bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential induction of Th1 versus Th2 cytokines by group A streptococcal toxic shock syndrome isolates
Authors: Norrby-Teglund A , Lustig R , Kotb M
Published: January 2020

The majority of group A streptococcal (GAS) isolates from patients with streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) express numerous virulence factors, including several superantigens (SAgs). Purified SAgs are potent inducers of inflammatory (Th1) cytokines that contribute to the pathogenesis of severe infections. However, GAS-infected individuals are likely to be exposed to a mixture of GAS SAgs as well as other virulence factors produced by the bacteria, and therefore, our goal was to characterize the mitogenic and cytokine induction profiles of this mixture. All GAS isolates tested had brisk mitogenic activity and induced potent cytokine responses, with higher frequencies of Th1 than Th2 cytokine-producing cells. The mitogenic activity produced in culture supernatants of three selected clinical GAS isolates was significantly different, but no marked difference was found in their overall cytokine induction profiles. However, significant differences (P < 0.0062) were noted in the induction of Th2 cytokines between GAS supernatants and recombinant streptococcal pyrogenic exotoxin A (rSpeA), suggesting that the presence of other SAgs and/or the production of additional virulence factors may alter the overall cytokine induction profile of SAgs. A significant individual variation in the level of proliferative and cytokine responses to the same GAS culture supernatants or to rSpeA was noted. Individuals with higher frequencies of cells producing Th2 cytokines mounted lower levels of Th1 cytokine responses, and vice versa. Furthermore, quantification of the intensity and cell area of interleukin-1beta (IL-1beta)-producing cells by image analysis revealed that individuals with higher Th2 responses had significantly lower IL-1beta production (P < 0.0001) than the individual with a strong Th1 response. Differences in the ability to induce Th1 versus Th2 cytokines, as well as the individual variations in cytokine responses to streptococcal SAgs, may play a central role in determining the severity of invasive GAS infections.
Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins
Authors: Gaur N K , Klotz S A
Published: January 2020

Adherence of Candida albicans to host tissues is a necessary step for maintenance of its commensal status and is likely a necessary step in the pathogenesis of candidiasis. The extracellular matrix (ECM) proteins are some of the host tissue and plasma proteins to which C. albicans adheres through adhesins located on the fungal cell surface. To isolate genes encoding ECM adhesins, an assay was developed based on the ability of yeast cells to adhere to magnetic beads coated with the ECM protein fibronectin, type IV collagen, or laminin. A C. albicans genomic library was constructed by cloning XbaI-partially-digested and size-selected fragments into pAUR112, an Escherichia coli-yeast low-copy-number shuttle vector. The C. albicans library was transformed into Saccharomyces cerevisiae YPH 499, and clones capable of adherence were selected by using ECM protein-coated magnetic beads. A plasmid containing an approximately 8-kb insert was isolated from 29 adherent clones. These clones exhibited adherence to all ECM protein-coated magnetic beads and to human buccal epithelial cells. The ALA1 gene (for agglutinin-like adhesin) was localized by subcloning it into a 5-kb XbaI fragment which retained the adherence phenotype in both orientations. The complete DNA sequence of the 5-kb insert was determined, and an open reading frame (ORF) encoding 1,419 amino acid residues was identified. Deletions from the 5' and 3' ends extending into the DNA sequence encoding the 1,419-amino-acid ORF product inactivated the adherence phenotype, suggesting that it is the coding region of the ALA1 gene. A database search identified ALA1 to be similar to the C. albicans ALS1 (for agglutinin-like sequence 1) protein and the S. cerevisiae agglutinin protein (AG alpha1), although the homology at the primary amino acid sequence level is limited to the first half of each of these proteins. ALA1 contains a central domain of six tandem repeats of 36 amino acids. We discuss the significance of various predicted ALA1 structural motifs and their relationships to function in the adherence process.
Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization
Authors: Lecuit M , Ohayon H , Braun L , Mengaud J , Cossart P
Published: January 2020

Listeria monocytogenes can use two different surface proteins, internalin (InlA) and InlB, to invade mammalian cells. The exact role of these invasiveness factors in vivo remains to be determined. In cultured cells, InlA is necessary to promote Listeria entry into human epithelial cells, such as Caco-2 cells, whereas InlB is necessary to promote Listeria internalization in several other cell types, including hepatocytes, fibroblasts, and epithelioid cells, such as Vero, HeLa, CHO, or Hep-2 cells. We have recently reported that the InlA receptor on Caco-2 cells is the cell adhesion molecule E-cadherin and demonstrated that nonpermissive fibroblasts become permissive for internalin-mediated entry when transfected with the gene coding for LCAM, the chicken homolog of the human E-cadherin gene. In this study, we demonstrate for the first time that the internalin protein alone is sufficient to promote internalization into cells expressing its receptor. Indeed, internalin confers invasiveness to both Enterococcus faecalis and internalin-coated latex beads. As shown by transmission electron microscopy, these beads were phagocytosed via a "zipper" mechanism similar to that observed during the internalin-E-cadherin-mediated entry of Listeria. Moreover, a functional analysis of internalin demonstrates that its amino-terminal region, encompassing the leucine-rich repeat (LRR) region and the inter-repeat (IR) region, is necessary and sufficient to promote bacterial entry into cells expressing its receptor. Several lines of evidence suggest that the LRR region would interact directly with E-cadherin, whereas the IR region would be required for a proper folding of the LRR region.
Association of Leishmania heat shock protein 83 antigen and immunoglobulin G4 antibody titers in Brazilian patients with diffuse cutaneous leishmaniasis
Authors: Skeiky Y A , Benson D R , Costa J L , Badaro R , Reed S G
Published: January 2020

Diffuse cutaneous leishmaniasis (DCL) is characterized by the presence of numerous nonulcerated nodules and plaques containing large numbers of Leishmania amazonensis parasites and few lymphoid elements. The immune responses of DCL patients reflect severe antigen-specific T-cell deficiencies, while the antibody response to Leishmania antigens is often accentuated. We report herein on the Leishmania antigen-specific antibody subclass distribution in DCL patients and demonstrate that a dominant antigen contributing to the biased immunoglobulin G4 antibody subclass in sera of DCL patients is Leishmania heat shock protein 83.
Salmonella typhi stimulation of human intestinal epithelial cells induces secretion of epithelial cell-derived interleukin-6
Authors: Weinstein D L , O'Neill B L , Metcalf E S
Published: January 2020

Interleukin 6 (IL-6) is a multifunctional cytokine that has been shown to be associated with both systemic and tissue-specific responses within the host. Moreover, IL-6 is produced by both lymphoid and nonlymphoid cells and has been identified as a growth-inducing, growth-inhibiting, and differentiation-inducing factor for these cells. Recent studies of uropathogenic and upper respiratory pathogens have suggested that epithelial cell-derived IL-6 plays a role in mucosal host-parasite interactions. Since many mucosal enteric pathogens enter the host through the epithelial cells of the distal small intestine, a role for intestinal epithelial cell-derived IL-6 in the initial interaction between bacteria and host might also be predicted. However, no studies to date have determined whether the interaction of any bacteria with the epithelial cells that line the small intestine of the host can induce IL-6. To address this issue, we have established an in vitro model to evaluate the capacity of the gram-negative bacterium Salmonella typhi to induce IL-6 in the small intestine epithelial cell line Int407 and in other intestinal epithelial cell lines. The results demonstrate that both wild-type and live, attenuated S. typhi vaccine strains induce small and large intestine epithelial cells to secrete IL-6, and kinetic analysis suggests that IL-6 may be one of the earliest responses following adherence and invasion of enteric organisms. Thus, these studies suggest a physiologic role for epithelial cell-derived IL-6 in the initial interactions between host and bacterium in the small intestine.
Passive immunity to infection with Yersinia spp. mediated by anti-recombinant V antigen is dependent on polymorphism of V antigen
Authors: Roggenkamp A , Geiger A M , Leitritz L , Kessler A , Heesemann J
Published: January 2020

The V antigen is a 37-kDa secreted polypeptide encoded on the 70-kb virulence plasmid of pathogenic Yersinia spp. Besides having regulatory functions, it is known to be a virulence factor and a protective antigen. DNA sequencing of the most common serotypes of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis revealed that two evolutionary distinct types of V antigen exist in Yersinia spp. One type is represented by Y. enterocolitica serotype 08 strains WA, WA-314, and NCTC 10938 (designated LcrV-YenO8); the other type comprises Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5,27 (LcrV-Yps). A hypervariable region between amino acids 225 and 232 represents the main difference between the two types. By raising monospecific antisera against both types of V antigen (anti-rVO8 and anti-rVO3), we were able to demonstrate that, in general, passive immunization of mice against a challenge with yersiniae was possible with both anti-Y. enterocolitica V antigen sera. However, anti-V antigen serum was protective only if the immunizing V antigen was the same type as the V antigen produced by the infective strain. The failure of the American V antigen type represented by Y. enterocolitica serotype O8 to protect against Yersinia spp. carrying the other V antigen type (LcrV-Yps) could be an explanation for the presence of plague foci in American countries.
Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice
Authors: Lima E C , Garcia I , Vicentelli M H , Vassalli P , Minoprio P
Published: January 2020

A possible role for tumor necrosis factor (TNF) alpha during Trypanosoma cruzi infection was explored by using transgenic mice expressing in blood high levels of a soluble TNFR1-FcIgG3 fusion protein, which neutralizes the effects of TNF in vivo. Nontransgenic littermates were used as controls. The transgenic mice showed high susceptibility to T. cruzi infection. Inocula sublethal for control mice resulted in over 80% mortality associated with higher levels of parasites in the blood. In histological sections of the hearts of transgenic mice, large parasitic clusters without inflammatory cell infiltrates around the parasites were seen, while smaller parasitic clusters associated with leukocytes were seen in control mice. No difference in specific antibody response or lymphocyte composition of the spleen was found between transgenic and control mice, although the unresponsiveness of spleen cells to concanavalin A stimulation in vitro, typical of the acute phase of T. cruzi infection, was less pronounced in transgenic mice. Infected transgenic mice produced higher levels of gamma interferon than did control mice. These results confirm that TNF is involved in mechanisms leading to parasite clearance and protection from death in the acute phase of T. cruzi infection. More importantly, the data reveal that TNF is necessary for the establishment of effective tissue inflammation and parasite load control in acute experimental Chagas' disease myocarditis.
Localization by site-directed mutagenesis of the site in human complement factor H that binds to Streptococcus pyogenes M protein
Authors: Sharma A K , Pangburn M K
Published: January 2020

M-protein receptors located on Streptococcus pyogenes cells are known to bind human plasma protein factor H. Human factor H is composed of 20 short consensus repeat (SCR) domains containing approximately 60 amino acids each. Factor H controls the activation of the alternative pathway of complement in plasma. We have scanned the entire human factor H molecule by site-directed deletion mutagenesis, expressed the recombinant proteins in insect cells using the baculovirus system, and measured the binding of different purified mutant proteins to three strains of S. pyogenes. These studies have revealed that recombinant factor H lacking SCR domains 6 to 10 does not bind to wild-type M+ S. pyogenes JRS4. Experiments performed with S. pyogenes JRS251, in which both C-repeat domains of M protein were deleted, demonstrated that all of the factor H mutant proteins bound weakly to these cells except those lacking the SCR region from domains 6 to 10. Neither human factor H nor any of the recombinant proteins bound to the M- strain JRS145. Our results indicate that the only binding site on human factor H that interacts with streptococcus M protein is located in SCR domains 6 to 10 of factor H and that regions of M protein outside the C-repeat domains are involved in binding factor H.
Th1 and Th2 cytokines in mice with invasive aspergillosis
Authors: Cenci E , Perito S , Enssle K H , Mosci P , Latgé J P , Romani L , Bistoni F
Published: January 2020

With a murine model of invasive aspergillosis we investigated cytokine production by CD4+ T helper cells and the effects of cytokine administration or neutralization on the course and outcome of infection. Patterns of susceptibility and resistance to infection were obtained with different strains of mice injected with different inocula of Aspergillus fumigatus conidia. Mice surviving the primary infection also resisted a subsequent lethal infection that was associated with production of gamma interferon by CD4+ T splenocytes. Impaired neutrophil antifungal activity, observed in susceptible mice, was concomitant with a predominant production of interleukin-4 (IL-4) by CD4+ splenocytes. In these mice, exogenous administration of IL-12 failed to induce resistance to infection; in contrast, treatment with soluble IL-4 receptor cured more than 70% of the mice from primary infection and resulted in the onset of acquired resistance to a subsequent lethal infection. These findings indicate that in murine invasive aspergillosis, production of IL-4 by CD4+ T cells may be one major factor discriminating susceptibility and resistance to infection.
Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi
Authors: Stevens M K , Klesney-Tait J , Lumbley S , Walters K A , Joffe A M , Radolf J D , Hansen E J
Published: January 2020

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.
Induction of cytotoxic T-cell responses against culture filtrate antigens in Mycobacterium bovis bacillus Calmette-Guérin-infected mice
Authors: Denis O , Lozes E , Huygen K
Published: January 2020

CD8+ T cells are essential for protection against mycobacteria, as is clearly demonstrated by the fatal outcome of experimental infection of beta-2 microglobulin knockout mice. However, the mechanisms and antigens (Ags) leading to CD8+ T-cell activation and regulation have been poorly characterized. Here we show that, upon immunization of major histocompatibility complex (MHC)-congenic mice with Mycobacterium bovis bacillus Calmette-Guérin (BCG), a cytotoxic response against BCG culture filtrate (CF) Ags (CFAgs) is induced in H-2b and H-2bxd haplotypes but not in H-2d haplotype. This response is mediated by CD8+ T cells and absolutely requires the activation of CD4+ T cells and their secretion of interleukin 2. The lack of cytotoxic response in H-2d mice cannot be explained by impaired cytokine production or by a defect in Ag presentation by H-2d macrophages. Using the MHC class I mutant B6.C-H-2bm13 mouse strain, we demonstrate that cytotoxic T lymphocytes (CTLs) recognize CFAgs exclusively in association with D(b) molecules. These Ags are cross-reactive in mycobacteria, since BCG-induced CTLs also recognize macrophages pulsed with CF from Mycobacterium tuberculosis H37Rv and H37Ra and from two virulent strains of M. bovis. Moreover, immunization with Mycobacterium kansasii induces CTLs able to lyse macrophages pulsed with BCG CF. Finally, we have found that these Ags can be characterized as hydrophilic proteins, since they do not bind to phenyl-Sepharose CL-4B. Our results indicate that MHC-linked genes exert a profound influence on the generation of CD8+ CTLs following BCG vaccination.
IpaB, a Shigella flexneri invasin, colocalizes with interleukin-1 beta-converting enzyme in the cytoplasm of macrophages
Authors: Thirumalai K , Kim K S , Zychlinsky A
Published: January 2020

Shigellae are the most prevalent etiological agents of dysentery. A crucial step in shigella pathogenesis is the induction of macrophage apoptosis. The invasion plasmid antigen B (IpaB) is necessary and sufficient to induce macrophage programmed cell death. IpaB activates apoptosis by binding to interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) or a highly homologous protease. Here, we show that IpaB is disseminated throughout the cytoplasm of shigella-infected macrophages as detected by both immunofluorescence and immunoelectron microscopy. The cytoplasmic distribution of IpaB requires phagosome escape, and it is specific to IpaB, since lipopolysaccharide, used here as a bacterial marker, remains closely associated with the bacteria. In double-labeling experiments, we show that IpaB and ICE colocalize in the cytoplasm of the macrophage, suggesting that soon after secretion, IpaB binds to ICE to initiate apoptosis and to promote the cleavage of IL-1 beta.
Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF-alpha) gene promoter and with elevated TNF-alpha levels in tear fluid
Authors: Conway D J , Holland M J , Bailey R L , Campbell A E , Mahdi O S , Jennings R , Mbena E , Mabey D C
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) may play a central role in the disease pathogenesis which occurs as a consequence of chlamydial infection. To investigate the importance of TNF-alpha gene promoter polymorphisms and TNF-alpha levels in tear fluid in scarring trachoma, a large matched-pair case-control study was performed in The Gambia. The -308A allele was present in a higher proportion of patients (28.4%) than controls (18.4%), with an increasing association for homozygotes (chi2 for trend, P = 0.032; allele frequency, 0.163 in patients and 0.099 in controls; chi2, P = 0.025). For the -238A allele, the association was similar but not significant. The disease association was highly significant when the number of either -308A or -238A sites in an individual was considered (P = 0.003). TNF-alpha promoter alleles are tightly linked to some HLA class I and II alleles, but multivariate analysis confirmed that the disease associations were independent of HLA, although a class I allele, A*6802, is also associated with disease. TNF-alpha was more frequently detected in tear samples from patients (27.6%) than from controls (15.9%), increasingly so for higher levels of detectable TNF-alpha (P = 0.015). Among patients, detectable TNF-alpha in tears was highly associated with the presence of ocular chlamydial infection (P < 0.001). The results indicate that TNF-alpha plays a major role in the tissue damage and scarring which occurs as a consequence of Chlamydia trachomatis infection.
Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response
Authors: Johansson M , Schön K , Ward M , Lycke N
Published: January 2020

CD4+ T cells have been found to play a critical role in immune protection against Chlamydia trachomatis infection. Since both humoral and cell-mediated antichlamydial immunity have been implicated in host protection, the crucial effector functions provided by the CD4+ T cells may rely on Th1 or Th2 functions or both. In the present study, we evaluated the development of natural immunity following vaginal infection with C. trachomatis serovar D in female gamma interferon receptor-deficient (IFN-gammaR-/-) mice with a disrupted Th1 effector system. We found that in comparison with wild-type mice, the IFN-gammaR-/- mice exhibited a severe ascending primary infection of prolonged duration which stimulated almost 10-fold-stronger specific local immunoglobulin A (IgA) and IgG responses in the genital tract. Following resolution of the primary infection and despite the augmented antibody responses to chlamydiae, the IFN-gammaR-/- mice were completely unprotected against reinfection, suggesting that local antibodies play a subordinate role in host protection against chlamydial infection. Immunohistochemical analysis of frozen sections of the genital tract revealed many CD4+ T cells in the IFN-gammaR-/- mice, with a dominance of interleukin 4-containing cells in mice following resolution of the secondary infection. However, in contrast to the findings with wild-type mice, the typical clusters of CD4+ T cells were not found in the IFN-gammaR-/- mice. Few and similarly distributed CD8+ T cells were observed in IFN-gammaR-/- and wild-type mice. Whereas chlamydia-infected macrophages from wild-type mice had no inclusion bodies (IB) and produced significant amounts of nitric oxide (NO) in the presence of IFN-gamma, macrophages from IFN-gammaR-/- mice contained many IB but no NO. These results indicate that CD4+ Th1 cells and IFN-gamma, rather than local antibodies, are critical elements in host immune protection stimulated by a natural ascending C. trachomatis infection in the female genital tract.
Humoral immune responses of Solomon Islanders to the merozoite surface antigen 2 of Plasmodium falciparum show pronounced skewing towards antibodies of the immunoglobulin G3 subclass
Authors: Rzepczyk C M , Hale K , Woodroffe N , Bobogare A , Csurhes P , Ishii A , Ferrante A
Published: January 2020

The immunoglobulin G (IgG) subclass distribution of antibodies to merozoite surface antigen 2 of Plasmodium falciparum in Solomon Islanders showed marked skewing towards the IgG3 subclass. This was not observed with crude P. falciparum schizont antigen. IgG3 responses may be short-lived and require repeated restimulation for their maintenance. This may be provided by persistent infection (premunition) or new infections.
Positive regulation of Clostridium difficile toxins
Authors: Moncrief J S , Barroso L A , Wilkins T D
Published: January 2020

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.
A novel malaria protein, Pfs28, and Pfs25 are genetically linked and synergistic as falciparum malaria transmission-blocking vaccines
Authors: Duffy P E , Kaslow D C
Published: January 2020

Antibodies to Pfs28 block Plasmodium falciparum transmission and when combined with antibodies to Pfs25 provide synergy in blocking transmission. Pfs28 and Pfs25 are immunogenic, have limited antigenic diversity, and are structurally similar and genetically linked on chromosome 10. Pfs28 may prove a useful addition to Pfs25 in an effective transmission-blocking vaccine.
Interleukin-12 suppresses immunoglobulin E production but enhances immunoglobulin G4 production by human peripheral blood mononuclear cells
Authors: de Boer B A , Kruize Y C , Rotmans P J , Yazdanbakhsh M
Published: January 2020

The effect of interleukin-12 (IL-12) on human immunoglobulin G4 (IgG4) and IgE production was examined with cells derived from filarial patients and European controls. IL-12 inhibited IgE release but enhanced IgG4 production in cultures of peripheral blood mononuclear cells stimulated with anti-CD2 plus IL-2. When purified T- and B-cell cocultures were examined, IL-12 again markedly enhanced IgG4, whereas IgE production was no longer inhibited.
Quantification of the relative contribution of major histocompatibility complex (MHC) and non-MHC genes to human immune responses to foreign antigens
Authors: Jepson A , Banya W , Sisay-Joof F , Hassan-King M , Nunes C , Bennett S , Whittle H
Published: January 2020

Understanding the extent to which genetic factors influence the immune response is important in the development of subunit vaccines. Associations with HLA gene polymorphisms appear insufficient to explain the range of variation in immune responses to vaccines and to infections by major pathogens. In this study of Gambian twins we report that regulation of the immune response to a variety of antigens from Plasmodium falciparum and Mycobacterium tuberculosis is controlled by factors which are encoded by genes that lie both within and outside the major histocompatibility complex (MHC). We define the relative contribution of these genes, which varies for different antigens. The cumulative genetic contribution of non-MHC genes to the total phenotypic variance exceeds that of the MHC-encoded genes.
Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates
Authors: Ziebuhr W , Heilmann C , Götz F , Meyer P , Wilms K , Straube E , Hacker J
Published: January 2020

Staphylococcus epidermidis is a common cause of catheter-associated infections and septicemia in immunocompromised patients. To answer the question whether S. epidermidis skin isolates differ from isolates causing septicemic diseases, 51 strains obtained from blood cultures, 1 strain from shunt-associated meningitis, and 36 saprophytic isolates were characterized. The study demonstrates that most of the blood culture strains formed a multilayered biofilm on plastic material, whereas skin and mucosal isolates did not. Moreover, biofilm-producing strains were found to generate large bacterial autoaggregates in liquid culture. Autoaggregation and biofilm formation on polymer surfaces was associated with the presence of a DNA sequence encoding an intercellular adhesion gene cluster (ica) that mediates the production of a polysaccharide intercellular adhesin. The presence of the intercellular adhesion genes in blood culture isolates was also found to be correlated with the exhibition of black colonies on Congo red agar, whereas the adhesin-negative strains formed red colonies. Upon subcultivation on Congo red agar, the black colony forms of the blood culture strains exhibited red colony variants which were biofilm and autoaggregation negative and occurred at a frequency of 10(-5). The DNA analysis of these S. epidermidis variants by pulsed-field gel electrophoresis and Southern hybridization with an ica-specific gene probe revealed no detectable difference between the black and red colony types. Moreover, after repeated passage, the phenotype of the parent strain could be restored. Therefore, these colony forms were regarded as phase variants. This phenotypic change was observed exclusively in adhesin-positive clinical isolates and not in adhesin-negative saprophytic strains of S. epidermidis.
Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies
Authors: Dunn B E , Vakil N B , Schneider B G , Miller M M , Zitzer J B , Peutz T , Phadnis S H
Published: January 2020

Helicobacter pylori is a spiral, gram-negative bacterium which causes chronic gastritis and plays a critical role in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori expresses significant urease activity which is an essential virulence factor. Since a significant fraction of urease activity is located on the surface of the bacterium, the urease molecule is a logical choice as an antigen for a vaccine; currently recombinant urease apoenzyme is being tested as a vaccine in phase II clinical trials. We have recently demonstrated that urease and HspB (a homolog of the GroEL heat shock protein) become associated with the surface of H. pylori in vitro in a novel manner: these cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria, reflecting the unique characteristics of the outer membrane. To determine if similar mechanisms are operative in vivo, we determined the ultrastructural locations of urease and HspB within bacteria present in human gastric biopsies. Our results demonstrate that both urease and HspB are located within the cytoplasm of all bacteria examined in human gastric biopsies. Interestingly, a significant proportion of the bacteria examined also possessed variable amounts of surface-associated urease and HspB antigen (from 5 to 50% of the total antigenic material), indicating that in vivo, H. pylori has surface characteristics which enable it to adsorb cytoplasmic proteins. This is consistent with our altruistic autolysis model in which H. pylori uses genetically programmed bacterial autolysis to release urease and other cytoplasmic proteins which are subsequently adsorbed onto the surface of neighboring viable bacteria. These observations have important implications regarding pathogenesis and development of vaccines for H. pylori.
Characterization of immune response to Eimeria tenella antigens in a natural immunity model with hosts which differ serologically at the B locus of the major histocompatibility complex
Authors: Brake D A , Fedor C H , Werner B W , Miller T J , Taylor R L , Clare R A
Published: January 2020

A model to simulate natural immunity to Eimeria tenella was developed in three chicken lines which differ at the B locus of the major histocompatibility complex. Homozygous, 1-day-old chicks of the B19B19, B24B24, or B30B30 genotype were trickle immunized by being orally fed a small infectious dose of E. tenella oocysts for 5 consecutive days. These naturally exposed birds were then challenged at different times between 5 and 24 days after the final dose, and the level of protection was assessed 6 days after challenge, using body weight gain and intestinal lesion scores. The duration of immunity in naturally exposed birds differed among the major histocompatibility complex lines. Trickle immunization of the B19B19 haplotype afforded the longest and strongest level of protection compared to the other two haplotypes tested. In addition, in vitro splenic and peripheral blood lymphocyte proliferative responses in trickle-immunized birds were measured against sporozoite, merozoite, and tissue culture-derived E. tenella parasite antigens isolated from the recently described SB-CEV-1/F7 established cell line. The lymphocytes obtained from B19B19 birds trickle immunized responded in vitro to the E. tenella-infected SB-CEV-1/F7 tissue culture-derived parasite antigen. Furthermore, antigen-specific immune responses appeared earlier in immune, challenged B19B19 birds than in their naive, challenged counterparts. The development of a model simulating natural immunization will serve as a foundation to further characterize both humoral and cell-mediated responses to E. tenella tissue culture-derived parasite antigens and to better understand host protective immune responses to avian coccidiosis.
Hemoglobin increases mortality from bacterial endotoxin
Authors: Su D , Roth R I , Yoshida M , Levin J
Published: January 2020

Cell-free hemoglobin (Hb) is being developed as an erythrocyte substitute. We have previously demonstrated that cell-free Hb is an endotoxin-binding protein which disaggregates endotoxin and subsequently increases the biological activity of endotoxin in several in vitro assays. Because much of the morbidity and mortality associated with gram-negative bacterial infection is the result of pathophysiologic responses to bacterial lipopolysaccharide (LPS; endotoxin), we studied the effect of Hb on LPS-mediated mortality. Hb infused intravenously into mice before, coincident with, or after intraperitoneal LPS injection substantially increased LPS-related mortality from <5% to 50 to 70% 24 h after administration of LPS and from 50% to 60 to 90% at 48 h. Enhanced mortality was observed over a range of doses of injected LPS. At a given LPS dose, enhancement of mortality was shown to be dependent on the dose of Hb administered. Unmodified native human Hb, alpha-alpha-cross-linked human Hb, and beta-beta-cross-linked human or bovine Hb all were shown to enhance LPS-mediated mortality. Depressed reticuloendothelial cell function may have contributed to the enhanced mortality from LPS in the presence of Hb. Therefore, Hb-based blood substitutes, which are currently undergoing clinical trials, may intensify the potentially fatal effects of the sepsis syndrome in patients with trauma, infection, or hypotension who receive Hb for erythrocyte replacement.
Proteus mirabilis mannose-resistant, Proteus-like fimbriae: MrpG is located at the fimbrial tip and is required for fimbrial assembly
Authors: Li X , Zhao H , Geymonat L , Bahrani F , Johnson D E , Mobley H L
Published: January 2020

The mannose-resistant, Proteus-like (MR/P) fimbria, responsible for mannose-resistant hemagglutination, is a virulence factor for uropathogenic Proteus mirabilis. Based on known fimbrial gene organization, we postulated that MrpG, a putative minor subunit of the MR/P fimbria, functions as an adhesin responsible for hemagglutination, while MrpA serves as the major structural subunit for the filamentous structure. To test this hypothesis, an mrpG mutant was constructed by allelic-exchange mutagenesis and verified by PCR and Southern blotting. The mrpG mutant was found to be negative for hemagglutination, while wild-type strain H14320 and the complemented mutant were positive. Western blots with antiserum raised against an overexpressed MrpG'-His6 fusion protein showed that MrpG was present in the fimbrial preparations of both the wild-type strain and the complemented mutant but absent in that of the mrpG mutant. The mrpG mutant was significantly less virulent in a CBA mouse model of ascending urinary tract infection. Western blots with antiserum to whole MR/P fimbriae showed that MrpA protein was also missing from the fimbrial preparation of the mrpG mutant. Using immunogold electron microscopy, we found that the normal MR/P-fimbrial structure was absent in the mrpG mutant, suggesting that MrpG is essential for initiation of normal fimbrial formation. In the wild-type strain, MrpG protein was localized to the tips of the fimbriae or at the surface of the cell when antiserum raised against overexpressed MrpG was used. Given the tip localization, MrpG may be required for initiation of assembly of MR/P fimbriae but does not appear to be the fimbrial adhesin.
Expression of a long pentraxin, PTX3, by monocytes exposed to the mycobacterial cell wall component lipoarabinomannan
Authors: Vouret-Craviari V , Matteucci C , Peri G , Poli G , Introna M , Mantovani A
Published: January 2020

PTX3 is a prototypic long pentraxin composed of a C-terminal domain similar to those of classical pentraxins (e.g., C reactive protein) and an unrelated N-terminal portion. PTX3 is expressed in a variety of cell types, notably mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The present study was designed to assess whether mycobacterial components were able to induce expression and production of PTX3. Mycobacterial lipoarabinomannan (LAM) induced expression of PTX3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside. Among mononuclear cells, monocytes were responsible for LAM-induced PTX3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced PTX3 expression. Pretreatment with actinomycin D abolished LAM-induced PTX3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced PTX3 expression was associated with the production of immunoreactive PTX3. IL-10 and IL-13 did not inhibit the induction of PTX3 by LAM. Under the same conditions, these anti-inflammatory cytokines inhibited MCP-1 expression. In contrast, gamma interferon inhibited LAM-induced PTX3 expression. Thus, in addition to IL-1, TNF, and lipopolysaccharide, mycobacterial cell wall components also induce expression and production of the long pentraxin PTX3. The significance of PTX3 in the immunobiology of mycobacterial infection and its relevance in relation to clinical involvement remain to be determined.
The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry
Authors: Rathman M , Barker L P , Falkow S
Published: January 2020

Although it has been known for some time that Salmonella typhimurium is able to survive and even replicate in the normally bactericidal environment of the macrophage phagosome, the mechanisms by which this organism accomplishes this feat remain obscure. In this study, a murine macrophage cell line and confocal immunofluorescence microscopy were used to more thoroughly define the specific nature of phagosomes containing latex beads or wild-type S. typhimurium (viable or heat-killed organisms). Live S. typhimurium organisms were observed to reside in phagosomes that diverge from the degradative pathway of the macrophage. These compartments contain lysosomal glycoproteins and lysosomal acid phosphatase, endocytic markers delivered to vacuoles by mannose 6-phosphate receptor-independent mechanisms, but are devoid of the mannose 6-phosphate receptor and cathepsin L. In contrast, phagosomes containing latex beads or heat-killed organisms appeared to be processed along the degradative pathway of the host cell; these compartments colocalized not only with lysosomal glycoproteins and lysosomal acid phosphatases but also with mannose 6-phosphate receptors and cathepsin L. The uniqueness of the phagosome containing viable S. typhimurium was confirmed by the observation that these compartments, in comparison to phagosomes containing latex beads, do not readily interact with incoming endocytic traffic. Finally, we show that an isogenic, noninvasive mutant of S. typhimurium, BJ66, ends up in an intracellular compartment identical to the wild-type S. typhimurium-containing phagosome. Thus, modifications of the Salmonella-containing compartment occur independently of the mechanism of bacterial entry.
Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive
Authors: Renzoni A , Klarsfeld A , Dramsi S , Cossart P
Published: January 2020

All virulence genes of Listeria monocytogenes identified to date are positively regulated by PrfA, a transcriptional activator belonging to the Crp-Fnr family. Low temperature and cellobiose are two environmental signals known to repress expression of virulence genes in L. monocytogenes. In the present work, we analyzed the effect of temperature and cellobiose on the expression of the PrfA protein. At low temperature, PrfA was undetected, although prfA monocistronic transcripts are present. In contrast, PrfA was fully expressed in the presence of cellobiose. These results strongly suggest that virulence gene activation depends on both the presence of PrfA and additional regulatory pathways that either modify PrfA or act synergistically with PrfA.
Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice
Authors: Sinha K , Mastroeni P , Harrison J , de Hormaeche R D , Hormaeche C E
Published: January 2020

Athymic (nu/nu) BALB/c mice and their euthymic (nu/+) littermates were inoculated intravenously with live attenuated vaccine strains of Salmonella typhimurium. All strains caused progressive infections in the athymic mice but not in their euthymic littermates. Athymic mice given strain SL3261, an aroA derivative of SL1344, in doses between log 4.7 and 5.7 CFU were all severely ill and were killed by weeks 4 to 5. Athymic mice given log 4.7 CFU of a derivative of S. typhimurium C5 carrying a mutation in htrA, encoding a stress protein, were ill and were killed by week 7 in one experiment but survived to week 13 in another. Athymic mice given log 4.6 CFU of a C5 aroD htrA double mutant were ill and were killed at week 7. Athymic mice given SL3261 had high bacterial counts in the reticuloendothelial system at 4 weeks. Athymic mice given SL3261 or C5 htrA made immunoglobulin G3 (IgG3) (and to a lesser extent IgM) antibody to lipopolysaccharide (LPS), whereas euthymic mice made IgM, IgG1, IgG2a, IgG2b, and IgG3 anti-LPS antibodies. The results indicate that both aroA and htrA strains will produce slow, progressively lethal infections in athymic mice, that the htrA strain is more attenuated than the aroA strain as measured by time to death in this model, and that IgG3 anti-LPS antibody alone cannot suppress the progress of infections by very attenuated strains in athymic mice.
Specific roles of alpha-toxin and beta-toxin during Staphylococcus aureus corneal infection
Authors: O'Callaghan R J , Callegan M C , Moreau J M , Green L C , Foster T J , Hartford O M , Engel L S , Hill J M
Published: January 2020

Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha- and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha- or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr-, Hlb-, or Hla- strains. Epithelial erosions produced by parent strains were not produced by Agr- or Hla- strains. Hlb+ strains, unlike Hlb- strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla-), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb-). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.
Antibody mapping to domains of botulinum neurotoxin serotype A in the complexed and uncomplexed forms
Authors: Chen F , Kuziemko G M , Amersdorfer P , Wong C , Marks J D , Stevens R C
Published: January 2020

The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggested that the binding domain is in direct contact with the nontoxic portion in the complex. Based on the antibody mapping to the different domains of the botulinum neurotoxin holotoxin and the entire complex, a model of the botulinum neurotoxin complex is proposed.