bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of a Campylobacter jejuni 29-kilodalton periplasmic binding protein
Authors: Garvis S G , Puzon G J , Konkel M E
Published: January 2020

Campylobacter jejuni, a gram-negative, microaerophilic, spiral bacterium, is a common cause of human gastrointestinal disease. Although investigators commonly use C. jejuni glycine-hydrochloride extracts in assays to determine the products that promote the binding of the organism to eukaryotic cells, the proteins contained within these extracts remain ill defined. Characterization of these proteins will provide a better understanding of C. jejuni gene regulation and organization. An antiserum was raised against a C. jejuni 29-kDa gel-purified protein detected in glycine-hydrochloride extracts. This antiserum was used to screen an expression library of C. jejuni. A reactive clone that contained an open reading frame of 256 amino acids was identified. The cloned gene was transcribed and translated, and the product was exported to the periplasmic space in Escherichia coli XL1-Blue. The translated C. jejuni product, designated P29, exhibited significant similarity to the histidine and lysine-arginine-ornithine periplasmic binding proteins (HisJ and LAO, respectively) of Salmonella typhimurium. The C. jejuni gene encoding the P29 protein complemented an S. typhimurium HisJ mutant but not a LAO mutant when provided in trans. These data suggest that the C. jejuni gene encoding the P29 protein is a homolog of the S. typhimurium hisJ gene.
Construction and characterization of a potential live oral carrier-based vaccine against Vibrio cholerae O139
Authors: Favre D , Cryz S J , Viret J F
Published: January 2020

The rfb region from Vibrio cholerae O139 strain MO45 was cloned from cosmid gene banks established in Escherichia coli HB101, using an immunoblot assay for screening of the correct clones. Immunoblot analysis of lipopolysaccharide (LPS) preparations revealed the presence of two types of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polymerized capsular polysaccharide (CPS) not bound to the E. coli K-12 LPS core. In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS and CPS chain length. Further characterization in E. coli showed that CPS constitutes a barrier against large particles such as the bacteriophage Ffm but not against bacteriophage lambda or P1. In addition, a portion of the K-12 LPS core may not be substituted with SOPS. Loci associated with the two clonal types were transferred into V. cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O polysaccharide. This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V. cholerae O139. Strains CH25 and CH26, which correspond to CH19 bearing the V. cholerae O139 rfb region integrated into the chromosome, were found to be genetically stable and essentially identical to the parent CVD103-HgR with respect to physiological properties such as cell motility, mercury resistance, toxicity, and production of the cholera toxin B subunit. Rabbits immunized with CH25 elicited high titers of anti-O139 SOPS- and CPS-specific serum antibodies. These strains possess characteristics desirable in candidate live oral vaccines against V. cholerae O139.
Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection
Authors: Burghaus P A , Wellde B T , Hall T , Richards R L , Egan A F , Riley E M , Ballou W R , Holder A A
Published: January 2020

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.
Tolerance to appetite suppression induced by peptidoglycan
Authors: Biberstine K J , Darr D S , Rosenthal R S
Published: January 2020

Physiologically realistic peptidoglycan (PG) fragments, derived from Neisseria gonorrhoeae, were shown previously to dose-dependently suppress food consumption and body weight gain in rats following single intraperitoneal injections. The present study, examining the effects of repeated daily injection of PG, provides additional support to our underlying hypothesis, i.e., that soluble PG fragments contribute to the loss of appetite commonly associated with bacterial infections. An initial intraperitoneal injection of purified, soluble, macromolecular, extensively O-acetylated PG fragments (S-O-PG) (240 micrograms/kg of body weight) decreased overnight food consumption in male Lewis rats (150 g) by approximately 35% relative to animals receiving diluent alone (P < 0.05). However, subsequent daily injections of S-O-PG resulted in progressively smaller effects on food consumption until, by the fourth day, rats were completely nonresponsive (tolerant) to S-O-PG-induced hypophagia. Rats that developed tolerance to the effects of S-O-PG on appetite were also tolerant to three other known hypophagic agents, lipopolysaccharide (LPS), muramyl dipeptide, and interleukin-1, when challenged one day after establishment of S-O-PG tolerance. Similarly, rats developed tolerance to repeated injections of muramyl dipeptide or LPS and were cross-tolerant to S-O-PG when challenged 1 day later. However, 30 days after establishment of S-O-PG tolerance, rats remained nonresponsive to S-O-PG but regained full responsiveness to LPS-mediated hypophagia. Thus, at least two mechanisms of tolerance to S-O-PG hypophagia exist: an early tolerance which is nonspecific and a late tolerance which is specific for S-O-PG. Late, but not early, tolerance to S-O-PG-mediated suppression of appetite was associated with an increase in specific anti-PG antibody activity as measured in an enzyme-linked immunosorbent assay.
Inhibition of human peripheral blood mononuclear cell proliferative response by glycosphingolipids from metacestodes of Echinococcus multilocularis
Authors: Persat F , Vincent C , Schmitt D , Mojon M
Published: January 2020

The effect on human peripheral blood mononuclear cells (PBMCs) of neutral glycosphingolipids extracted from metacestodes of the parasite Echinococcus multilocularis was investigated. Neutral glycosphingolipids inhibited [3H]thymidine uptake by human PBMCs upon stimulation by mitogens such as phytohemagglutinin A and pokeweed mitogen or by allogeneic Burkitt B cells. This effect was dose dependent and was related to a decrease in interleukin 2 (IL-2) synthesis, the expression of IL-2 receptors (CD25) being unmodified. Addition of exogenous recombinant IL-2 restored the cell proliferation. Partial inhibition of immunoglobulin G (IgG), IgA, and IgM synthesis was observed in the supernatant of cell culture in association with the inhibitory effect. Identification of active subfractions contained in the neutral glycosphingolipid fraction was also studied in relation to cell viability. The free ceramide fraction had an inhibitory effect, in part related to cell lysis, particularly at high concentration, while the monogalactosylceramides had a paradoxical effect: as an activator at low concentrations and as an inhibitor at high concentrations, with limited cell survival. The immunogenic neutral glycosphingolipids containing at least two carbohydrate residues, all having a structure based on Gal beta 1-->6Gal, were inhibitors of PBMC proliferation and showed good cell survival. These results suggest that parasite neutral glycosphingolipids may play an immunologically relevant role in alveolar hydatid disease.
Elimination of the listeriolysin O-directed immune response by conservative alteration of the immunodominant listeriolysin O amino acid 91 to 99 epitope
Authors: Bouwer H G , Moors M , Hinrichs D J
Published: January 2020

A major H2-Kd-presented epitope for antilisterial cytotoxic T lymphocytes (CTLs) is the nanomer peptide which corresponds to the amino acid 91 to 99 (aa91-99) sequence from listeriolysin O (LLO). Although the LLO sequence contains at least five additional nanomer peptides which also satisfy the H2-Kd binding motif, aa91-99 is the only LLO-derived target peptide that is recognized by antilisterial CTLs following infection of BALB/c mice with Listeria monocytogenes. In order to investigate further the immunodominance of the LLO aa91-99 epitope following endogenous processing of LLO, we introduced a point mutation in hly (the gene for LLO) which results in a conservative Y-to-F substitution for the anchor residue at position 2 within the aa91-99 sequence. This "92F" L. monocytogenes mutant produces biologically active LLO and is phenotypically indistinct from wild-type L. monocytogenes in terms of intracellular growth in vitro and virulence in vivo. BALB/c mice actively immunized with the 92F L. monocytogenes mutant are protected against challenge with wild-type L. monocytogenes. Antilisterial CTLs from mice immunized with the 92F mutant lyse targets infected with L. monocytogenes; however, these CTLs do not lyse target cells pulsed with either the LLO aa91-99 peptide, other LLO-derived peptides which satisfy the H2-Kd binding motif, or a peptide corresponding to the LLO aa91-92F-99 sequence. Target cells pulsed with the LLO aa91-92F-99 peptide are, however, lysed by wild-type LLO aa91-99-specific cytotoxic cells. Thus, a conservative amino acid change in the first anchor residue of the immunodominant aa91-99 sequence of LLO eliminates the induction of the cytotoxic cell response to this epitope as well as to any of the other candidate LLO-derived peptides which fit the H2-Kd binding motif. The lack of anti-LLO-specific CTLs following immunization with the 92F mutant does not appear, however, to influence the protective antilisterial immune response.
Binding of resting platelets to Candida albicans germ tubes
Authors: Robert R , Mahaza C , Miegeville M , Ponton J , Marot-Leblond A , Senet J M
Published: January 2020

The binding of resting platelets to Candida albicans germ tubes was studied by means of an affinity column in which germ tubes were physically immobilized. Adhesion of platelets to the column was dependent on both the germ tube concentration and the number of platelets applied. It was found that the interaction of C. albicans germ tubes with platelets is specific and should be mediated by a fungal protein receptor. The results obtained by scanning electron microscopy confirmed that resting platelets can fix directly onto germ tubes. In addition, this study showed that attachment of platelets onto C. albicans is associated with morphological changes. Platelets lost their discoid shape, became globular, generated spikes or pseudopods, and then flattened on the yeast cells.
Attenuated Salmonella vaccine-induced suppression of murine spleen cell responses to mitogen is mediated by macrophage nitric oxide: quantitative aspects
Authors: Huang D , Schwacha M G , Eisenstein T K
Published: January 2020

Previous reports from our laboratory have shown that 7 days after infection of C3HeB/FeJ mice with an attenuated strain of Salmonella typhimurium, there is profound suppression of responses to B- and T-cell mitogens and suppression of the capacity of spleen cells to mount a primary, in vitro plaque-forming-cell (PFC) response to sheep erythrocytes. Inhibition of the PFC response was shown to be mediated by nitric oxide (NO), as NG-monomethyl-L-arginine (NMMA) gave complete reversal of suppression. The experiments reported here examined the role of NO in suppression of the response to the mitogen concanavalin A (ConA). In contrast to the PFC system, it was found that addition of NMMA to ConA-stimulated immune spleen cells resulted in less than 20% reversal of suppression. However, addition to NMMA resulted in a 50% reversal of suppression in cocultures of immune and normal spleen cells at a ratio of 1:4. A complete restoration of ConA-induced responses was achieved in cocultures incubated in medium containing a reduced concentration of L-arginine plus 1.25 mM NMMA. Investigation of why NMMA alone was not 100% effective in reversing suppression showed that addition of ConA significantly augmented production of nitrite and gamma interferon (IFN-gamma) in cocultures containing immune cells. Addition of anti-IFN-gamma reduced nitrite levels in the cultures, although results with the combination of anti-IFN-gamma and NMMA were not significantly better than results with NMMA alone. These findings suggest that suppression in cultures stimulated with ConA is difficult to reverse completely with NMMA alone because of an overproduction of NO, which can be offset by either reducing the L-arginine concentration or blocking IFN-gamma. The quantitative relationship between nitrite levels and suppression in cocultures was examined. It was found that suppression did not correlate directly with the nitrite concentration but rather with the log10 of the nitrite concentration. Nitrite levels above 15 microM gave almost complete suppression, and levels between 1 and 10 microM gave a wide range of suppression. These results strongly support NO as the suppressor factor in Salmonella-induced immunosuppression of responses to ConA and, by inference, suppression of responses to mitogens induced by other microbes. The results show that involvement of NO cannot always be demonstrated by simple addition of NMMA to suppressed mitogen-stimulated spleen cell cultures.
Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells
Authors: van de Kerkhof A , Haesebrouck F , Chiers K , Ducatelle R , Kamp E M , Smits M A
Published: January 2020

The effect of Actinobacillus pleuropneumoniae and its metabolites on the viability of porcine alveolar epithelial cells was studied by using a neutral-red uptake test. Alveolar epithelial cells were obtained from 5-week-old colostrum-deprived pigs. The purity of these cells as assessed by the modified Papanicolaou stain was 90 to 95%. Incubation of these cells with 10(6) CFU of a biotype 1 serotype 1 strain resulted in death of the alveolar epithelial cells within 1.5 h. A cytotoxic effect was also seen when alveolar epithelial cells were incubated with sterile culture supernatants of biotype 1 serotype 1, biotype 1 serotype 10, and biotype 2 serotype 2 strains or with ApxI, ApxII, or ApxIII produced by recombinant Escherichia coli. Incubation of alveolar epithelial cells with a knockout mutant of the biotype 1 serotype 1 parent strain which is unable to secrete Apx toxins or with its supernatant did not result in death of these cells. These results indicate that cytotoxicity is at least in part due to production of Apx toxins.
Influence of anti-Actinobacillus actinomycetemcomitans Y4 (serotype b) lipopolysaccharide on severity of generalized early-onset periodontitis
Authors: Califano J V , Gunsolley J C , Nakashima K , Schenkein H A , Wilson M E , Tew J G
Published: January 2020

The objective of this study was to determine if a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) and severity of periodontal disease in generalized early-onset periodontitis (G-EOP). The concentration of antibody reactive with A. actinomycetemcomitans serotype b LPS was determined for 102 G-EOP subjects. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans serotype b LPS and measures of periodontal attachment loss indicated that the patients with the highest concentrations of antibody reactive with A. actinomycetemcomitans serotype b LPS had significantly less attachment loss. These high-responder subjects also had anti-A. actinomycetemcomitans serotype b LPS with significantly higher relative avidity. The results suggest that antibody reactive with A. actinomycetemcomitans serotype b LPS is protective in G-EOP patients.
Immune response to Shigella sonnei in U.S. Marines
Authors: Mikhail M M , Mansour M M , Oyofo B A , Malone J D
Published: January 2020

Pre- and postdeployment serum samples obtained from U.S. marines in Operations Desert Shield and Desert Storm were tested for antibodies to Shigella sonnei. High predeployment levels of immunoglobulin A (IgA) and/or IgG antibodies to S. sonnei lipopolysaccharide antigen in serum and seroconversions were accompanied by higher IgA and/or IgG antibody response to Shigella invasion plasmids (Ipa). The results suggest exposure to S. sonnei in predeployment troops and frequent exposure to this bacterial agent during deployment.
Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria
Authors: Perlmann P , Perlmann H , Flyg B W , Hagstedt M , Elghazali G , Worku S , Fernandez V , Rutta A S , Troye-Blomberg M
Published: January 2020

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.
Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum
Authors: Wyatt C R , Brackett E J , Perryman L E , Rice-Ficht A C , Brown W C , O'Rourke K I
Published: January 2020

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.
Sequestration of Mycobacterium tuberculosis in tight vacuoles in vivo in lung macrophages of mice infected by the respiratory route
Authors: Moreira A L , Wang J , Tsenova-Berkova L , Hellmann W , Freedman V H , Kaplan G
Published: January 2020

Following aerosol infection of mice with Mycobacterium tuberculosis, single mycobacteria or pairs of bacilli were observed within individual phagocytic vacuoles bound by tightly apposed vacuolar membranes. The virulent organism was not observed free in the cytoplasm of the parasitized cells or in the extracellular space of the lung granulomata. This study indicates that in vivo, virulent mycobacteria survive and probably replicate within a unique tight vacuole in the infected phagocyte within the lung.
Lipopeptides of Borrelia burgdorferi outer surface proteins induce Th1 phenotype development in alphabeta T-cell receptor transgenic mice
Authors: Infante-Duarte C , Kamradt T
Published: January 2020

Induction of the appropriate T helper cell (Th) subset is crucial for the resolution of infectious diseases and the prevention of immunopathology. Some pathogens preferentially induce Th1 or Th2 responses. How microorganisms influence Th phenotype development is unknown. We asked if Borrelia burgdorferi, the spirochete which causes Lyme arthritis, can promote a cytokine milieu in which T cells which are not specific for B. burgdorferi are induced to produce proinflammatory cytokines. Using alphabeta T-cell receptor transgenic mice as a source of T cells with a defined specificity other than for B. burgdorferi, we found that B. burgdorferi induced Th1 phenotype development in ovalbumin-specific transgenic T cells. Small synthetic lipopeptides corresponding to the N-terminal sequences of B. burgdorferi outer surface lipoproteins had similar effects. B. burgdorferi and its lipopeptides induced host cells to produce interleukin-12. When the peptides were used in delipidated form, they did not induce Th1 development. These findings may be of pathogenic importance, since it is currently assumed that a Th2-mediated antibody response is protective against B. burgdorferi. Bacteria associated with reactive arthritis, namely, Yersinia enterocolitica, Shigella flexneri, and Salmonella enteritidis, had different effects. The molecular definition of pathogen-host interactions determining cytokine production should facilitate rational therapeutic interventions directing the host response towards the desired cytokine response. Here, we describe small synthetic molecules capable of inducing Th1 phenotype development.
Differential response of human monocytes to Neisseria gonorrhoeae variants expressing pili and opacity proteins
Authors: Knepper B , Heuer I , Meyer T F , van Putten J P
Published: January 2020

Experiments in vitro suggest that Neisseria gonorrhoeae surface variation plays a key role in gonococcal pathogenesis by providing the appropriate bacterial phenotypes to go through different stages of the infection. Here we report on the effects of phase and antigen variation of two major gonococcal adhesins, pili and opacity (Opa) outer membrane proteins, on the interaction of the gonococci with human monocytes. Using a set of recombinants of gonococcus strain MS11 that each express 1 of 11 genetically defined Opa proteins or a defined type of pilus, we found that both Opa proteins and pili promote bacterial phagocytosis by monocytes in the absence of serum and that this feature largely depends on the type of protein that is expressed. One of the Opa proteins (Opa[50]) strongly promoted uptake by monocytes but had little effect on the interaction with polymorphonuclear leukocytes under the conditions employed. Similarly, the phagocytosis-promoting effect of the pili was much more pronounced in monocytes than in neutrophils (4-fold versus 22-fold stimulation of uptake, respectively). Only a subpopulation of both types of phagocytes actively ingested bacteria, as has been observed during natural infections. Measurements of luminol-enhanced chemiluminescence demonstrated that phagocytosis of opaque but not piliated gonococci was accompanied by an increase in oxygen-reactive metabolites. These findings demonstrate that the monocyte response towards gonococci is highly dependent on the bacterial phenotype and differs from the neutrophil response. This diversity in bacterial behavior towards various types of human phagocytic cells underlines the biological impact of gonococcal surface variation and may explain previous contradictory results on this subject.
Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli
Authors: Czeczulin J R , Balepur S , Hicks S , Phillips A , Hall R , Kothary M H , Navarro-Garcia F , Nataro J P
Published: January 2020

Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.
Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats
Authors: Lee J C , Park J S , Shepherd S E , Carey V , Fattom A
Published: January 2020

The protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide (CP5) was examined in a modified model of catheter-induced endocarditis. Rats were catheterized by surgically passing a polyethylene catheter through the right carotid artery and aortic valve into the left ventricle. The following day, the rats were injected by the intraperitoneal (i.p.) route with immunoglobulin G (IgG) purified from nonimmunized rabbits or from rabbits immunized with a conjugate vaccine composed of CP5 and CP8 linked covalently to recombinant Pseudomonas aeruginosa exotoxoid A. One day after passive immunization, the animals were challenged i.p. with one of three serotype 5 S. aureus isolates (strain Reynolds, Lowenstein, or VP) or nontypeable strain 521. Protection was evaluated by comparing quantitative cultures of blood, endocardial vegetations, and kidneys from control and immune animals. For experiments performed with S. aureus Reynolds and Lowenstein, rats given capsular antibodies (645 microg of CP5-specific IgG) showed a significantly (P < 0.05) lower prevalence of endocarditis than rats injected with nonimmune IgG. Similarly, quantitative cultures of the blood, kidneys, and aortic valve vegetations revealed that fewer S. aureus cells were recovered from rats given capsule-specific IgG than from rats administered nonimmune IgG. Rats challenged with strain VP were protected with 1.145 mg of CP5-specific IgG. Capsular antibodies did not protect against infection elicited by a nontypeable strain. These results demonstrate that capsular antibodies elicited by immunization with a polysaccharide-protein conjugate vaccine protect experimental animals against serotype 5 S. aureus infection in a modified model of endocarditis.
Redundant contribution of myeloperoxidase-dependent systems to neutrophil-mediated killing of Escherichia coli
Authors: Rosen H , Michel B R
Published: January 2020

Neutrophil microbicidal activity is a consequence of overlapping antimicrobial systems that vary in prominence according to the conditions of the neutrophil-microbe interaction, the nature of the microbe, and its metabolic state. In this study, normal, myeloperoxidase-deficient, and respiratory burst-deficient (chronic granulomatous disease [CGD]) neutrophils killed Escherichia coli with equivalent, high efficiencies. Killing by CGD and myeloperoxidase-deficient neutrophils was not augmented by supplements, such as exogenous H2O2 and myeloperoxidase, directed at ameliorating their metabolic defects, suggesting that nonoxidative microbicidal systems were sufficient for a full microbicidal effect. Neutrophils with an intact myeloperoxidase antimicrobial system (normal or appropriately supplemented deficient cells) were capable of rapidly suppressing E. coli DNA synthesis, while unsupplemented CGD or myeloperoxidase-deficient cells were far less effective, indicating that the myeloperoxidase system was active in normal neutrophils. The degree of DNA synthesis inhibition by myeloperoxidase-sufficient neutrophils could account, in a cell-free system, for most of the observed microbicidal activity. While the myeloperoxidase system was active and probably bactericidal, it was not rate limiting for microbicidal activity and appears to have been redundant with other microbicidal systems in the cell. Rapid and extensive inhibition of bacterial DNA synthesis appears to be an indicator of myeloperoxidase activity in neutrophils.
Survival of a bacterioferritin deletion mutant of Brucella melitensis 16M in human monocyte-derived macrophages
Authors: Denoel P A , Crawford R M , Zygmunt M S , Tibor A , Weynants V E , Godfroid F , Hoover D L , Letesson J J
Published: January 2020

A bacterioferritin (BFR) deletion mutant of Brucella melitensis 16M was generated by gene replacement. The deletion was complemented with a broad-host-range vector carrying the wild-type bfr gene, pBBR-bfr. The survival and growth of the mutant, B. melitensis PAD 2-78, were similar to those of its parental strain in human monocyte-derived macrophages (MDM). These results suggest that BFR is not essential for the intracellular survival of B. melitensis in human MDM.
Attachment of piliated, Opa- and Opc- gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins
Authors: Merz A J , So M
Published: January 2020

Attachment of piliated Neisseria gonorrhoeae or Neisseria meningitidis cells to A431, Chang, HEC-1-B, or polarized T(84) cells triggers rearrangements of cortical microfilaments and the accumulation of phosphotyrosine-containing proteins at sites of bacterial contact. Actin stress fibers and the microtubule network remain unaltered in infected cells. The rearrangements reported here are triggered by piliated, Opa- and Opc- strains and also by nonpiliated gonococci (GC) that produce the invasion-associated OpaA protein. Thus, neisserial adhesion via either of at least two different adhesins can trigger cortical rearrangements. In contrast, these rearrangements are not triggered by nonadherent GC or meningococcal strains, by heat-killed or chloramphenicol-treated GC strains, or by Escherichia coli recombinants that adhere to cells via GC OpaA or Opal fusion proteins, suggesting that additional neisserial components are involved. Immunoblotting experiments did not detect consistent increases in the phosphorylation of specific proteins. Possible biological implications of these Neisseria-induced cortical rearrangements are discussed.
Identification of a 35-kilodalton serovar-cross-reactive flagellar protein, FlaB, from Leptospira interrogans by N-terminal sequencing, gene cloning, and sequence analysis
Authors: Lin M , Surujballi O , Nielsen K , Nadin-Davis S , Randall G
Published: January 2020

During the screening of antibodies to pathogenic leptospires, a murine monoclonal antibody (designated M138) was found to react with various serovars. An antigen of approximately 35 kDa from Leptospira interrogans serovar pomona, which reacted strongly with M138, was characterized by N-terminal amino acid sequencing and identified as a flagellin, a class B polypeptide subunit (FlaB) of the periplasmic flagella. The gene encoding the FlaB protein, flaB, was amplified from the genomic DNA of several pathogenic serovars by PCR with a single pair of oligonucleotide primers, suggesting that FlaB is highly conserved among these serovars. Cloning and sequence analysis of flaB from serovar pomona revealed that it contains an 849-bp open reading frame with a G + C content of 46.88% which encodes a 283-amino-acid protein with a calculated molecular mass of 31.297 kDa and a predicted pI of 9.065. A sequence comparison of flagellin proteins revealed that the amino acid sequence is most variable in the central portion of the serovar pomona FlaB, which is believed to contain specific sequence information and which may thus be useful in the design of DNA or synthetic peptide probes suitable for the detection of infection with pathogenic leptospires.
Interleukin-12 is indispensable for protective immunity against Leishmania major
Authors: Mattner F , Di Padova K , Alber G
Published: January 2020

Interleukin-12 (IL-12)-deficient mice derived from a strain genetically resistant to infection with Leishmania major were recently shown to be susceptible toward this parasite, developing a strong Th2 response after injection of a large number of parasites. We further investigated the role of IL-12 in L. major infection by studying the responses of mutant mice against smaller numbers of parasites. IL-12-deficient mice infected with only small numbers of parasites showed the progressive lesion development and high parasite burden associated with a polarized Th2 response. Our data show that IL-12 is indispensable for protective immunity against L. major. Even at low inocula, no salvage pathway appears to compensate for the lack of IL-12. However, genetically susceptible BALB/c mice infected with small numbers of parasites were able to resolve lesions and restrict the parasite burden to levels which were 10(5)-fold lower than those in IL-12-deficient mice. In contrast to mutant mice, BALB/c mice mounted a type 1 response against low inocula of L. major. IL-12-deficient BALB/c mice, however, developed a type 2 response. These data emphasize the essential role of IL-12 in resistance against L. major. In addition, this study suggests that in the absence of IL-12, susceptibility to L. major is determined by the inability to induce a Th1 response rather than the development of a Th2 response. Our results are relevant for potential vaccination strategies that use low inocula of infective microorganisms which fail to induce a protective type 1 response at higher inocula.
Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence
Authors: Kvaal C A , Srikantha T , Soll D R
Published: January 2020

Candida albicans WO-1 switches between a white- and an opaque-colony-forming phenotype. The gene WH11 is expressed differentially in the white phase. The WH11 open reading frame was inserted downstream of the promoter of the opaque-phase-specific gene OP4 in the transforming vector pCWOP16, and resulting transformants were demonstrated to misexpress WH11 in the opaque phase. Misexpression had no effect on the ability to switch from the white to the opaque or the opaque to the white phase, and it had no effect on the genesis of the unique opaque-phase cellular phenotype, even though the Wh11 protein was distributed throughout the cytoplasm in a manner similar to that observed for the endogenous gene product in the white phase. Misexpression did, however, increase the frequency of the opaque-to-white transition 330-fold and markedly increased the virulence of cells in the opaque phase in a mouse tail injection model.
Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein
Authors: Plum G , Brenden M , Clark-Curtiss J E , Pulverer G
Published: January 2020

Mycobacterium avium is an intracellular pathogen that has evolved to be a frequent cause of disseminated infection in immunocompromised patients. Although these bacilli are readily phagocytized, they are able to survive and even multiply within human macrophages. The process whereby mycobacteria circumvent the lytic functions of the macrophages is currently not well understood, but this is a key aspect in the pathogenicity of all pathogenic mycobacteria. Previously, we identified a gene in M. avium, designated mig (for macrophage-induced gene), the expression of which is induced when the bacilli grow in human macrophages (G. Plum and J. E. Clark-Curtiss, Infect. Immun. 62:476-483, 1994). In the present study we show that (i) the nucleotide sequence of the mig gene has an open reading frame of 295 amino acids with a strong bias for mycobacterial codon usage, (ii) the mig gene also codes for a putative signal peptide of 19 amino acid residues, (iii) mig is induced by acidity to be expressed as an early-secreted 30-kDa protein, and (iv) the Mig protein exhibits an AMP-binding domain signature. However, beyond this motif which is common to enzymes that activate a large variety of substrates, no homologies to known sequences are found. We also show that (v) Mycobacterium smegmatis strains expressing the Mig protein have a limited advantage for survival in macrophages. These findings may be concordant with a role of the mig gene in the virulence of M. avium.
Catalase, a novel antigen for Helicobacter pylori vaccination
Authors: Radcliff F J , Hazell S L , Kolesnikow T , Doidge C , Lee A
Published: January 2020

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 microg) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.
Gamma interferon induces Fas-dependent apoptosis of Peyer's patch T cells in mice following peroral infection with Toxoplasma gondii
Authors: Liesenfeld O , Kosek J C , Suzuki Y
Published: January 2020

Since we previously observed a remarkable decrease in the numbers of T cells in the Peyer's patches of the small intestines in C57BL/6 mice following peroral infection with Toxoplasma gondii, we performed studies to examine the mechanism(s) whereby this decrease in numbers of the T cells occurs. We found that apoptotic cell death of CD4+ and CD8+ alphabeta T cells occurred in Peyer's patches following infection. Upregulation of Fas expression was observed in these T cells. C57BL/6-background mutant mice which lack functional Fas antigen did not develop apoptosis in their Peyer's patches following infection. Treatment of infected C57BL/6 mice with anti-gamma interferon (IFN-gamma) monoclonal antibodies prevented the upregulation of Fas on their Peyer's patch T cells and inhibited the occurrence of apoptosis of these T cells. These results indicate that IFN-gamma induces Fas-dependent apoptosis in CD4+ and CD8+ alphabeta T cells in Peyer's patches in C57BL/6 mice following peroral infection with T. gondii.
Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor alpha-dependent shock
Authors: Mattner F , Ozmen L , Podlaski F J , Wilkinson V L , Presky D H , Gately M K , Alber G
Published: January 2020

The role of interleukin-12 (IL-12) was investigated in different shock models using anti-IL-12 reagents. IL-12 is composed of two disulfide-bonded subunits, p35 and p40. The IL-12 p40 homodimer (p40)2 has been shown to be a potent IL-12 antagonist in vitro. We investigated its in vivo inhibitory capacity in different shock models of mice. We could demonstrate that (p40)2 is able to protect mice from septic shock in primarily IL-12-dependent models such as the Shwartzman reaction and lipopolysaccharide (LPS)-induced shock, whereas (p40)2 has no effect in the tumor necrosis factor alpha-dependent LPS/D-GalN shock model. In IL-12-dependent shock models, (p40)2 inhibits IL-12-induced gamma interferon production and thereby interferes with the cascade of cytokine release, finally leading to death.
Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene
Authors: Schwan W R , Kopecko D J
Published: January 2020

A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive.
Profiles of healing and nonhealing Cryptosporidium parvum infection in C57BL/6 mice with functional B and T lymphocytes: the extent of gamma interferon modulation determines the outcome of infection
Authors: Theodos C M , Sullivan K L , Griffiths J K , Tzipori S
Published: January 2020

This study describes healing and nonhealing models of Cryptosporidium parvum infection with adult mice that have functional T and B lymphocytes. In our nonhealing model, mice on a C57BL/6 background which have a targeted disruption in the gamma interferon (IFN-gamma) gene (GKO mice) are utilized. C. parvum-infected GKO mice shed extremely high levels of oocysts and displayed overwhelming infection of the entire small intestine. The majority of these mice succumbed within 2 to 3 weeks due to severe acute infection and profound mucosal destruction. In our healing murine model, C57BL/6J mice treated with a single injection of the neutralizing anti-IFN-gamma monoclonal antibody XMG 1.2 prior to infection were used. These mice developed two peaks of oocyst shedding but were ultimately free of parasites on day 30 of infection. Again, the small intestine was the primary site of infection. Mesenteric lymph node (MLN) cells isolated from C. parvum-infected nonhealing GKO mice proliferated and secreted interleukin 2 (IL-2) but not IFN-gamma or IL-4 in response to ex vivo restimulation with intact C. parvum sporozoites or a C. parvum sporozoite antigen preparation. In contrast, parasite-specific MLN cells isolated from healing C57BL/6J mice secreted IL-2 and IFN-gamma but not IL-4. These results suggest that IFN-gamma, either directly or indirectly, is important for resistance to and resolution of cryptosporidiosis. Moreover, these models now allow the analysis of parasite-specific cell-mediated and humoral mucosal immune responses to determine what constitutes protective immunity to C. parvum.
Study of the role of the htrB gene in Salmonella typhimurium virulence
Authors: Jones B D , Nichols W A , Gibson B W , Sunshine M G , Apicella M A
Published: January 2020

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.