bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain
Authors: Nardelli-Haefliger D , Kraehenbuhl J P , Curtiss R , Schodel F , Potts A , Kelly S , De Grandi P
Published: January 2020

An attenuated strain of Salmonella typhi delta(cya) delta(crp-cdt) delta(asd) expressing a gene encoding a hepatitis B virus core-pre-S protein was tested in female adult volunteers for its ability to elicit a systemic and a mucosal immune response. Specifically, our purpose was to evaluate the potential of such a vaccine strain to induce specific secretory immunoglobulin A (sIgA) at genital and rectal surfaces. Oral and rectal routes of immunization were compared: oral immunization induced seroconversion against the bacterial lipopolysaccharide (LPS) in six out of seven volunteers, while after rectal immunization only one out of six volunteers seroconverted against LPS. To our disappointment, the latter volunteer was also the only one who seroconverted against the carried antigen (pre-S1), demonstrating the poor ability of this live vaccine to induce an immune response against the carried antigen. Anti-LPS sIgA was found in both the vaginal and cervical secretions of a volunteer who presented a strong seroconversion after oral immunization (16-fold increase in anti-LPS IgG). Smaller amounts of anti-LPS sIgA were found in the rectal secretions of one orally and one rectally immunized volunteer and in the saliva of three orally and one rectally immunized woman. Our data show for the first time that it is possible to induce specific sIgA in the genital and rectal tracts of women by using an S. typhi vaccine strain.
In vivo alterations in cytokine production following interleukin-12 (IL-12) and anti-IL-4 antibody treatment of CB6F1 mice with chronic cutaneous leishmaniasis
Authors: Li J , Scott P , Farrell J P
Published: January 2020

CB6F1 mice exhibit intermediate resistance to infection with Leishmania major compared to their highly susceptible (BALB/c) and resistant (C57BL/6) parental strains. Unlike the C57BL/6 and BALB/c strains, which rapidly develop dominant Th1- or Th2-type responses, respectively, after infection, CB6F1 mice develop responses in which both Th1- and Th2-type cytokines are elevated through at least the first month of infection before Th1 responses become dominant as cutaneous lesions gradually heal. We have examined the effects of interleukin-12 (IL-12) and/or anti-IL-4 antibody treatment on cytokine production and the course of disease in CB6F1 mice with chronic L. major infections. When administered at 1 month of infection, IL-12 treatment led to a rapid decrease in mRNA levels for IL-4 within parasitized lesions and a moderate increase in gamma interferon (IFN-gamma) transcript levels in lymph nodes draining the site of infection. When IL-12 and anti-IL-4 antibody were administered together, they induced a marked decrease in IL-4 and transforming growth factor beta mRNA expression within lesions and a more dramatic increase in lymph node IFN-gamma transcript levels within 4 days after treatment. In comparison, similar treatment of infected BALB/c mice led to only a moderate increase in IFN-gamma transcripts but no decrease in mRNA levels for Th2-type cytokines. Treatment of CB6F1 mice with either IL-12 or anti-IL-4 antibody had no significant effect on the subsequent course of infection, whereas combined IL-12 plus anti-IL-4 treatment resulted in a decrease in lesion size and parasite numbers and a shift towards a Th1-dominant response. These results suggest that the immediate effects of cytokine or anti-cytokine therapy may be predictive of the long-term effects on the course of infection and that down-regulation of Th-2 type cytokines may be critical to the development of a Th1-dominant response.
Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of Th1 cells
Authors: Mahon B P , Ryan M S , Griffin F , Mills K H
Published: January 2020

Using a murine respiratory infection model, we have demonstrated previously that infection with Bordetella pertussis or immunization with a whole-cell pertussis vaccine induced antigen-specific Th1 cells, which conferred a high level of protection against aerosol challenge. In contrast, immunization with an acellular vaccine, consisting of the B. pertussis components detoxified pertussis toxin, filamentous hemagglutinin, and pertactin adsorbed to alum, generated Th2 cells and was associated with delayed bacterial clearance following challenge. In this study, we demonstrated that addition of interleukin-12 (IL-12) either in vitro or in vivo enhanced type 1 T-cell cytokine responses induced with an acellular vaccine. Furthermore, the rate of bacterial clearance in mice coinjected with IL-12 and the acellular vaccine was similar to that observed following immunization with a potent whole-cell vaccine. Analysis of IL-12 secretion by murine macrophages suggested that this cytokine is produced in vivo following B. pertussis infection or immunization with the whole-cell vaccine. IL-12 was detected in the supernatants of lung, splenic, and peritoneal macrophages infected with live B. pertussis or stimulated with heat-killed whole B. pertussis or B. pertussis lipopolysaccharide. In contrast, IL-12 could not be detected following stimulation of macrophages with the bacterial antigens filamentous hemagglutinin, detoxified pertussis toxin, and pertactin, the components of acellular vaccines. Our findings suggest that induction of endogenous IL-12 may contribute to the high efficacy of pertussis whole-cell vaccines and also demonstrate that it is possible to attain these high levels of protection with a less reactogenic acellular vaccine incorporating IL-12 as an adjuvant.
Phenotypic variation and persistence of Histoplasma capsulatum yeasts in host cells
Authors: Eissenberg L G , Poirier S , Goldman W E
Published: January 2020

Many Histoplasma capsulatum strains spontaneously give rise to variants during broth culture or subsequent to ingestion by epithelial cells. Unlike their parents, these variants are defective in killing macrophages and lack a major cell wall constituent, alpha-(1,3)-glucan. Inside macrophages, where the variants can persist for several weeks, they adopted an unusual morphology strikingly similar to that reported in the tissues of persistently infected humans or animals. These yeasts were often enlarged or misshapen (allomorphic), but were viable. Decreased cytotoxicity for macrophages was more strongly associated with allomorph formation than was the absence of cell wall alpha-(1,3)-glucan. Allomorphs were also formed in rat and mouse resident macrophages, but not in hamster trachea epithelial cells, indicating that host cell type influences the morphology of these yeasts. We propose that during H. capsulatum infection of mammalian hosts, spontaneous variants arise which can be recognized by their unusual morphologies. In contrast with their virulent parents, such variants "peacefully coexist" within macrophages, potentially contributing to the establishment of latency in vivo.
Nonadherent cultures of human monocytes kill Mycobacterium smegmatis, but adherent cultures do not
Authors: Barker K , Fan H , Carroll C , Kaplan G , Barker J , Hellmann W , Cohn Z A
Published: January 2020

Human peripheral blood monocytes are permissive for the growth of Mycobacterium tuberculosis, but the fate of nonpathogenic Mycobacterium smegmatis in these cells is not known. Since M. smegmatis may be used as a host with which to express and screen for M. tuberculosis genes needed for survival in monocytes, we determined whether human peripheral blood monocytes could restrict the growth of Mycobacterium smegmatis. Adherent human peripheral blood monocytes were permissive for the growth of M. smegmatis, as measured by ex vivo [3H]uracil uptake. However, human peripheral blood monocytes which were cultured nonadherently in Teflon wells were able to restrict the growth of M. smegmatis while remaining permissive for the growth of M. tuberculosis H37Ra. The loss of viability of M. smegmatis in nonadherent cells was correlated with an increase in nonspacious phagocytic vacuoles. The killing of M. smegmatis was not blocked by NG-monomethyl-L-arginine, suggesting that it was not due to the production of reactive nitrogen intermediates. Incubation of the monocytes for 1 to 7 days before infection had no effect on the fate of M. smegmatis, suggesting that adherence versus nonadherence, and not differentiation, was the key determinant for the difference in functional ability. Nonadherent human peripheral blood monocytes may be a more appropriate model than adherent cells for the study of factors employed by bacterial to survive within monocytes and for selection screening of bacterial genes needed for intracellular survival.
Elevated aspartic proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus
Authors: De Bernardis F , Chiani P , Ciccozzi M , Pellegrini G , Ceddia T , D'Offizzi G , Quinti I , Sullivan P A , Cassone A
Published: January 2020

Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomatic patients secreted, on average, up to eightfold more proteinase than the isolates from uninfected or HIV-infected but asymptomatic subjects. This differential property was stably expressed by the strains even after years of maintenance in stock cultures. Moreover, representative high-proteinase isolates were significantly more pathogenic for mice than low-proteinase isolates of C. albicans. The characters high proteinase and increased virulence were not associated with a single molecular type or category identifiable through DNA fingerprinting or pulsed-field electrophoretic karyotype, and both SAP1 and SAP2 genes were present in both categories of isolates, on the same respective chromosomes. In conclusion, our data suggest that during HIV infection more-virulent strains or biotypes of C. albicans which are identifiable by direct analysis of virulence determinants are selected. It also appears that the biotype switch to increased aspartic proteinase and virulence properties occurs before the HIV-infected subject enters the symptomatic stage and overt AIDS.
A novel 95-kilodalton antigen of Wuchereria bancrofti infective larvae identified by species-specific monoclonal antibodies
Authors: Burkot T R , Kwan-Lim G E , Maizels R M
Published: January 2020

CBA and BALB/c mice produced polyspecific and monospecific polyclonal antibody responses, respectively, following immunization with Wuchereria bancrofti stage-3 larvae. Two monoclonal antibodies (MAbs) were produced from the immunized BALB/c mouse. These MAbs (both isotype M) recognized a previously undescribed highly expressed W. bancrofti antigen present in stage-3 larvae. The epitopes bound by the MAbs appear to be species specific for W. bancrofti since the MAbs did not bind to antigens of either nine other nematode species or two vector species in Western blots (immunoblots). Phosphorylcholine epitopes, responsible for immunological cross-reactivity among nematodes, were identified only on a 200-kDa antigen and not on the 95-kDa molecule. The targets of these immunoglobulin M MAbs are not carbohydrate epitopes.
Pathogenicity of Theileria parva is influenced by the host cell type infected by the parasite
Authors: Morrison W I , MacHugh N D , Lalor P A
Published: January 2020

Theileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro. However, the majority of parasitized cells in the tissues of infected cattle are alpha/beta T cells. The aim of this study was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes, failed to resolve the issue, because of prolonged period of culture required to clone and characterize the cell lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as 3 x 10(4) peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations of CD2+, CD4+, and CD8+ T cells. By contrast, infected B cells gave rise to mild self-limiting infections even when administered at a 10-fold-higher dose. In animals that received infected CD4+ or CD8+ T cells, the parasitized cells in the lymph nodes on day 11 of infection were all within the CD4+ and CD8+ populations, respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.
Development of Shigella sonnei live oral vaccines based on defined rfbInaba deletion mutants of Vibrio cholerae expressing the Shigella serotype D O polysaccharide
Authors: Favre D , Cryz S J , Viret J F
Published: January 2020

Previous experimentation has highlighted a number of difficulties in the development of carrier-based bivalent vaccines (J.-F. Viret and D. Favre, Biologicals 22:361-372, 1994) In an attempt to obviate these carrier strains. Toward this aim, a series of defined rfbInaba deletion (delta rfbInaba) mutants of the cholera vaccine strain V. cholerae CVD103-HgR (O1 Inaba serotype) and derivative bearing the chromosomally integrated locus encoding the S. sonnei O-PS were constructed and characterized. The various mutations disrupt genes thought to be involved in either the synthesis of perosamine, the synthesis of 3-deoxy-L-glycero tetronic acid, or the O-PS transport functions together with synthesis of the perosamine synthetase. Some deletions were obtained only in strains expressing the heterologous lipopolysaccharide (LPS). Viable delta rfbInaba deletions in CVD103-HgR profoundly altered some of its phenotypic properties. The same deletions present in CVD103-HgR derivatives expressing the heterologous LPS affected their phenotypes only to a lesser extent. Only in strains in which perosamine synthesis was specifically abolished could high amounts of core-bound S. sonnei O-PS be synthesized. Two such strains (CH21, which expresses both the R1 core and the S. sonnei O-PS, and CH22, which expresses only the latter antigenic determinant) were further analyzed and were found to be indistinguishable from CVD103-HgR with regard to lack of enterotoxin activity, choleragenoid production, mercury resistance, pilin production, and, for CH22, motility. Mice immunized with CH22 produced high titers of S. sonnei O-PS-specific antibodies.
Identification of T-cell determinants in natural immune responses to the Plasmodium falciparum apical membrane antigen (AMA-1) in an adult population exposed to malaria
Authors: Lal A A , Hughes M A , Oliveira D A , Nelson C , Bloland P B , Oloo A J , Hawley W E , Hightower A W , Nahlen B L , Udhayakumar V
Published: January 2020

AMA-1 of Plasmodium falciparum is a promising candidate antigen in malaria vaccine development. In this study, we have mapped the immunodominant T-cell determinants in this antigen by using synthetic peptides. From the amphipathic scores, 17 putative T-cell determinants were identified. Nine of the 17 peptides complementary to the putative T-cell determinants induced proliferation of peripheral blood mononuclear cells (PBMC) from Kenyan residents who had lifelong exposure to malaria; none of these peptides induced proliferation of PBMC from donors who were not previously exposed to malaria. This indicates that AMA-1 peptides were stimulating T cells that were previously primed by prior exposure to P. falciparum. Many positive responders showed reactivity to more than one peptide, and some of the potent proliferative T epitopes were found to be localized in the highly conserved regions of AMA-1, suggesting that it may be possible to induce T-cell memory that can recognize different variant forms of the parasite. This information on the natural immune responses against the AMA-1 vaccine antigen in clinically immune adults will be helpful in the development of an AMA-1 antigen-based malaria vaccine and may also guide testing of AMA-1-based vaccine formulations.
Increased numbers of interleukin-12-producing cells in human tuberculosis
Authors: Munk M E , Mayer P , Anding P , Feldmann K , Kaufmann S H
Published: January 2020

Numbers of interleukin-12 (IL-12) producing cells were quantitated in the peripheral blood of healthy donors and tuberculosis patients by the ELISPOT assay. We observed that (i) stimulation with mycobacteria increases numbers of IL-12 producers from healthy donors and (ii) tuberculosis patients have larger numbers of IL-12 producers than healthy donors. Our data emphasize the importance of Il-12 in immunity to tuberculosis.
Susceptibility of Chlamydia trachomatis to protegrins and defensins
Authors: Yasin B , Harwig S S , Lehrer R I , Wagar E A
Published: January 2020

We compared the susceptibilities of Chlamydia trachomatis elementary bodies (EBs) to human defensin HNP-2 and porcine protegrin PG-1, cysteine-rich beta-sheet antimicrobial peptides produced by mammalian leukocytes. Although both peptides protected McCoy cell monolayers from infection by chlamydial EBs, protegrins were especially potent. Protegrin-mediated inactivation of chlamydiae occurred rapidly, was relatively independent of the presence of serum, and was effective against serovars L2, D, and H. Protegrin-treated EBs showed striking morphological changes, with obvious damage to their limiting membranes and loss of their cytoplasmic contents and nucleoid. Their effectiveness against chlamydial EBs and other sexually transmitted pathogens combined with their relative lack of cytotoxicity suggests that protegrins and related molecules could serve as prototypes for topical agents to prevent sexually transmitted chlamydial infection.
Induction of adherence and degranulation of polymorphonuclear leukocytes: a new expression of the invasive phenotype of Shigella flexneri
Authors: Renesto P , Mounier J , Sansonetti P J
Published: January 2020

In the present study, the ability of Shigella flexneri to activate polymorphonuclear neutrophils (PMN) was examined. The invasive serotype 5 strain M90T induced strong PMN adherence, which was dependent on both the multiplicity of infection and the duration of incubation. When tested under the same experimental conditions, the noninvasive strain BS176 (cured of the 220-kb virulence plasmid) was less efficient. Indeed, incubation of PMN for 2 h with either M90T or BS176 (multiplicity of infection, 100) induced 51.8% +/- 10.5% and 15.2% +/- 4.2% adherence, respectively (n = 3; P < 0.05). Stronger PMN activation by M90T was confirmed by evaluating PMN degranulation induced by the two strains. Whereas M90T triggered significant PMN secretion, BS176 did not. M90T strains with mutations in ipa genes were then analyzed. When PMN were incubated with these mutants, their activation was of the same intensity as that obtained with BS176. These data provide the first evidence for PMN activation induced by S. flexneri, a process which appears to be mediated by Ipa invasins.
Differential contribution of Yersinia enterocolitica virulence factors to evasion of microbicidal action of neutrophils
Authors: Ruckdeschel K , Roggenkamp A , Schubert S , Heesemann J
Published: January 2020

The differential contribution of the virulence factors invasin, protein tyrosine phosphatase (YopH), cytotoxin (YopE), and adhesin (YadA) of Yersinia enterocolitica to evasion of the antibacterial activities of polymorphonuclear leukocytes (PMNs) (oxidative burst, phagocytosis, killing) was analyzed. We constructed virulence gene knockout mutants and a novel two-plasmid system allowing production and secretion of individual virulence factors. Wild-type Y. enterocolitica WA-314 harboring the virulence plasmid pYV08 resisted phagocytosis and killing by PMNs. Moreover, strain WA-314 was able to inhibit the neutrophil oxidative burst upon stimulation with opsonized zymosan independently on preincubation with normal human serum or YadA-specific serum. These phenotypic properties of strain WA-314 were differentially affected when mutants impaired in YadA production or Yop secretion were used. A more detailed analysis revealed that YopH plays the dominant role in suppression of the antibacterial action of PMNs without damaging the cells. The YopH suppressing effect could be enhanced by coproduction of YopE and YadA. The contribution of YadA is attributed to the adhesin function promoting interaction with PMNs under both opsonizing and nonopsonizing conditions. In contrast, invasin seems to mediate only opsonin-independent interaction with PMNs. Taken together, our results demonstrate that YopH, YopE, and YadA act in concert towards neutrophil attack to enable extracellular survival of Y. enterocolitica in host tissue.
Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen
Authors: Birkelund S , Mygind P , Holm A , Larsen B , Beck F , Christiansen G
Published: January 2020

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.
Anti-Hsp65 antibodies recognize M proteins of group A streptococci
Authors: Quinn A , Shinnick T M , Cunningham M W
Published: January 2020

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.
Gamma interferon and interleukin-10 gene expression in innately susceptible and resistant mice during the early phase of Salmonella typhimurium infection
Authors: Pie S , Matsiota-Bernard P , Truffa-Bachi P , Nauciel C
Published: January 2020

Previous studies have shown that gamma interferon (IFN-gamma) plays a major role in natural resistance to Salmonella typhimurium during the early phase of infection. To assess whether the level of natural resistance in mice is related to the level of IFN-gamma gene expression, we compared IFN-gamma mRNA levels by means of reverse transcriptase-PCR in the spleens of genetically susceptible Itys (C57BL/6 and BALB/c) and resistant Ityr (CBA and DBA/2) mice during the first 5 days of infection. The mRNA expression of interleukin-10 (IL-10), a cytokine which antagonizes IFN-gamma effects, was also investigated. Mice were infected with 10(3) CFU of the virulent strain S. typhimurium C5, a dose which is lethal within a week for susceptible mice only. IFN-gamma mRNA increased to similar levels in both susceptible and resistant mice, suggesting that susceptibility to S. typhimurium infection is not related to defective IFN-gamma gene expression. In contrast, IL-10 mRNA reached much higher levels in susceptible than in resistant mice. Similar results were found in Ity congenic mice, confirming a link between the presence of the Itys allele and a high level of IL-10 gene expression during infection. High levels of IL-10 mRNA in susceptible mice correlated with high IL-10 serum levels (on day 5), whereas IL-10 was not detectable in the sera of resistant mice. However, administration of neutralizing anti-IL-10 monoclonal antibodies did not modify the course of infection. To evaluate the influence of bacterial multiplication on IL-10 mRNA expression, susceptible mice were infected with an attenuated strain of S. typhimurium. This strain induced a low level of IL-10 mRNA expression. When susceptible mice were immunized with an attenuated strain and challenged with the virulent strain, they inhibited the growth of the challenge bacteria and exhibited a low level of IL-10 mRNA. In contrast, when resistant mice were infected with a high (lethal) dose of the virulent strain, they exhibited a high level of IL-10 mRNA. Taken together, these results indicate that the level of IL-10 gene expression correlates with the level of bacterial multiplication in the organs and that the high level of IL-10 mRNA in Itys mice is a consequence rather than the cause of their susceptibility to S. typhimurium infection.
Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells
Authors: Rosenshine I , Ruschkowski S , Finlay B B
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) induces tyrosine phosphorylation of a 90-kDa protein (Hp90) in infected epithelial cells. This in turn facilitates intimate binding of EPEC via the outer membrane protein intimin, effacement of host cell microvilli, cytoskeletal rearrangement, and bacterial uptake. This phenotype has been commonly referred to as attaching/effacing (A/E). The ability of EPEC to induce A/E lesions was dependent on bacterial growth phase and temperature. Early-logarithmic-phase EPEC grown at 37 degrees C elicits strong A/E activity within minutes after infection of HeLa epithelial cells. EPEC de novo protein syntheses during the first minutes of interaction with the host cell was required to elicit A/E lesions. However, once formed, bacterial viability was not needed to maintain A/E lesions. The type of growth media and partial O2 pressure level do not seem to affect the ability of EPEC to cause A/E lesions. These results indicates that the A/E activity of EPEC is tightly regulated by environmental and host factors.
Phylogenetic distribution of streptococcal superantigen SSA allelic variants provides evidence for horizontal transfer of ssa within Streptococcus pyogenes
Authors: Reda K B , Kapur V , Goela D , Lamphear J G , Musser J M , Rich R R
Published: January 2020

Phylogenetic analyses recently found the gene encoding the streptococcal superantigen SSA of Streptococcus pyogenes to occur in several well-differentiated clones comprising 10 (12.5%) of 80 clonal lineages examined. To determine if distinct clonal lineages carried the same ssa coding sequence or harbored a group of allelic variants, ssa was sequenced from 23 S. pyogenes strains representing the 10 clones identified by multilocus enzyme electrophoresis. Three alleles of ssa were found in natural populations of S. pyogenes. ssa-1 and ssa-3 differed by a single synonymous substitution in codon 94; both encoded SSA-1. Each of these alleles was present in phylogenetically diverse clones that had not shared a recent common ancestor. ssa-2 was present in a single clonal lineage. It was identical to ssa-3 at codon 94 but had a nonsynonymous substitution at codon 28 that changed the second amino acid of the mature protein from serine to arginine. This substitution altered the predicted isoelectric point and affected the apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite sequence variation both upstream of and within the ssa locus, all ssa-positive lineages expressed either SSA-1 or SSA-2. The observed patterns of ssa allele-clone distribution provide evidence for individual incidences of horizontal transfer and recombination of ssa among distinct group A streptococcal lineages. Although the extensive homology of SSA to the staphylococcal superantigen SEB raises the possibility of intergeneric gene transfer, a search for ssa in 68 genetically diverse clones of Staphylococcus aureus did not identify the gene. Moreover, the absence of ssa among 119 representative strains of Lancefield group B, C, or G streptococci suggests that ssa is confined to S. pyogenes.
Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier
Authors: Barzu S , Fontaine A , Sansonetti P , Phalipon A
Published: January 2020

The capacity of Shigella flexneri to act as a delivery system for stimulation of local immunity was investigated by use of an S. Flexneri 2a vaccine candidate (SC602). This vaccine strain was constructed by deletion of virulence genes responsible for dissemination (icsA) and survival (iuc:iut) of bacteria within the colonic mucosa. Among the most immunogenic S. flexneri 2a proteins inducing a local immunoglobulin A antibody response, the IpaC invasion was selected as a potential protein carrier. Overexpression of IpaC from a plasmid in trans within SC602 (SC602/pIpaC) was shown to be required for the induction of optimal anti-IpaC antibody response in the murine pulmonary infection model. A weak anti-IpaC antibody response was obtained after intranasal inoculations with SC602/pIpaC live bacteria. This response was enhanced by combining systemic priming with SC602/pIpaC killed bacteria and local boosting with SC602/pIpaC live bacteria. These results suggest that naive B cells primed systemically could account for local antibody production. This immunization protocol will allow further evaluation of the immunogenicity of IpaC hybrid proteins expresses in SC602.
The structural gene encoding human enterotoxigenic Escherichia coli PCFO20 is homologous to that for porcine 987P
Authors: Viboud G I , Jonson G , Dean-Nystrom E , Svennerholm A M
Published: January 2020

Putative colonization factor PCFO20 was recently identified in an enterotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated from a child with diarrhea in Argentina. The gene encoding the structural subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in the expression phage vector lambda ZAP Express. One positive clone, pGV29, that carried a 3.3-kb fragment was identified on the basis of fimbrillin production by using a monospecific rabbit anti-PCFO20 serum. Nucleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pGV292 containing the region coding for PCFO20 fimbrillin revealed two open reading frames of which one was complete. A western blot (immunoblot) showed that the cloned protein, FotA, migrated like the PCFO20 fimbrial subunit protein did. Fimbriae were not detected on the surface of E. coli host bacteria containing pGV292 or pGV29, suggesting that the genes needed for assembly of PCFO20 fimbriae are lacking in both clones. The fotA gene encodes a 20,574-Da prefimbrillin protein which contains a 21-amino-acid signal sequence; the mature protein has a size of 18.1 kDa. The subunit protein FotA was found to be more homologous to the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC. Alignments of the amino acid sequences of the two proteins indicate that they are partly identical, with an overall similarity of 82%. FotA fimbrillin was shown to be transported and assembled by the fimbria assembly machinery in porcine ETEC strain 987. PCFO20 and 987P may have evolved from a common ancestral gene. They are immunologically related but have affinity for different host cell receptors, since PCFO20-producing bacteria do not bind to neonatal piglet enterocytes.
Immunization with high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae modifies experimental otitis media in chinchillas
Authors: Barenkamp S J
Published: January 2020

Prevention of nontypeable Haemophilus influenzae otitis media by vaccination is an important health care goal. Proteins important in bacterial adherence deserve consideration as potential vaccine candidates. Two colleagues and I previously identified a family of immunogenic high-molecular-weight proteins important in adherence of nontypeable H. influenzae to human epithelial cells (J.W. St. Geme III, S. Falkow, and S.J. Barenkamp, Proc. Natl. Acad. Sci. USA, 90:2875-2879, 1993). In the work described here, I determined whether immunization with two such adherence proteins, HMW1 and HMW2, purified from prototype nontypeable Haemophilus strain 12, would modify the course of experimental otitis media caused by the homologous strain. Chinchillas received three monthly subcutaneous injections with 40 microgram of an HMW1/HMW2 protein mixture in Freud's adjuvant. One month after the last injection, animals were challenged by intrabullar inoculation with 300 CFU of nontypeable H. influenzae 12. Infection developed in five of five control animals versus 5 of 10 immunized animals (P = 0.08, Fisher exact, one-tailed). Among infected animals, bacterial counts in middle ear fluid specimens 7 days postchallenge were significantly greater in control animals than in immunized animals (P = 0.014, Mann-Whitney U test). Serum antibody titers following immunization were comparable in uninfected and infected animals. However, infection in immunized animals was uniformly associated with the appearance of bacteria downregulated in expression of the high-molecular-weight proteins, suggesting bacterial selection in response to immunologic pressure. Although protection following immunization was incomplete, these data suggest that the high-molecular-weight adhesion proteins are potentially important protective antigens which might represent one component of a multicomponent nontypeable Haemophilus vaccine.
Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells
Authors: Vanrompay D , Charlier G , Ducatelle R , Haesebrouck F
Published: January 2020

In order to find an explanation for the observed differences in levels of pathogenicity in turkeys of Chlamydia psittaci 84/55 (avian serovar A), 89/1326 (avian serovar B), 92/1293 (avian serovar D), and the Texas Turkey strain (avian serovar D) (P.B. Wyrick, J. Choong, S.T. Knight, D. Goyeau, E.S. Stuart, and A.B. MacDonald, Immunol. Infect. Dis. 4:131-141, 1994), the reproductive cycles of organisms of the four strains were studied in Buffalo Green Monkey cells by transmission electron microscopy, immunoelectron microscopy, and flow cytometry. Organisms of strains most pathogenic in turkeys, namely, the serovar A strain and the 92/1293 serovar D strain, (i) replicated faster, since at 50 h postinoculation significantly larger inclusions with more numerous infectious organisms were observed than with the less pathogenic strains; (ii) were often found devoid of inclusion membranes scattered throughout the cytoplasms; and (iii) induced severe degenerative changes in Buffalo Green Monkey cells. By immunoelectron microscopy and flow cytometry, chlamydial antigens could not be detected in the plasma membranes of infected host cells. However, the presence of chlamydial antigens in inclusion membranes was demonstrated by immunoelectron microscopy.
Interleukin-12 and tumor necrosis factor alpha mediate innate production of gamma interferon by group B Streptococcus-treated splenocytes of severe combined immunodeficiency mice
Authors: Derrico C A , Goodrum K J
Published: January 2020

The existence of interleukin-12-mediated innate immune responses to group B streptococci (GBS) was tested by examining T-lymphocyte-independent gamma interferon (IFN) production in cultured splenocytes from severe combined immunodeficiency mice. Splenocytes were cultured with killed or living GBS for 48 h, and then IFN was measured by enzyme-linked immunosorbent assay. Type III GBS as well as other extracellular bacterial agents of neonatal sepsis (staphylococci and enterococci) induced IFN production, which was enhanced by interleukin-2 and was inhibited by neutralizing antibodies to tumor necrosis factor alpha and to mouse interleukin-12. Interleukin-12 bioactivity was present in conditioned medium from GBS-treated adherent macrophages. Adherent peritoneal macrophages and bone marrow-derived natural killer cells from severe combined immunodeficiency mice cultured separately with GBS did not produce IFN, whereas cocultures did produce IFN. Functional macrophage activation was evident by nitric oxide production in GBS-treated splenocyte cultures. The results show that extracellular pathogens such as GBS, similarly to intracellular microbes, induce macrophage interleukin-12 and tumor necrosis factor alpha, which promote natural killer cell secretion of IFN, which then enhances innate phagocyte resistance mechanisms.
Human pro- and anti-inflammatory cytokine patterns induced by Streptococcus pyogenes erythrogenic (pyrogenic) exotoxin A and C superantigens
Authors: Müller-Alouf H , Alouf J E , Gerlach D , Ozegowski J H , Fitting C , Cavaillon J M
Published: January 2020

The superantigenic streptococcal erythrogenic toxins A and C (ETA/SPEA and ETC/SPEC) elicit the production by human peripheral blood mononuclear cells of substantial amounts of Th1-derived cytokines (interleukin-2 [IL-2] and gamma interferon) as well as anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist). In contrast, very low levels of IL-4 and no alpha interferon were induced. The production of these cytokines after stimulation with Streptococcus pyogenes heat-killed bacteria and lipopolysaccharide from gram negative bacteria differed qualitatively and quantitatively from that elicited by the superantigens.
The Legionella pneumophila hel locus encodes intracellularly induced homologs of heavy-metal ion transporters of Alcaligenes spp
Authors: McClain M S , Hurley M C , Brieland J K , Engleberg N C
Published: January 2020

We continued characterization of the Legionella pneumophila hel locus. Mutagenesis and DNA sequencing identified three genes similar to the czc and cnr loci of Alcaligenes eutrophus and the ncc locus of Alcaligenes xylosoxidans. On the basis of their similarity to these loci, we designated the L. pneumophila genes helC, helB, and helA. Mutations in the hel genes led to reduced cytopathicity towards U937 cells, although the mutant strains did not appear defective in other assays of virulence. Transcription of the hel locus was induced by the intracellular environment but was not induced by any of a variety of in vitro stress conditions. The function of the hel gene products remains to be determined.
Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells
Authors: de Bentzmann S , Roger P , Dupuit F , Bajolet-Laudinat O , Fuchey C , Plotkowski M C , Puchelle E
Published: January 2020

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.
De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells
Authors: Susa M , Hacker J , Marre R
Published: January 2020

Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila.
Relative importance of three iron-regulated outer membrane proteins for in vivo growth of Vibrio cholerae
Authors: Tashima K T , Carroll P A , Rogers M B , Calderwood S B
Published: January 2020

Iron is an essential nutrient to support the growth of most bacterial species. However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme. In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced outer membrane proteins. Previous data on the role of iron acquisition systems for the intraintestinal growth of mucosal pathogens such as V. cholerae are conflicting. In this report, we isolated mutants of V. cholerae with TnphoA fusions in each of viuA, hutA, and irgA, as well as strains mutant in each pair of these genes and all three simultaneously, to analyze the role of these iron-induced outer membrane protein receptors for in vivo growth of V. cholerae. The fusion between hutA and TnphoA in a single copy on the chromosome allowed the study of in vitro regulation of hutA in response to iron, fur, and irgB; transcription of hutA was tightly iron regulated (70-fold) and dependent on a functional Fur but did not require IrgB. To investigate the effects of mutations in these iron-induced outer membrane proteins on in vivo growth, we inoculated ileal loops in a rabbit model of infection. This avoids exposure of organisms to the potential killing effects of gastric acid, allows several logarithmic increases in growth in the in vivo environment, and facilitates direct comparison of multiple strains in the same animal to avoid any differences between animals. We grew each mutant to be tested in competition with the wild-type strain in the same loop, to provide an internal control. We confirmed that the inocula for these experiments were grown under conditions of iron stress prior to in vivo inoculation, by measuring the alkaline phosphatase activity of the iron-regulated fusion in each strain. The results confirmed that mutation of irgA produced a much more substantial in vivo growth defect than mutation of either hutA or viuA alone. Double mutants of irgA with either viuA or hutA, or the strain mutant in all three genes, showed an in vivo growth defect comparable to the strain mutant in irgA only, suggesting that mutation of irgA was the most relevant for in vivo growth. The strain mutant in both hutA and viuA was also markedly impaired for in vivo growth, suggesting that mutation of both of these iron uptake systems simultaneously can also produce a substantial in vivo growth defect.
Spectrum of Legionella species whose intracellular multiplication in murine macrophages is genetically controlled by Lgn1
Authors: Miyamoto H , Maruta K , Ogawa M , Beckers M C , Gros P , Yoshida S
Published: January 2020

We examined the intracellular growth of 20 strains within six species of Legionella in thioglycolate-elicited peritoneal macrophages from A/J and C57BL/6 mice and a congenic strain derived from them (A.B Lgn1). With the exception of Legionella pneumophila Togus-1 and Bloomington-2, the intracellular growth of the 15 L. pneumophila strains tested was controlled by Lgn1. However, the intracellular growth of five Legionella species other than L. pneumophila was not under Lgn1's control.