bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 10- and Fcγ Receptor-Deficient Mice ResolveLeishmania mexicanaLesions
Authors: Buxbaum Laurence U. , Scott Phillip
Published: March 2005

Infection of C57BL/6 (B6) mice withLeishmania mexicanais associated with a minimal immune response and chronic disease. Here we show that B6 interleukin 10−/−(IL-10−/−) mice resolve their lesions and exhibit increased gamma interferon (IFN-γ), nitric oxide production, and delayed-type hypersensitivity. This enhanced resistance was dependent upon IL-12p40, since treatment ofL. mexicana-infected IL-10−/−mice with anti-IL-12p40 monoclonal antibody abrogated healing. Antibody-opsonizedL. mexicanainduced IL-10 production by B6 macrophages in vitro, implicating antibody binding to Fc receptors as a mechanism involved in IL-10 production in this infection. Furthermore, B6 FcRγ−/−mice resolveL. mexicanalesions, and lymph node cells from these mice produced less IL-10 and more IFN-γ than cells from infected wild-type mice. These data demonstrate that removal of IL-10 or FcγR leads to resolution ofL. mexicanadisease and support a model in which ligation of FcγR byL. mexicana-bound immunoglobulin G promotes IL-10 production, leading to chronic disease.
Mode of Action of Invasion-Inhibitory Antibodies Directed against Apical Membrane Antigen 1 of
Plasmodium falciparum
Authors: Dutta Sheetij , Haynes J. David , Barbosa Arnoldo , Ware Lisa A. , Snavely Jeffrey D. , Moch J. Kathleen , Thomas Alan W. , Lanar David E.
Published: March 2005

Antibodies against apical membrane antigen 1 (AMA-1) of
Plasmodium falciparum
inhibit merozoite invasion into erythrocytes. Invasion-inhibitory polyclonal AMA-1 antibodies inhibit secondary proteolytic processing and surface redistribution of AMA-1 on merozoites. We present evidence supporting inhibition of processing and redistribution as probable causes of inhibition of invasion by polyclonal antibodies. Polyclonal anti-AMA-1 was much more inhibitory than monoclonal antibody (MAb) 4G2dc1 in an invasion assay. Although both polyclonal and monoclonal immunoglobulin G (IgG) inhibited secondary processing of the 66-kDa form of AMA-1, only polyclonal IgG caused its anomalous processing, inhibited its redistribution, and cross-linked soluble forms of AMA-1 on merozoites. Moreover, Fab fragments of polyclonal IgG that fail to cross-link did not show the enhancement of inhibitory effect over intact IgG, as observed in the case of Fab fragments of MAb 4G2dc1. We propose that although blocking of biologically important sites is a common direct mode of action of anti-AMA-1 antibodies, blocking of AMA-1 secondary processing and redistribution are additional indirect inhibitory mechanisms by which polyclonal IgG inhibits invasion. We also report a processing inhibition assay that uses a C-terminal AMA-1-specific MAb, 28G2dc1, to detect merozoite-bound remnants of processing (∼20 kDa from normal processing to 48 and 44 kDa and ∼10 kDa from anomalous processing to a 52-kDa soluble form of AMA-1). The ratio of intensity of 10-kDa bands to the sum of 10- and 20-kDa bands was positively correlated with inhibition of invasion by polyclonal antibodies. This assay may serve as an important immunochemical correlate for inhibition of invasion.
Epitopes Recognized by a Nonautoreactive Murine Anti-N-Propionyl Meningococcal Group B Polysaccharide Monoclonal Antibody
Authors: Moe Gregory R. , Dave Apurva , Granoff Dan M.
Published: March 2005

The capsular polysaccharide ofNeisseria meningitidisgroup B (MBPS) is a polymer of alpha (2→8)N-acetyl neuraminic acid. The polysaccharide is chemically identical to an autoantigen, polysialic acid (PSA), and is a poor immunogen, even when conjugated to protein carriers. Immunization of mice with MBPS-protein conjugate vaccines, in whichN-acetyl groups have been replaced by propionyl groups (N-Pr MBPS), elicits serum bactericidal antibodies. A subpopulation of these antibodies do not cross-react with human PSA. The reasons for the increased immunogenicity ofN-Pr MBPS and the antigenic targets of the bactericidal nonautoreactive antibodies are unknown. In this study, we investigated the antigenic targets of a protective murine monoclonal antibody (MAb) prepared against aN-Pr MBPS-tetanus toxoid conjugate vaccine. Binding of the MAb toN-Pr MBPS (as demonstrated by an enzyme-linked immunosorbent assay) and bactericidal activity were inhibited by de-N-acetylated MBPS and re-N-acetylated MBPS, which indicate thatN-propionyl groups are not obligatory determinants for binding. The results of affinity selection from a preparation ofN-Pr MBPS and matrix-assisted laser desorption ionization-time of flight mass spectroscopic analysis indicated that the minimal epitope recognized by the MAb is a MBPS disaccharide containing one de-N-acetylated residue. Thus, the bacterial capsular epitope recognized by this bactericidal, nonautoreactive, anti-group-B MAb likely contains de-N-acetyl residues.
Regulation of Type III Secretion Hierarchy of Translocators and Effectors in Attaching and Effacing Bacterial Pathogens
Authors: Deng Wanyin , Li Yuling , Hardwidge Philip R. , Frey Elizabeth A. , Pfuetzner Richard A. , Lee Sansan , Gruenheid Samantha , Strynakda Natalie C. J. , Puente Jose L. , Finlay B. Brett
Published: March 2005

Human enteropathogenic
Escherichia coli
(EPEC), enterohemorrhagic
E. coli
(EHEC), and the mouse pathogen
Citrobacter rodentium
(CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their
sepL
and
sepD
mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in
sepL
and
sepD
. However, the secretion profile of the
sepD
and
sepL
mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.
Lipopolysaccharide Preparation Extracted fromPorphyromonas gingivalisLipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling
Authors: Asai Yasuyuki , Hashimoto Masahito , Fletcher Hansel M. , Miyake Kensuke , Akira Shizuo , Ogawa Tomohiko
Published: March 2005

We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from aPorphyromonas gingivalislipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that ofEscherichia coliLPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant ofP. gingivalisstrain 381 by allelic exchange mutagenesis using anermF-ermAMantibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (ΔPG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well asLimulusamoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the ΔPG1828-LPS preparation to activate NF-κB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the ΔPG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A ofP. gingivalisby TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the ΔPG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor ofP. gingivalis.
The Innate Immune System Is Activated by Stimulation of Vaginal Epithelial Cells withStaphylococcus aureusand Toxic Shock Syndrome Toxin 1
Authors: Peterson Marnie L. , Ault Kevin , Kremer Mary J. , Klingelhutz Aloysius J. , Davis Catherine C. , Squier Christopher A. , Schlievert Patrick M.
Published: March 2005

Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSSStaphylococcus aureusand TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSSS. aureusMN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that35S-TSST-1 bound to 5 × 104receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determinedS. aureusMNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed withS. aureusMN8. TSST-1 (100 μg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response toS. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines—MIP-3α, 478-fold; GRO-α, 26-fold; GRO-β, 14-fold; and GRO-γ, 30-fold—suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced byS. aureusand TSST-1.S. aureusMN8, when incubated with porcine vaginal tissue, increased the flux of35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.
Increased Expression of Interleukin-5 (IL-5), IL-13, and Tumor Necrosis Factor Alpha Genes in Intestinal Lymph Cells of Sheep Selected for Enhanced Resistance to Nematodes during Infection withTrichostrongylus colubriformis
Authors: Pernthaner Anton , Cole Sally-Ann , Morrison Lilian , Hein Wayne R.
Published: March 2005

Cytokine gene expression in cells migrating in afferent and efferent intestinal lymph was monitored for extended time periods in individual sheep experimentally infected with the nematodeTrichostrongylus colubriformis. Animals from stable selection lines with increased levels of either genetic resistance (R) or susceptibility (S) to nematode infection were used. Genes for interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha (TNF-α), but not for IL-4, IL-10, or gamma interferon (IFN-γ), were consistently expressed at higher levels in both afferent and efferent lymph cells of R sheep than in S sheep. However, only minor differences were observed in the surface phenotypes and antigenic and mitogenic responsiveness of cells in intestinal lymph between animals from the two selection lines. The IL-4 and IL-10 genes were expressed at higher levels in afferent lymph cells than in efferent lymph cells throughout the course of the nematode infection in animals of both genotypes, while the proinflammatory TNF-α gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-α genes declined significantly in afferent lymph cells but not in efferent lymph cells during infection. Collectively, the results showed that R-line sheep developed a strong polarization toward a Th2-type cytokine profile in immune cells migrating in lymph from sites where the immune response to nematodes was initiated, although the IFN-γ gene was also expressed at moderate levels. Genes or alleles that predispose an animal to develop this type of response appear to have segregated with the R selection line and may contribute to the increased resistance of these animals.
Yersinia pestisV Protein Epitopes Recognized by CD4 T Cells
Authors: Parent Michelle A. , Berggren Kiera N. , Mullarky Isis K. , Szaba Frank M. , Kummer Lawrence W. , Adamovicz Jeffrey J. , Smiley Stephen T.
Published: March 2005

Pneumonic plague, an often-fatal disease for which no vaccine is presently available, results from pulmonary infection by the bacteriumYersinia pestis. TheY. pestisV protein is a promising vaccine candidate, as V protein immunizations confer to mice significant protection against aerosolizedY. pestis. CD4 T cells play central roles during vaccine-primed immune responses, but their functional contributions toY. pestisvaccines have yet to be evaluated and optimized. Toward that end, we report here the identification of three distinct epitopes within theY. pestisV protein that activate CD4 T cells in C57BL/6 mice. To our knowledge, these are the first identified CD4 T-cell epitopes in anyY. pestisprotein. The epitopes are restricted by the I-Abclass II major histocompatibility complex molecule and are fully conserved betweenY. pestis,Yersinia pseudotuberculosis, andYersinia enterocolitica. Immunizing mice with a V protein-containing vaccine or with short peptides containing the identified epitopes primes antigen-specific production of interleukin 2 and gamma interferon by CD4 T cells upon their restimulation in vitro. Consistent with prior studies documenting protective roles for CD4 T cells duringY. enterocoliticainfection, vaccinating mice with a 16-amino-acid peptide encoding one of the epitopes suffices to protect against an otherwise lethalY. enterocoliticachallenge. The identification of these epitopes will permit quantitative assessments of V-specific CD4 T cells, thereby enabling researchers to evaluate and optimize the contribution of these cells to vaccine-primed protection against pneumonic plague.
Natural History of Gastric Mucosal Cytokine Expression inHelicobacter pyloriGastritis in Mongolian Gerbils
Authors: Yamaoka Yoshio , Yamauchi Kazuyoshi , Ota Hiroyoshi , Sugiyama Atsushi , Ishizone Satoshi , Graham David Y. , Maruta Fukuto , Murakami Maki , Katsuyama Tsutomu
Published: March 2005

Data regarding the chronological changes in gastric mucosal cytokines in the different phases ofHelicobacter pyloriinfection are unavailable. We examined Mongolian gerbils for up to 52 weeks afterH. pylori(ATCC 43504) inoculation. Levels of mRNAs of mucosal cytokines (interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-4, IL-6, and IL-10) were assessed using real-time reverse transcription-PCR. Starting 26 weeks afterH. pyloriinoculation, two clinicohistologic patterns appeared: gastric ulcers in 32% and hyperplastic polyps in 68% of gerbils. High levels of mucosal IL-1β mRNA were observed early in the infection, reaching maximum at 4 weeks and then rapidly declining. Mucosal IFN-γ mRNA also reached maximal levels at 4 weeks but remained high thereafter. Both IL-1β and IFN-γ mRNA levels were consistently higher in the pyloric mucosa than in the fundic mucosa. In contrast, IL-4, IL-6, and IL-10 mRNA levels peaked at 8 to 26 weeks and levels were similar in the pyloric mucosa and the fundic mucosa. IFN-γ mRNA levels were significantly higher in gerbils with ulcers than in those with hyperplastic polyps (median IFN-γ/glyceraldehyde-3-phosphate dehydrogenase ratio × 100,000 = 650 versus 338, respectively [antrum], and 172 versus 40, respectively [corpus]) (P< 0.05). We propose that the different outcomes (e.g., ulcers or hyperplastic polyps) might relate to imbalances among cytokines.
Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance ofYersinia enterocoliticaSerotype O:3
Authors: Biedzka-Sarek Marta , Venho Reija , Skurnik Mikael
Published: March 2005

Complement attack is a host strategy leading to elimination of pathogens.Yersinia enterocoliticaexpresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival ofY. enterocoliticaserotype O:3 in nonimmune human serum, we constructed 23 mutant strains ofY. enterocoliticaO:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed.
Fluid- or Surface-Phase Human Salivary Scavenger Protein gp340 Exposes Different Bacterial Recognition Properties
Authors: Loimaranta V. , Jakubovics N. S. , Hytönen J. , Finne J. , Jenkinson H. F. , Strömberg N.
Published: March 2005

Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g.,
Streptococcus gordonii
and
Streptococcus mutans)
, non-viridans group streptococci (e.g.,
Streptococcus pyogenes
and
Streptococcus suis
), and oral
Actinomyces
. Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of
Streptococcus
tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for
S. gordonii
was correlated with a variant of sialic acid specificity. Adherence of
S. gordonii
DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of
S. mutans
NG8 involved antigen I/II polypeptide. Deletion of the
mga
virulence regulator gene in
S. pyogenes
resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.
The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases
Authors: Bertin Patrícia B. , Lozzi Silene P. , Howell Jerrilyn K. , Restrepo-Cadavid Glória , Neves David , Teixeira Antonio R. L. , de Sousa Marcelo V. , Norris Steven J. , Santana Jaime M.
Published: March 2005

Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirocheteBorrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts fromB. burgdorferiby using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified fromB. burgdorferiby a two-step chromatographic procedure, yielding a ∼300-kDa homo-oligomeric enzyme formed by monomers of ∼50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II ofB. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60°C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase ofB. burgdorferi(TAPBb) depends on Zn2+, and temperatures over 70°C promoted its inactivation through a transition from the hexameric state to the monomeric state. SinceB. burgdorferiis deficient in pathways for amino acid synthesis, TAPBbcould play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.
Virulence ofPseudomonas aeruginosain a Murine Model of Gastrointestinal Colonization and Dissemination in Neutropenia
Authors: Koh Andrew Y. , Priebe Gregory P. , Pier Gerald B.
Published: March 2005

Pseudomonas aeruginosabacteremia in cancer patients develops from initial gastrointestinal (GI) colonization with translocation into the bloodstream in the setting of chemotherapy-induced neutropenia and GI mucosal damage. We established a reproducible mouse model ofP. aeruginosaGI colonization and systemic spread during neutropenia. Mice received 2 mg of streptomycin/ml of drinking water and 1,500 U of penicillin G/ml for 4 days and then ingested 107CFU ofP. aeruginosaper ml of drinking water for 5 days. After GI colonization levels were determined, cyclophosphamide (Cy) was then injected intraperitoneally (i.p.) three times every other day or an antineutrophil monoclonal antibody, RB6-8C5, was injected i.p. once. Dissemination was defined by the presence ofP. aeruginosain spleens of moribund or dead mice. In this mouse model,P. aeruginosacolonizes the GI tract and then disseminates systemically once Cy or RB6-8C5 is administered. The duration and intensity of neutropenia, related to Cy dose, was found to be a means to compare the virulence of differentP. aeruginosastrains, as exhibited by comparisons of strains lacking or producing the virulence-enhancing ExoU cytotoxin. The lipopolysaccharide outer core polysaccharide and O side chains were critical in establishing GI colonization, andP. aeruginosamutants lacking thearoAgene (necessary for synthesizing aromatic amino acids) were able to establish GI colonization but unable to disseminate. Both the colonization and dissemination phases ofP. aeruginosapathogenesis can be studied in this model, which should prove useful for evaluating pathogenesis, therapies, and associated means to controlP. aeruginosanosocomial infections.
Immunoglobulin G Isotype Responses to Erythrocyte Surface-Expressed Variant Antigens of
Plasmodium falciparum
Predict Protection from Malaria in African Children
Authors: Yone Clarisse L. R. P. , Kremsner Peter G. , Luty Adrian J. F.
Published: March 2005

We assessed immunoglobulin G (IgG) isotype responses to variant surface antigens (VSA) expressed on parasite-infected erythrocytes of a panel of heterologous isolates during and after acute episodes in groups of Gabonese children presenting with either mild or severe
Plasmodium falciparum
malaria. In the acute and convalescent phases IgG3 and IgG1 anti-VSA antibodies, respectively, predominated. In the absence of infection, the levels of both cytophilic isotypes waned, while those of IgG4 increased, particularly in those admitted with severe malaria. Prospective analyses showed significantly longer delays between malaria attacks associated both (i) with increasing IgG1 responses with specificity for VSA of isolates from children with mild malaria and (ii) with increasing IgG4 responses with specificity for VSA of isolates from children with severe malaria. These findings imply that the predictive value of prospectively measured cross-reactive VSA-specific IgG antibodies with respect to protection against malaria in African children depends both on their isotype and on their fine specificity.
DNA Immunization with Na
+
-K
+
ATPase (
Sseat-6
) Induces Protective Immunity to Larval
Strongyloides stercoralis
in Mice
Authors: Kerepesi Laura A. , Keiser Paul B. , Nolan Thomas J. , Schad Gerhard A. , Abraham David , Nutman Thomas B.
Published: March 2005

Strongyloides stercoralis
causes chronic asymptomatic infections which can be maintained in the human host for many decades. Identification and treatment of S.
stercoralis
-infected individuals is required because immunosuppression can lead to fatal hyperinfection. In this study, human immunoglobulin G (IgG) that had previously been shown to transfer protective immunity to mice was used to identify potential protective antigens. Three antigens or genes from
S. stercoralis
larvae were identified as tropomyosin (
Sstmy-1
), Na
+
-K
+
ATPase (
Sseat-6
), and LEC-5 (
Sslec-5
). The genes were cloned into plasmids for DNA immunization, and mice were immunized intradermally with the three plasmids individually in combination with a plasmid containing murine granulocyte-macrophage colony-stimulating factor. Only Na
+
-K
+
ATPase induced a significant reduction in larval survival after DNA immunization. Immunization with a combination of all three plasmids, including Na
+
-K
+
ATPase, did not induce protective immunity. Serum from mice immunized with DNA encoding Na
+
-K
+
ATPase was transferred to naïve mice and resulted in partial protective immunity. Therefore, DNA immunization with Na
+
-K
+
ATPase induces protective immunity in mice, and it is the first identified vaccine candidate against infection with larval
S. stercoralis
.
Processing and Major Histocompatibility Complex Class II Presentation ofLegionella pneumophilaAntigens by Infected Macrophages
Authors: Neild Annie , Murata Takahiro , Roy Craig R.
Published: March 2005

To better understand interactions between the intracellular pathogenLegionella pneumophilaand macrophages (Mφs), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Mφs (BMφs) infected with wild-typeL. pneumophilawere higher than the responses to avirulentdotAmutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication ofL. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMφs infected with wild-typeL. pneumophilathat did not respond to Mφ infection withdotAmutant bacteria. Endocytic processing was required for presentation ofL. pneumophilaantigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BMφ endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMφs infected withL. pneumophila icmR,icmW, andicmSmutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type anddotAmutant bacteria. In addition, it was found that antigens fromdotandicmmutants are presented earlier than antigens from wild-typeL. pneumophila. Although immune CD4 T-cell responses to proteins secreted by theL. pneumophilaLsp system were not detected, it was found that the Lsp system is important for primingL. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis ofL. pneumophilaproteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.
Identification and Functional Characterization of Chicken Toll-Like Receptor 5 Reveals a Fundamental Role in the Biology of Infection with
Salmonella enterica
Serovar Typhimurium
Authors: Iqbal Muhammad , Philbin Victoria J. , Withanage G. S. K. , Wigley Paul , Beal Richard K. , Goodchild Marianne J. , Barrow Paul , McConnell Ian , Maskell Duncan J. , Young John , Bumstead Nat , Boyd Yvonne , Smith Adrian L.
Published: March 2005

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of
Salmonella
serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5
+
cells to flagellin induced elevated levels of chicken interleukin-1β (chIL-1β) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar
Salmonella enterica
serovar Typhimurium
fliM
mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the
fliM
mutant induced less IL-1β mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated
Salmonella
into systemic sites of the chicken.
Identification and Functional Characterization of Complement Regulator-Acquiring Surface Protein 1 of the Lyme Disease SpirochetesBorrelia afzeliiandBorrelia garinii
Authors: Wallich Reinhard , Pattathu Joseph , Kitiratschky Veronique , Brenner Christiane , Zipfel Peter F. , Brade Volker , Simon Markus M. , Kraiczy Peter
Published: March 2005

Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein ofBorrelia burgdorferiand is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 ofB. burgdorferisensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistantBorrelia afzeliistrains express a unique FHL-1 and factor H-binding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Baof 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1BaofB. afzeliispirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Bamolecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Bais shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the correspondingcspA-like gene(s) of a serum-sensitiveBorrelia gariniistrain has also been cloned and characterized. Most notably, two CRASP-1-relatedB. gariniiproteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Barepresents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenicBorreliastrains.
Clinical and Microbiological Responses of Volunteers to Combined Intranasal and Oral Inoculation with a
Streptococcus gordonii
Carrier Strain Intended for Future Use as a Group A Streptococcus Vaccine
Authors: Kotloff Karen L. , Wasserman Steven S. , Jones Kevin F. , Livio Sofie , Hruby Dennis E. , Franke Christine A. , Fischetti Vincent A.
Published: March 2005

Streptococcus gordonii
shows promise as a live mucosal vaccine vector for immunization against respiratory pathogens. In preparation for clinical trials to evaluate
S. gordonii
engineered to express group A streptococcal M protein antigens, we characterized the responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5 × 10
9
CFU of SP204(1-1), an
S. gordonii
strain not bearing vaccine antigens. SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, we performed serial culturing of nose, mouth, and saliva samples from 120 subjects treated with azithromycin beginning 5 days after inoculation to determine whether SP204(1-1) could be rapidly eliminated should safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were monitored with repeated culturing for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within 5 days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected by culturing in 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a 5-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within 7 days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of
S. gordonii
as a live mucosal vaccine vector is a feasible strategy.
Involvement of theEscherichia coliO157:H7(pO157)ecfOperon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs
Authors: Yoon Jang W. , Lim Ji Youn , Park Yong H. , Hovde Carolyn J.
Published: March 2005

Escherichia coliO157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157ecf(E. coliattaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is thelpxMgene product.E. coliO157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of theecfoperon by intrinsic DNA curvature and the genetic redundancy oflpxMthat is associated with lipid A modification led us to hypothesize that the pO157ecfoperon andlpxMare associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenicecfoperon and/orlpxMdeletion mutants ofE. coliO157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in theecfandlpxMgenes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.
Haemophilus ducreyi
Outer Membrane Determinants, Including DsrA, Define Two Clonal Populations
Authors: White Catherine Dinitra , Leduc Isabelle , Olsen Bonnie , Jeter Chrystina , Harris Chavala , Elkins Christopher
Published: March 2005

The
Haemophilus ducreyi
outer membrane component DsrA (for ducreyi serum resistance A) is necessary for complete resistance to normal human serum (NHS). When DsrA expression in 19 temporally and geographically diverse clinical isolates of
H. ducreyi
was examined by Western blotting, 5 of the strains expressed a different immunotype of the DsrA protein (DsrA
II
) than the well-characterized prototypical strain 35000HP (DsrA
I
). The predicted DsrA proteins expressed by the DsrA
II
strains were 100% identical to each other but only 48% identical to that of strain 35000HP. In addition to the DsrA
II
protein, class II strains also expressed variant forms of other outer membrane proteins (OMPs) including NcaA (necessary for collagen adhesion A), DltA (ducreyi lectin A), Hlp (
H. ducreyi
lipoprotein), major OMP, and/or OmpA2 (for OMP A2) and synthesized a distinct, faster-migrating lipooligosaccharide. Based on these data, strains expressing DsrA
I
were termed class I, and those expressing DsrA
II
were termed class II. Expression of
dsrA
II
from strain CIP 542 ATCC in the class I
dsrA
I
mutant FX517 (35000HP background), which does not express a DsrA protein, rendered this strain resistant to 50% NHS. This demonstrates that DsrA
II
protein is also critical to serum resistance. Taken together, these results indicate that there are two clonal populations of
H. ducreyi
. The implications of two classes of
H. ducreyi
strains differing in important antigenic outer membrane components are discussed.
Staphylococcus aureusDeficient in Lipidation of Prelipoproteins Is Attenuated in Growth and Immune Activation
Authors: Stoll Hartmut , Dengjel Jörn , Nerz Christiane , Götz Friedrich
Published: March 2005

A lipoprotein diacylglyceryl transferase (lgt) deletion mutant ofStaphylococcus aureusSA113 was constructed. The lipoprotein and prelipoprotein expression, the growth behavior, and the ability of the mutant to elicit an immune response in various host cells were studied. In the wild type, the majority of [14C]palmitate-labeled lipoproteins were located in the membrane fraction, although some lipoproteins were also present on the cell surface and in the culture supernatant. Thelgtmutant completely lacked palmitate-labeled lipoproteins and released high amounts of some unmodified prelipoproteins, e.g., the oligopeptide-binding protein OppA, the peptidyl-prolylcis-transisomerase PrsA, and the staphylococcal iron transporter SitC, into the culture supernatant. The growth of thelgtmutant was hardly affected in rich medium but was retarded under nutrient limitation. Thelgtmutant and its crude lysate induced much fewer proinflammatory cytokines and chemokines in human monocytic (MonoMac6), epithelial (pulmonary A549), and endothelial (human umbilical vein endothelial) cells than the wild type. However, in whole blood samples, the culture supernatant of thelgtmutant was equal or even superior to the wild-type supernatant in tumor necrosis factor alpha induction. Lipoprotein fractionation experiments provided evidence that a small proportion of the mature lipoproteins are released by theS. aureuswild type despite the lipid anchor and are trapped in part by the cell wall, thereby exposing the immune-activating lipid structure on the cell surface. Bacterial lipoproteins appear to be essential for a complete immune stimulation by gram-positive bacteria.
Genetic Diversity and Carriage Dynamics ofNeisseria lactamicain Infants
Authors: Bennett Julia S. , Griffiths David T. , McCarthy Noel D. , Sleeman Karen L. , Jolley Keith A. , Crook Derrick W. , Maiden Martin C. J.
Published: March 2005

Neisseria lactamica, a harmless human commensal found predominantly in the upper respiratory tracts of infants, is closely related toNeisseria meningitidis, a pathogen of global significance. Colonization withN. lactamicamay be responsible for the increase in immunity to meningococcal disease that occurs during childhood, when rates of meningococcal carriage are low. This observation has led to the suggestion thatN. lactamicawhole cells or components are potential constituents of novel meningococcal vaccines. However, the dynamics of carriage and population diversity ofN. lactamicain children are poorly understood, presenting difficulties for the choice of representative isolates for use in vaccine development. This problem was addressed by the multilocus sequence typing ofN. lactamicaisolates from two longitudinal studies of bacterial carriage in infants. The studies comprised 100 and 216 subjects, withN. lactamicacarriage monitored from age 4 weeks until age 96 weeks and from age 2 weeks until age 24 weeks, respectively. The maximum observed carriage rate was 44% at 56 weeks of age, with isolates obtained on multiple visits for the majority (54 of 75, 72%) of carriers. TheN. lactamicaisolates were genetically diverse, with 69 distinct genotypes recovered from the 75 infants. Carriage was generally long-lived, with an average rate of loss of under 1% per week during the 28 weeks following acquisition. Only 11 of the 75 infants carried more than one genotypically unique isolate during the course of the study. Some participants shared identical isolates with siblings, but none shared identical isolates with their parents. These findings have implications for the design of vaccines based on this organism.
Role of Predicted Transmembrane Domains for Type III Translocation, Pore Formation, and Signaling by the
Yersinia pseudotuberculosis
YopB Protein
Authors: Ryndak Michelle B. , Chung Hachung , London Erwin , Bliska James B.
Published: March 2005

YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic
Yersinia
species. YopB is required for
Yersinia
spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed.
Yersinia pseudotuberculosis
strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.
Functional Analysis of
Plasmodium falciparum
Apical Membrane Antigen 1 Utilizing Interspecies Domains
Authors: Healer Julie , Triglia Tony , Hodder Anthony N. , Gemmill Alan W. , Cowman Alan F.
Published: March 2005

Plasmodium falciparum
apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of
P. falciparum
(PfAMA1) with that of
P. chabaudi
(PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against
Escherichia coli
expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal
pro
region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.
Enterococcus faecalisTropism for the Kidneys in the Urinary Tract of C57BL/6J Mice
Authors: Kau Andrew L. , Martin Steven M. , Lyon William , Hayes Ericka , Caparon Michael G. , Hultgren Scott J.
Published: March 2005

Enterococcus faecalisis a gram-positive bacterium that can cause a variety of nosocomial infections of which urinary tract infections are the most common. These infections can be exceptionally difficult to treat because of drug resistance of manyE. faecalisisolates. Despite their troublesome nature, little is known about the host or bacterial factors necessary forE. faecalisto cause disease in the urinary tract. Using a mouse model of urinary tract infection, we have shown thatE. faecalisis capable of persisting in the kidneys of mice for at least 2 weeks. In contrast, bacterial titers from the bladders of the same mice were inconsistent and tended to be much lower than those recovered from the kidney. This preference for the kidney over the bladder is also observed in other clinicalE. faecalisstrains. Histologic examination of bladder and kidney tissues demonstrated thatE. faecalisinduced an inflammatory response in the kidney but not in the bladder. This inflammatory response was TLR2 independent and did not induce inflammatory markers typically associated with uropathogenicEscherichia coli. Using a competition assay, we demonstrated that a pyelonephritis clinical isolate had a growth advantage over a laboratory strain ofE. faecalisin the kidneys but not in the bladders of mice. Taken together, these results demonstrate thatE. faecalishas tropism for the kidneys in the urinary tracts of mice and that this system can be used to study factors involved in the pathogenesis of urinary tract infections.