedege Elisabeth , Bolstad Karin , Michaelsen Terje E. , Aase Audun , Käyhty Helena
Published: July 2005

Neisseria meningitidis, an important cause of bacterial meningitis and septicemia worldwide, is associated with high mortality and serious sequelae. Natural immunity against meningococcal disease develops with age, but the specificity and functional activity of natural antibodies associated with protection are poorly understood. We addressed this question by using a selected subset of prevaccination sera (n= 26) with convergent or discrepant serum bactericidal activity (SBA) and infant rat protective activity (IRPA) against the serogroup B meningococcal strain 44/76-SL (B:15:P1.7,16) from Icelandic teenagers (B. A. Perkins et al., J. Infect. Dis. 177:683-691, 1998). The sera were analyzed by opsonophagocytic activity (OPA) assay, immunoblotting, immunoglobulin G (IgG) quantitation against live meningococcal cells by flow cytometry, and enzyme immunosorbent assay (EIA). High levels of SBA and OPA were reflected in distinct IgG binding to major outer membrane proteins and/or lipopolysaccharide in immunoblots. However, we could not detect any specific antibody patterns on blots that could explain IRPA. Only IgM antibody to group B capsular polysaccharide (B-PS), measured by EIA, correlated positively (r= 0.76,P< 0.001) with IRPA. Normal human sera (NHS;n= 20) from healthy Finnish children of different ages (7, 14, and 24 months and 10 years) supported this finding and showed an age-related increase in IRPA that coincided with the acquisition of B-PS specific IgM antibody. The protection was independent of complement-mediated bacterial lysis, as detected by the inability of NHS to augment SBA in the presence of human or infant rat complement and the equal protective activity of NHS in rat strains with fully functional or C6-deficient complement.
Fungal Antigens Expressed during Invasive Aspergillosis
Authors: Denikus Nicole , Orfaniotou Foteini , Wulf Gerald , Lehmann Paul F. , Monod Michel , Reichard Utz
Published: July 2005

Rabbits that had been infected intravenously with conidiospores ofAspergillus fumigatuswere used as sources of antibody for screening a λ phage cDNA expression library. The cDNA was derived fromA. fumigatusmRNA that had been extracted from newly formed, germling hyphae. Thirty-six antigens were identified using antisera from six rabbits. Though many of these antigens were expected to be intracellular proteins because their genes did not encode a signal sequence, the antisera showed consistently a stronger immunoblot reaction with a cell fraction enriched for the fungal cell wall than with a fraction of predominantly intracellular components. Antibodies to eight antigens, including the glycosylhydrolase Asp f 16, were produced by more than one rabbit. In current vaccine studies, Asp f 16 is the only single antigen which has been reported to be capable of inducing protection against invasive aspergillosis in mice (S. Bozza et al., Microb. Infect.4:1281-1290, 2002). Enolase andAspergillusHSP90 were detected also; their homologues inCandida albicanshave been tested as vaccines and have been reported to provide a partially protective response against invasive candidiasis in mice. TheAspergillusantigens reported here may have value both in diagnostic tests for different forms of aspergillosis and as vaccine candidates for protection against invasive disease.
Conditions Influencing the Efficacy of Vaccination with Live Organisms againstLeishmania majorInfection
Authors: Tabbara Khaled S. , Peters Nathan C. , Afrin Farhat , Mendez Susana , Bertholet Sylvie , Belkaid Yasmine , Sacks David L.
Published: July 2005

Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasiteLeishmania major, yet inoculation with live, wild-typeL. majorremains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with liveL. majororganisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-γ)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-γ+CD4+cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4+T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines againstLeishmaniasisand other intracellular pathogens.
Growth Characteristics of and Virulence Factor Production by Group A
Streptococcus
during Cultivation in Human Saliva
Authors: Shelburne Samuel A. , Granville Chanel , Tokuyama Maria , Sitkiewicz Izabela , Patel Payal , Musser James M.
Published: July 2005

Group A
Streptococcus
(GAS) commonly infects the human oropharynx, but the initial molecular events governing this process are poorly understood. Saliva is a major component of the innate and acquired immune defense in this anatomic site. Although landmark studies were done more than 60 years ago, investigation of GAS-saliva interaction has not been addressed extensively in recent years. Serotype M1 GAS strain MGAS5005 cultured in human saliva grew to ∼10
7
CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A
2
. Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface.
Characterization of theHelicobacter pyloriCysteine-Rich Protein A as a T-Helper Cell Type 1 Polarizing Agent
Authors: Deml Ludwig , Aigner Michael , Decker Jochen , Eckhardt Alexander , Schütz Christian , Mittl Peer R. E. , Barabas Sascha , Denk Stefanie , Knoll Gertrud , Lehn Norbert , Schneider-Brachert Wulf
Published: July 2005

Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-γ) levels have been proposed to play an important role inHelicobacter pylori-induced gastritis and peptic ulceration. However, bacterial factors contributing to the initiation of Th1 polarization ofH. pylori-specific immune responses have not been characterized in detail thus far. We report here on the identification ofHelicobactercysteine-rich protein A (HcpA) as a novel proinflammatory and Th1-promoting protein. The capacity of HcpA to induce immune activation was studied in splenocyte cultures of naiveH. pylori-negative mice. HcpA stimulated the release of high concentrations of the proinflammatory and Th1-promoting cytokines interleukin-6 (IL-6) and IFN-γ, in addition to significant levels of IL-12, tumor necrosis factor alpha, and IL-10. The observed cytokine profile was comparable to that induced by lipopolysaccharide but differed in the kinetics and maximum levels of cytokine production. In addition, HcpA-induced cytokine release resembled that observed upon incubation withH. pyloriexcept for IL-10, which was only moderately released upon HcpA stimulation. Both HcpA- andH. pylori-mediated IFN-γ production was drastically reduced by a neutralizing antibody against IL-12 but not by an anti-IL-2 antibody. Thus, HcpA seems to represent a novel bacterial virulence factor triggering the release of a concerted set of cytokines to instruct the adaptive immune system for the initiation of proinflammatory and Th1-biased immunity.
Influence of Na+, Dicarboxylic Amino Acids, and pH in Modulating the Low-Calcium Response ofYersinia pestis
Authors: Brubaker Robert R.
Published: July 2005

The virulence of yersiniae is promoted in part by shared ≈70-kb plasmids (pCD inYersinia pestisand pYV in enteropathogenicYersinia pseudotuberculosisandYersinia enterocolitica) that mediate a low-calcium response. This phenotype is characterized at 37°C by either bacteriostasis in Ca2+-deficient medium with expression of pCD/pYV-encoded virulence effectors (Yops and LcrV) or vegetative growth and repression of Yops and LcrV with ≥2.5 mM Ca2+(Lcr+). Regulation of Yops and LcrV is well defined but little is known about bacteriostasis other than that Na+plusl-glutamate promotes prompt restriction ofY. pestis. As shown here,l-aspartate substituted forl-glutamate in this context but only Na+exacerbated the nutritional requirement for Ca2+. Bacteriostasis ofY. pestis(but not enteropathogenic yersiniae) was abrupt in Ca2+-deficient medium at neutral to slightly alkaline pH (7.0 to 8.0), although increasing the pH to 8.5 or 9.0, especially with added Na+(but notl-glutamate), facilitated full-scale growth. Addedl-glutamate (but not Na+) favored Ca2+-independent growth at acidic pH (5.0 to 6.5). Yops and LcrV were produced in Ca2+-deficient media at pH 6.5 to 9.0 regardless of the presence of added Na+orl-glutamate, although their expression at alkaline pH was minimal. Resting Ca2+-starved Lcr+cells ofY. pestissupplied withl-glutamate first excreted and then destroyedl-aspartate. These findings indicate that expression of Yops and LcrV is necessary but not sufficient for bacteriostasis of Ca2+-starved yersiniae and suggest that abrupt restriction ofY. pestisrequires Na+and the known absence of aspartate ammonia-lyase in this species.
Identification and Characterization of the Locus for Diffuse Adherence, Which Encodes a Novel Afimbrial Adhesin Found in Atypical EnteropathogenicEscherichia coli
Authors: Scaletsky Isabel C. A. , Michalski Jane , Torres Alfredo G. , Dulguer Michelle V. , Kaper James B.
Published: July 2005

The O26 serogroup of enteropathogenicEscherichia coli(EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagicE. coli(EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negativeE. colistrains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in anstx-negativebfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated thelocus fordiffuseadherence (lda), confers diffuse adherence on HEp-2 cells when expressed inE. coliK-12. Sequence analysis ofldarevealed a G+C content of 46.8% and 15 open reading frames sharing homology with theE. coliK88faeand CS31Aclpfimbrial operons. Theldaregion is part of a putative 26-kb genomic island inserted into theproPgene of theE. colichromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene,ldaH, acrossE. coliserogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of theE. coliDH5αlda+clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.
Pathoadaptive Mutation That Mediates Adherence of Shiga Toxin-Producing
Escherichia coli
O111
Authors: Torres Alfredo G. , Vazquez-Juarez Roberto C. , Tutt Christopher B. , Garcia-Gallegos J. Gerardo
Published: July 2005

The adherence of pathogenic
Escherichia coli
strains to intestinal epithelium is essential for initiation of infection. The
cad
operon encodes the lysine decarboxylase (LDC) system responsible for
metabolizing lysine, and this operon has been proposed as an
antivirulence mechanism in enteroinvasive
E. coli
and
Shigella flexneri
and as a factor mediating
E. coli
O157:H7 adherence. We sought to determine whether the LDC activity was
present in a phylogenetically characterized collection of diarrheagenic
E. coli
(DEC) strains and to establish whether its expression
was associated with their adherence to tissue culture cells. LDC
activity was found in most of the pathogenic
E. coli
strains
tested and was absent from Shiga toxin-producing
E. coli
(STEC) O111 strains (DEC pathotype 8). Analysis of the
cad
region in these O111 strains indicates that the operon has been
rearranged and some of the genes are either missing or disrupted. A
similar rearrangement was found in an
E. coli
O111:H8 strain
recently isolated from an outbreak in Texas. Complementation of the
LDC-negative strains with the
cad
operon in
trans
restored the LDC activity and resulted in a reduction in adherence to
tissue culture cells. Initial analysis of the protein profiles on the
surface of the O111 strains indicates that the LDC activity has an
effect on the expression of the adhesin intimin. Cadaverine had a
slight effect on LDC-negative strain adhesion but none on intimin
expression. Our data suggest that this pathoadaptive mutation is an
important mechanism to control functions potentially implicated in the
pathogenesis of these
organisms.
Species-Specific Inhibition of Cerebral Malaria in Mice Coinfected withPlasmodiumspp
Authors: Voza Tatiana , Vigário Ana M. , Belnoue Elodie , Grüner Anne Charlotte , Deschemin Jean-Christophe , Kayibanda Michèle , Delmas Florian , Janse Chris J. , Franke-Fayard Blandine , Waters Andrew P. , Landau Irène , Snounou Georges , Rénia Laurent
Published: July 2005

Recent epidemiological observations suggest that clinical evolution ofPlasmodium falciparuminfections might be influenced by the concurrent presence of anotherPlasmodiumspecies, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected withP. bergheiANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence ofP. yoelii yoeliibut not that ofP. vinckeior another line ofP. berghei. In the protected coinfected mice, the accumulation of CD8+T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixedPlasmodiumspecies infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria.
Independent Protective Effects for Tumor Necrosis Factor and Lymphotoxin Alpha in the Host Response toListeria monocytogenesInfection
Authors: Roach D. R. , Briscoe H. , Saunders B. M. , Britton W. J.
Published: July 2005

Although the essential role of tumor necrosis factor (TNF) in resistance toListeria monocytogenesinfection is well established, the roles of the related cytokines lymphotoxin alpha (LTα) and lymphotoxin beta (LTβ) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTα, and LTβ in the host response toListeria. To overcome the lack of peripheral lymph nodes in LTα−/−and LTβ−/−mice, bone marrow chimeras were constructed. TNF−/−and LTα−/−chimeras that lacked both secreted LTα3and membrane-bound LTα1β2and LTα2β1were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTβ−/−chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. TheListeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTα−/−and LTβ−/−chimeras, WT chimeras, and TNF−/−and WT mice). TNF−/−mice and LTα−/−chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF−/−and LTα−/−chimeras. Therefore, in addition to TNF, soluble LTα3plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.
The Multifunctional
Staphylococcus aureus
Autolysin Aaa Mediates Adherence to Immobilized Fibrinogen and Fibronectin
Authors: Heilmann Christine , Hartleib Jörg , Hussain Muzaffar S. , Peters Georg
Published: July 2005

Staphylococci can cause a wide spectrum of infections, including endocarditis, osteomyelitis, and sepsis, which is reflected by the numerous virulence factors they produce, among them a recently identified new class of adhesins, namely, the multifunctional autolysins/adhesins. Here we report the identification and molecular characterization of Aaa, a novel autolysin/adhesin from
Staphylococcus aureus
. The gene encoding Aaa was cloned from the clinical isolate
Staphylococcus aureus
4074. DNA sequence analysis revealed that
aaa
encodes a deduced protein of 334 amino acids with a predicted molecular mass of 35.8 kDa. Aaa contains three N-terminal repetitive sequences that comprise features of a peptidoglycan-binding domain, the LysM domain. The expression of
aaa
by
Escherichia coli
and its subsequent characterization revealed that Aaa possesses bacteriolytic activity as well as adhesive properties, such as binding to extracellular matrix proteins. Real-time biomolecular interaction analysis demonstrated that the interaction of Aaa with fibrinogen, fibronectin, and vitronectin is dose dependent and saturable and occurs with a high affinity. Furthermore, we demonstrate that Aaa binds to the Aα and Bβ chains of fragment D of fibrinogen. Immunofluorescence microscopy revealed that Aaa is located at the cell surface. Finally, an
aaa
knockout mutant showed reduced adherence to surface-adsorbed fibrinogen and fibronectin, strongly suggesting a role for Aaa in the colonization of host factor-coated polymer surfaces and/or host tissue.
Effect of Skin Barrier Disruption on Immune Responses to Topically Applied Cross-Reacting Material, CRM197, of Diphtheria Toxin
Authors: Godefroy S. , Peyre M. , Garcia N. , Muller S. , Sesardic D. , Partidos C. D.
Published: July 2005

The high accessibility of the skin and the presence of immunocompetent cells in the epidermis makes this surface an attractive route for needle-free administration of vaccines. However, the lining of the skin by the stratum corneum is a major obstacle to vaccine delivery. In this study we examined the effect of skin barrier disruption on the immune responses to the cross-reacting material CRM197, a nontoxic mutant of diphtheria toxin (DTx) that is considered as a vaccine candidate. Application of CRM197, together with cholera toxin (CT), onto the tape-stripped skin of mice elicited antibody responses that had anti-DTx neutralizing activity. Vaccine delivery onto mildly ablated skin or intact skin did not elicit any detectable anti-CRM197antibodies. Mice immunized with CRM197alone onto the tape-stripped skin mounted a vigorous antigen-specific proliferative response. In contrast, the induction of cellular immunity after CRM197deposition onto mildly ablated or intact skin was adjuvant dependent. Furthermore, epidermal cells were activated and underwent apoptosis that was more pronounced when the stratum corneum was removed by tape stripping. Overall, these findings highlight the potential for transcutaneous delivery of CRM197and establish a correlation between the degree of barrier disruption and levels of antigen-specific immune responses. Moreover, these results provide the first evidence that the development of a transcutaneous immunization strategy for diphtheria, based on simple and practical methods to disrupt the skin barrier, is feasible.
Bacterial Ghosts as an Oral Vaccine: a Single Dose of
Escherichia coli
O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge
Authors: Mayr Ulrike Beate , Haller Christoph , Haidinger Wolfgang , Atrasheuskaya Alena , Bukin Eugenij , Lubitz Werner , Ignatyev Georgy
Published: July 2005

Enterohemorrhagic
Escherichia coli
(EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.
Slow Genetic Divergence of
Helicobacter pylori
Strains during Long-Term Colonization
Authors: Lundin Annelie , Björkholm Britta , Kupershmidt Ilya , Unemo Magnus , Nilsson Peter , Andersson Dan I. , Engstrand Lars
Published: July 2005

The genetic variability of
Helicobacter pylori
is known to be high compared to that of many other bacterial species.
H. pylori
is adapted to the human stomach, where it persists for decades, and adaptation to each host results in every individual harboring a distinctive bacterial population. Although clonal variants may exist within such a population, all isolates are generally genetically related and thus derived from a common ancestor. We sought to determine the rate of genetic change of
H. pylori
over 9 years in two asymptomatic adult patients. Arbitrary primed PCR confirmed the relatedness of individual subclones within a patient. Furthermore, sequencing of 10 loci (∼6,000 bp) in three subclones per time and patient revealed only two base pair changes among the subclones from patient I. All sequences were identical among the patient II subclones. However, PCR amplification of the highly divergent gene
amiA
revealed great variation in the size of the gene between the subclones within each patient. Thus, both patients harbored a single strain with clonal variants at both times. We also studied genetic changes in culture- and mouse-passaged strains, and under both conditions no genetic divergence was found. These results suggest that previous estimates of the rate of genetic change in
H. pylori
within an individual might be overestimates.