bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigenic Organization of the N-Terminal Part of the Polymorphic Outer Membrane Proteins 90, 91A, and 91B ofChlamydophilaabortus
Authors: Vretou Evangelia , Giannikopoulou Panagiota , Longbottom David , Psarrou Evgenia
Published: May 2003

A series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) ofChlamydophilaabortusand two recombinant peptides spanning gene fragmentp91Bf99of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former speciesC. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genusChlamydophila. Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.
Killing of
Candida albicans
by Human Salivary Histatin 5 Is Modulated, but Not Determined, by the Potassium Channel TOK1
Authors: Baev Didi , Rivetta Alberto , Li Xuewei S. , Vylkova Slavena , Bashi Esther , Slayman Clifford L. , Edgerton Mira
Published: May 2003

Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen
Candida albicans
, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxin's fungicidal activity. Hst 5 action on
Candida
resembles, in many respects, the action of the K1 killer toxin on
Saccharomyces cerevisiae
, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the
Candida
homologue of
Saccharomyces
Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the
C. albicans TOK1
gene. The resultant strains (
TOK1/tok1
) and (
tok1/tok1
) were compared with wild-type
Candida
(
TOK1/TOK1
) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on
Candida
protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in
Candida
. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in
Saccharomyces
. Knockout of
CaTOK1
(
tok1/tok1
) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (
tok1/tok1
) increases residual viability of
Candida
after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (
TOK1/tok1
) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type
TOK1
genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type
TOK1
, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.
Characterization of
Brucella abortus
O-Polysaccharide and Core Lipopolysaccharide Mutants and Demonstration that a Complete Core Is Required for Rough Vaccines To Be Efficient against
Brucella abortus
and
Brucella ovis
in the Mouse Model
Authors: Monreal D. , Grilló M. J. , González D. , Marín C. M. , De Miguel M. J. , López-Goñi I. , Blasco J. M. , Cloeckaert A , Moriyón I.
Published: May 2003

Brucella abortus
rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two
wbk
genes (
wbkA
[putative glycosyltransferase; formerly
rfbU
] and
per
[perosamine synthetase]), into
manB
(
pmm
[phosphomannomutase; formerly
rfbK
]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-
manno
-d-
octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the
wbk
mutants. The rough LPS of
manB
mutant lacked the outer core epitope and the gene was designated

manB
core

to distinguish it from the

wbk manB
O-Ag

. The fourth gene (provisionally designated
wa**
) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in
B. abortus
outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against
B. abortus
in mice. The mutants
per
and
wbkA
induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against
B. ovis
, S19, RB51, and the
wbkA
and
per
mutants afforded similar levels of protection. These results suggest that rough
Brucella
vaccines should carry a complete core for maximal effectiveness.
Neospora caninum-Infected Cattle Develop Parasite-Specific CD4+Cytotoxic T Lymphocytes
Authors: Staska Lauren M. , McGuire Travis C. , Davies Christopher J. , Lewin Harris A. , Baszler Timothy V.
Published: May 2003

Cattle infected withNeospora caninumreadily experience transplacental parasite transmission, presumably after maternal parasitemia, leading to abortion or birth of congenitally infected calves. Cytotoxic T lymphocytes (CTL) are important mediators of protective immunity againstToxoplasma gondii, an intracellular apicomplexan protozoan closely related toN. caninum. In this study,N. caninum-specific CTL expanded from peripheral blood mononuclear cells of two major histocompatibility complex-mismatched, experimentally infected cattle were identified by using a51Cr release cytotoxicity assay. Enrichment and blocking of CD4+- and CD8+-T-lymphocyte effector subsets indicated that CD4+CTL killedN. caninum-infected, autologous target cells and that killing was mediated through a perforin/granzyme pathway. Detection and characterization of CTL responses toN. caninumin the natural, outbred, bovine host will facilitate identification of immunogens and design of immunization strategies to induce parasite-specific CTL against transplacentalN. caninumtransmission in cattle.
Distribution and Kinetics of Lipoprotein-Bound Lipoteichoic Acid
Authors: Levels Johannes H. M. , Abraham Philip R. , van Barreveld Erik P. , Meijers Joost C. M. , van Deventer Sander J. H.
Published: May 2003

Lipoteichoic acid (LTA), a major cell wall component of gram-positive bacteria, is an amphipathic anionic glycolipid with structural similarities to lipopolysaccharide (LPS) from gram-negative bacteria. LTA has been implicated as one of the primary immunostimulatory components that may trigger the systemic inflammatory response syndrome. Plasma lipoproteins have been shown to sequester LPS, which results in attenuation of the host response to infection, but little is known about the LTA binding characteristics of plasma lipid particles. In this study, we have examined the LTA binding capacities and association kinetics of the major lipoprotein classes under simulated physiological conditions in human whole blood (ex vivo) by using biologically active, fluorescently labeled LTA and high-performance gel permeation chromatography. The average distribution of an LTA preparation fromStaphylococcus aureusin whole blood from 10 human volunteers revealed that >95% of the LTA was associated with total plasma lipoproteins in the following proportions: high-density lipoprotein (HDL), 68% ± 10%; low-density lipoprotein (LDL), 28% ± 8%; and very low density lipoprotein (VLDL), 4% ± 5%. The saturation capacity of lipoproteins for LTA was in excess of 150 μg/ml. The LTA distribution was temperature dependent, with an optimal binding between 22 and 37°C. The binding of LTA by lipoproteins was essentially complete within 10 min and was followed by a subsequent redistribution from HDL and VLDL to LDL. We conclude that HDL has the highest binding capacity for LTA and propose that the loading and redistribution of LTA among plasma lipoproteins is a specific process that closely resembles that previously described for LPS (J. H. M. Levels, P. R. Abraham, A. van den Ende, and S. J. H. van Deventer, Infect. Immun.68:2821-2828, 2001).
Characterization of an
iroBCDEN
Gene Cluster on a Transmissible Plasmid of Uropathogenic
Escherichia coli
: Evidence for Horizontal Transfer of a Chromosomal Virulence Factor
Authors: Sorsa Liisa Johanna , Dufke Severin , Heesemann Jürgen , Schubert Sören
Published: May 2003

The chromosomal
iroBCDEN
gene cluster first described for
Salmonella enterica
is involved in the uptake of catecholate-type siderophore compounds. An orthologous gene cluster has recently been detected in
Escherichia coli
strains which cause extraintestinal disease. This
E. coli iroBCDEN
gene cluster has an impact on virulence and has been reported to be located in a pathogenicity island on the chromosome. In this study we characterized an
iro
gene cluster of a uropathogenic
E. coli
isolate which is located on a transmissible plasmid related to the R64 plasmid of
S. enterica
. This cluster is highly homologous to the chromosomal
iro
cluster of
E. coli
. When introduced into an
E. coli fepA cir fiu aroB
mutant, IroN, but not IroBCDE, mediated the utilization of structurally related catecholate siderophores, including 2,3-dihydroxybenzoyl-
l
-serine, 2,3-dihydroxybenzoyl-
d
-ornithine, 2,3-dihydroxybenzoic acid, and enterochelin. This study supports the idea of an ongoing horizontal transfer of putative virulence factors and the mobilization of single virulence gene clusters, which lead to a modular assembly of virulence determinants such as pathogenicity islands.
Mutational Analysis of the Enzymatic Domain ofClostridium difficileToxin B Reveals Novel Inhibitors of the Wild-Type Toxin
Authors: Spyres Lea M. , Daniel Jeremy , Hensley Amy , Qa'Dan Maen , Ortiz-Leduc William , Ballard Jimmy D.
Published: May 2003

Toxin B (TcdB), a majorClostridium difficilevirulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB1-556were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused tolfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB1-556was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated withClostridium sordelliilethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection againstClostridium novyialpha-toxin (Τcnα), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.
The Immunoglobulin D-Binding Protein MID fromMoraxella catarrhalisIs Also an Adhesin
Authors: Forsgren Arne , Brant Marta , Karamehmedovic Mirela , Riesbeck Kristian
Published: May 2003

TheMoraxella catarrhalisimmunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD. MID is found in the majority ofM. catarrhalisstrains. In the present paper, we show that MID-expressingM. catarrhalisstrains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast,M. catarrhalisisolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity. To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule. The truncated MID proteins were expressed inEscherichia coli, purified, and used for raising polyclonal antibodies in rabbits. Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID764-913. In addition, antibodies against full-length MID, MID764-913, or a 30-amino-acid consensus sequence (MID775-804) inhibited adhesion to alveolar epithelial cells. Antibodies against UspA1, an outer membrane protein expressed in essentially allM. catarrhalisstrains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells. Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine againstM. catarrhalis.
Secretin of the EnteropathogenicEscherichia coliType III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization
Authors: Gauthier Annick , Puente Jose Luis , Finlay B. Brett
Published: May 2003

At least 16 proteins are thought to be involved in forming the enteropathogenicEscherichia coli(EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered inescVorescNmutants, where EscC accumulated in the periplasm. EscC was correctly localized in theescFneedle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on thesec-dependent secretion pathway but also on other type III apparatus components.
Association ofActinobacillus pleuropneumoniaeCapsular Polysaccharide with Virulence in Pigs
Authors: Bandara Aloka B. , Lawrence Mark L. , Veit Hugo P. , Inzana Thomas J.
Published: May 2003

The capsular polysaccharide (CP) ofActinobacillus pleuropneumoniaeis required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affectsA. pleuropneumoniaevirulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export inA. pleuropneumoniaeserotype 1 was cloned and sequenced. Three open reading frames, designatedcps1A, cps1B,andcps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kanr) was inserted into a 750-bp deletion spanningcps1ABor into a 512-bp deletion incps1Bonly, and the constructs were cloned in a suicide vector. The Kanrgene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Δcps1N and strain 4074Δcps1B, respectively. Strain 4074Δcps1N produced no detectable CP, but strain 4074Δcps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. Thecps1ABCgenes of strain 4074 and thecps5ABCandcps5ABCDEgenes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Δcps1N to produce 4074Δcps1N(pABcps101), 4074Δcps1N(pJMLcps53), and 4074Δcps1N(pABcps55), respectively. Strain 4074Δcps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Δcps1N(pJMLcps53) and 4074Δcps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Δcps1N(pABcps101) ≥ strain 4074Δcps1N > strain 4074Δcps1B. Strain 4074Δcps1N(pJMLcps53) was less virulent than strain 4074Δcps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains ofA. pleuropneumoniaecorrelated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.
Study of Capsular Polysaccharide from
Vibrio
parahaemolyticus
Authors: Hsieh Yu-Chi , Liang Shu-Mei , Tsai Wan-Ling , Chen Yee-Hsiung , Liu Teh-Yung , Liang Chi-Ming
Published: May 2003

The leading cause of food poisoning in both Taiwan and Japan is
Vibrio
parahaemolyticus
infection, whose mechanism of enteropathogenesis is still unclear. To evaluate whether surface components are responsible for the intestinal adhesion of
V
.
parahaemolyticus
, we have developed a novel method for isolating the capsular polysaccharide (CPS) from
V
.
parahaemolyticus
(serotype O4:K8). We found that culturing of
V
.
parahaemolyticus
in broth for 1 week or more changed the colony form of the bacteria on an agar plate from opaque to translucent. The translucent colonies of
V
.
parahaemolyticus
contained little CPS and exhibited a much lower level of adherence to epithelial cells (Int-407) than the opaque colonies of the bacteria. Incubation of
V
.
parahaemolyticus
in medium supplemented with bile increased the levels of CPS and adherence. Treatment of
V
.
parahaemolyticus
with anti-CPS but not anti-LPS serum decreased the level of bacterial adherence. In addition, purified CPS bound to epithelial cells in a dose-dependent manner. Intranasal administration of CPS to mice in the presence of adjuvants such as immunostimulatory sequence oligodeoxynucleotides or cholera toxin elicited CPS-specific mucosal and systemic immune responses. These results indicate that CPS plays an important role in the adherence of
V
.
parahaemolyticus
to its target cells and may be considered a potential target for the development of a vaccine against this pathogen.
Differential Induction of Interleukin-10 and Interleukin-12 in Dendritic Cells by Microbial Toll-Like Receptor Activators and Skewing of T-Cell Cytokine Profiles
Authors: Qi Hai , Denning Timothy L. , Soong Lynn
Published: May 2003

Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of appropriate effector phenotypes accordingly. Microbial recognition and differentiation are mediated in part by pattern recognition receptors such as Toll-like receptors (TLRs), whereas the development of T-cell effector functions is critically dependent on DC-derived cytokines such as interleukin-12 (IL-12) and IL-10. However, it is not entirely clear to what extent various microbial TLR activators could induce different functional states of DCs that favor different T-cell effector phenotypes. Toward a better understanding of this issue, we examined IL-10 and IL-12 production and T-cell-polarizing potentials of murine bone marrow-derived DCs after stimulation by three microbial TLR activators, namely, lipopolysaccharide (LPS), peptidoglycan (PGN), and zymosan. We found that the three stimuli induced drastically different profiles of IL-10 and IL-12 production in DCs. Further, these stimuli differentially conditioned CD40-dependent IL-10 and IL-12 production by DCs. Finally, LPS-, PGN-, and zymosan-stimulated DCs primed distinct T-cell cytokine profiles. Our results support the notion that microbe-specific information sensed through different TLRs by DCs is linked to differential Th priming through DC-derived cytokines.
A Mosaic Pathogenicity Island Made Up of the Locus of Enterocyte Effacement and a Pathogenicity Island of
Escherichia
coli
O157:H7 Is Frequently Present in Attaching and Effacing
E
.
coli
Authors: Morabito Stefano , Tozzoli Rosangela , Oswald Eric , Caprioli Alfredo
Published: May 2003

Enteropathogenic
Escherichia
coli
(EPEC) and enterohemorragic
E
.
coli
(EHEC) possess a pathogenicity island (PAI), termed the locus of enterocyte effacement (LEE), which confers the capability to cause the characteristic attaching and effacing lesions of the brush border. Due to this common property, these organisms are also termed attaching and effacing
E
.
coli
(AEEC). Sequencing of the EHEC O157 genome recently revealed the presence of other putative PAIs in the chromosome of this organism. In this article, we report on the presence of four of those PAIs in a panel of 133
E
.
coli
strains belonging to different pathogroups and serotypes. One of these PAIs, termed O122 in strain EDL 933 and SpLE3 in strain Sakai, was observed in most of the AEEC strains examined but not in the other groups of
E
.
coli
. It was also found to contain the virulence-associated gene
efa1
/
lifA
. In EHEC O157, PAI O122 is located 0.7 Mb away from the LEE. Conversely, we demonstrated that in many EHEC non-O157 strains and EPEC strains belonging to eight serogroups, PAI O122 and the LEE are physically linked to form a cointegrated structure. This structure can be considered a mosaic PAI that could have been acquired originally by AEEC. In some clones, such as EHEC O157, the LEE-O122 mosaic PAI might have undergone recombinational events, resulting in the insertion of the portion referred to as PAI O122 in a different location.
Structural and Functional Consequences of Cleavage of Human Secretory and Human Serum Immunoglobulin A1 by Proteinases from
Proteus
mirabilis
and
Neisseria
meningitidis
Authors: Almogren Adel , Senior Bernard W. , Loomes Lesley M. , Kerr Michael A.
Published: May 2003

The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of
Neisseria
meningitidis
and cleavage by the proteinase from
Proteus
mirabilis
have been compared. For serum IgA1, both proteinases cleaved only the α chain.
N
.
meningitidis
proteinase cleaved only in the hinge.
P
.
mirabilis
proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by
N
.
meningitidis
proteinase occurred only in the hinge and was as rapid as that of serum IgA1.
P
.
mirabilis
proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the α1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by
N
.
meningitidis
proteinase stimulated a respiratory burst in neutrophils through Fcα receptors, whereas the (Fcα1)
2
-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with
P
.
mirabilis
proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcα receptors. S-IgA1 treated with
P
.
mirabilis
proteinase under the same conditions retained the ability to bind to Fcα receptors. The results are consistent with the Fcα receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcα receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.
Helicobacter pylori
Infection Induces Interleukin-8 Receptor Expression in the Human Gastric Epithelium
Authors: Bäckhed Fredrik , Torstensson Elisabeth , Seguin Delphine , Richter-Dahlfors Agneta , Rokbi Bachra
Published: May 2003

The gastric pathogen
Helicobacter pylori
is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in
H. pylori
-infected human gastric biopsy samples. Biopsy samples from patients infected with
H. pylori
strains harboring the
cag
pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the
cag
PAI, while a strain lacking the
cag
PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to
H. pylori
infection.
c-Jun NH2-Terminal Kinase-Mediated Signaling Is Essential forPseudomonas aeruginosaExoS-Induced Apoptosis
Authors: Jia Jinghua , Alaoui-El-Azher Mounia , Chow Marie , Chambers Timothy C. , Baker Henry , Jin Shouguang
Published: May 2003

As an opportunistic bacterial pathogen,Pseudomonas aeruginosamainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS ofP.aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH2-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochromecis released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2 leading ultimately to cytochromecrelease and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction betweenP.aeruginosaand host cells.
Global Analysis of
Borrelia burgdorferi
Genes Regulated by Mammalian Host-Specific Signals
Authors: Brooks Chad S. , Hefty P. Scott , Jolliff Sarah E. , Akins Darrin R.
Published: May 2003

Lyme disease is a tick-borne infection that can lead to chronic, debilitating problems if not recognized or treated appropriately.
Borrelia burgdorferi
, the causative agent of Lyme disease, is maintained in nature by a complex enzootic cycle involving
Ixodes
ticks and mammalian hosts. Many previous studies support the notion that
B. burgdorferi
differentially expresses numerous genes and proteins to help it adapt to growth in the mammalian host. In this regard, several studies have utilized a dialysis membrane chamber (DMC) cultivation system to generate “mammalian host-adapted” spirochetes for the identification of genes selectively expressed during mammalian infection. Here, we have exploited the DMC cultivation system in conjunction with microarray technology to examine the global changes in gene expression that occur in the mammalian host. To identify genes regulated by only mammal-specific signals and not by temperature, borrelial microarrays were hybridized with cDNA generated either from organisms temperature shifted in vitro from 23°C to 37°C or from organisms cultivated by using the DMC model system. Statistical analyses of the combined data sets revealed that 125 genes were expressed at significantly different levels in the mammalian host, with almost equivalent numbers of genes being up- or down-regulated by
B. burgdorferi
within DMCs compared to those undergoing temperature shift. Interestingly, during DMC cultivation, the vast majority of genes identified on the plasmids were down-regulated (79%), while the differentially expressed chromosomal genes were almost entirely up-regulated (93%). Global analysis of the upstream promoter regions of differentially expressed genes revealed that several share a common motif that may be important in transcriptional regulation during mammalian infection. Among genes with known or putative functions, the cell envelope category, which includes outer membrane proteins, was found to contain the most differentially expressed genes. The combined findings have generated a subset of genes that can now be further characterized to help define their role or roles with regard to
B. burgdorferi
virulence and Lyme disease pathogenesis.
Prior Exposure to LiveMycobacterium bovisBCG DecreasesCryptococcus neoformans-Induced Lung Eosinophilia in a Gamma Interferon-Dependent Manner
Authors: Walzl Gerhard , Humphreys Ian R. , Marshall Ben G. , Edwards Lorna , Openshaw Peter J. M. , Shaw Rory J. , Hussell Tracy
Published: May 2003

Some common childhood infections appear to prevent the development of atopy and asthma. In someMycobacterium bovisBCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allergen-induced lung eosinophilia in animal models, little attention has been given to the effect of immunity to BCG on responses against live pathogens. We used the murineCryptococcus neoformansinfection model to investigate whether prior BCG infection can alter such responses. The present study shows that persistent pulmonary BCG infection of C57BL/6 mice induced an increase in gamma interferon, a reduction in interleukin-5, and a decrease in lung eosinophilia during subsequentCryptococcusinfection. This effect was long lasting, depended on the presence of live bacteria, and required persistence of mycobacterial infection in the lung. Reduction of eosinophilia was less prominent after infection with a mutant BCG strain (ΔhspR), which was rapidly cleared from the lungs. These observations have important implications for the development of vaccines designed to prevent Th2-mediated disease and indicate that prior lung BCG vaccination can alter the pattern of subsequent host inflammation.
Contribution of Proton-Translocating Proteins to the Virulence of
Salmonella enterica
Serovars Typhimurium, Gallinarum, and Dublin in Chickens and Mice
Authors: Turner A. K. , Barber L. Z. , Wigley P. , Muhammad S. , Jones M. A. , Lovell M. A. , Hulme S. , Barrow P. A.
Published: May 2003

We investigated the attenuating effects of a range of respiratory chain mutations in three
Salmonella
serovars which might be used in the development of live vaccines. We tested mutations in
nuoG
,
cydA
,
cyoA
,
atpB
, and
atpH
in three serovars of
Salmonella enterica:
Typhimurium, Dublin, and Gallinarum. All three serovars were assessed for attenuation in their relevant virulence assays of typhoid-like infections. Serovar Typhimurium was assessed in 1-day-old chickens and the mouse. Serovar Gallinarum 9 was assessed in 3-week-old chickens, and serovar Dublin was assessed in 6-week-old mice. Our data show variation in attenuation for the
nuoG
,
cydA
, and
cyoA
mutations within the different serovar-host combinations. However, mutations in
atpB
and
atpH
were highly attenuating for all three serovars in the various virulence assays. Further investigation of the mutations in the
atp
operon showed that the bacteria were less invasive in vivo, showing reduced in vitro survival within phagocytic cells and reduced acid tolerance. We present data showing that this reduced acid tolerance is due to an inability to adapt to conditions rather than a general sensitivity to reduced pH. The data support the targeting of respiratory components for the production of live vaccines and suggest that mutations in the
atp
operon provide suitable candidates for broad-spectrum attenuation of a range of
Salmonella
serovars.
Disparity in Functional Activity between Serum Anticapsular Antibodies Induced in Adults by Immunization with an Investigational Group A and CNeisseria meningitidis-Diphtheria Toxoid Conjugate Vaccine and by a Polysaccharide Vaccine
Authors: Harris Shannon L. , Finn Adam , Granoff Dan M.
Published: May 2003

Polysaccharide-protein conjugate vaccines elicit higher concentrations of serum anticapsular antibody in infants and children than do unconjugated polysaccharide vaccines. The conjugate-induced antibodies also have higher avidity and complement-mediated bactericidal activity. Similar vaccine-related differences in the magnitude or functional activity of antibody are observed infrequently in immunized adults. We compared the antibody responses of adults immunized with an investigational group A and C meningococcal conjugate vaccine to those elicited by an unconjugated meningococcal polysaccharide vaccine. Although there were no significant differences between the respective geometric mean bactericidal titers of the two vaccine groups, it took, on average, three- to fourfold higher concentrations of polysaccharide-induced serum anticapsular antibody to achieve 50% complement-mediated bacteriolysis than conjugate-induced antibody (P< 0.001 for groups A and C). At limiting doses, the polysaccharide-induced anticapsular antibodies also were less effective in conferring passive protection against meningococcal bacteremia in infant rats challenged with a group C strain (P< 0.04). The avidity index of the group C antibodies was higher in the conjugate vaccine group than in the polysaccharide vaccine group (P< 0.005). The disparities in the functional activity of the anticapsular antibodies elicited in adults by the two vaccines imply fundamental differences in the respective B-cell populations stimulated.
Immunity Profiles of Wild-Type and Recombinant Shiga-Like Toxin-Encoding Bacteriophages and Characterization of Novel Double Lysogens
Authors: Allison Heather E. , Sergeant Martin J. , James Chloë E. , Saunders Jon R. , Smith Darren L. , Sharp Richard J. , Marks Trevor S. , McCarthy Alan J.
Published: May 2003

The pathogenicity of Shiga-like toxin (stx)-producing
Escherichia coli
(STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (
stx
1
and/or
stx
2
) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either

stx
1

or

stx
2

genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the
stx
genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of
stx
genes across a diverse range of
E. coli
serotypes.
Antigenic Composition of
Borrelia burgdorferi
during Infection of SCID Mice
Authors: Crother Timothy R. , Champion Cheryl I. , Wu Xiao-Yang , Blanco David R. , Miller James N. , Lovett Michael A.
Published: May 2003

The general concept that during infection of mice the
Borrelia burgdorferi
surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted
Borrelia
(HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated
Borrelia
(IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic
B. burgdorferi
antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.
Experimental Validation of Low Virulence in Field Strains of
Listeria monocytogenes
Authors: Roche S. M. , Gracieux P. , Albert I. , Gouali M. , Jacquet C. , Martin P. M. V. , Velge P.
Published: May 2003

Several reports have described
Listeria monocytogenes
strains which were nonpathogenic or weakly pathogenic, but little is known about these low-virulence strains. We found that 9 field
L. monocytogenes
strains were hypovirulent and 17 were avirulent, based on the number of mice contaminated and the colonization of their spleens after subcutaneous inoculation. All these strains possessed the known virulence genes. We have now assessed the low virulence of these strains in other assays before determining how they differ from virulent strains. We have shown that the low-virulence strains exhibited a phenotypic stability and were not a mixture of virulent and avirulent bacteria. They did not recover virulence after many passages in mice and colonized the spleens of mice more poorly than virulent strains after i.v. inoculation. Their lethal capacities, determined by 50% lethal dose (LD
50
), were lower than those of virulent strains. Like
Listeria innocua
, 14 of 17 avirulent strains had no LD
50
and were eliminated by the lymph nodes after subcutaneous inoculation. The virulent, hypovirulent, and avirulent strains were always significantly different, whatever the tests of virulence used, confirming the importance of these low-virulence field strains in identifying the proteins involved in virulence.
Inhibition of Interleukin-17 Prevents the Development of Arthritis in Vaccinated Mice Challenged withBorrelia burgdorferi
Authors: Burchill Matthew A. , Nardelli Dean T. , England Douglas M. , DeCoster David J. , Christopherson John A. , Callister Steven M. , Schell Ronald F.
Published: May 2003

We showed thatBorrelia burgdorferi-vaccinated interferon gamma-deficient (IFN-γ0) mice challenged with the Lyme spirochete developed a prominent chronic severe destructive osteoarthropathy. The immune response underlying the development of the severe destructive arthritis involves interleukin-17 (IL-17). Treatment of vaccinated IFN-γ0mice challenged withB. burgdorferiwith anti-IL-17 antibody delayed the onset of swelling of the hind paws but, more importantly, inhibited the development of arthritis. Histopathologic examination confirmed that treatment with anti-IL-17 antibody prevented the destructive arthropathy seen in vaccinated and challenged IFN-γ0mice. Similar preventive results were obtained when vaccinated and challenged IFN-γ0mice were treated with anti-IL-17 receptor antibody or sequentially with anti-IL-17 antibody followed by anti-IL-17 receptor antibody. By contrast, treatment of vaccinated and challenged IFN-γ0mice with recombinant IL-17 (rIL-17) did not alter the development and progression of arthritis found in vaccinated and challenged IFN-γ0mice without treatment with rIL-17. Therapeutic intervention may be a realistic approach to prevent arthritis, especially if IL-17 is involved in the perpetuation of chronic or intermittent arthritis.
Host Susceptibility to the Attaching and Effacing Bacterial PathogenCitrobacter rodentium
Authors: Vallance Bruce A. , Deng Wanyin , Jacobson Kevan , Finlay B. Brett
Published: May 2003

Many studies have shown that genetic susceptibility plays a key role in determining whether bacterial pathogens successfully infect and cause disease in potential hosts. Surprisingly, whether host genetics influence the pathogenesis of attaching and effacing (A/E) bacteria such as enteropathogenic and enterohemorrhagicEscherichia colihas not been examined. To address this issue, we infected various mouse strains withCitrobacter rodentium, a member of the A/E pathogen family. Of the strains tested, the lipopolysaccharide (LPS) nonresponder C3H/HeJ mouse strain experienced more rapid and extensive bacterial colonization than did other strains. Moreover, the high bacterial load in these mice was associated with accelerated crypt hyperplasia, mucosal ulceration, and bleeding, together with very high mortality rates. Interestingly, the basis for the increased susceptibility was not due to LPS hyporesponsiveness, as the genetically related but LPS-responsive C3H/HeOuJ and C3H/HeN mouse strains were also susceptible to infection. Analysis of the intestinal pathology in these susceptible strains revealed significant crypt epithelial cell apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining) as well as bacterial translocation to the mesenteric lymph nodes. Further studies with infection of SCID (T- and B-lymphocyte-deficient) C3H/HeJ mice demonstrated that loss of lymphocytes had no effect on bacterial numbers but did reduce crypt cell apoptosis and delayed mortality. These studies thus identify the adaptive immune system, crypt cell apoptosis, and bacterial translocation but not LPS responsiveness as contributing to the tissue pathology and mortality seen duringC.rodentiuminfection of highly susceptible mouse strains. Determining the basis for these strains' susceptibility to intestinal colonization by an A/E pathogen will be the focus of future studies.
Nontypeable
Haemophilus influenzae
Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media
Authors: Mason Kevin M. , Munson Robert S. , Bakaletz Lauren O.
Published: May 2003

The gram-negative bacterium nontypeable
Haemophilus influenzae
(NTHI) is the predominant pathogen in chronic otitis media with effusion and, with
Streptococcus pneumoniae
and
Moraxella catarrhalis
, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless
gfpmut3
gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of
H. influenzae
strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.