bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the superantigenic activity of mutant and allelic forms of streptococcal pyrogenic exotoxin A
Authors: Kline J B , Collins C M
Published: January 2020

Infections with Streptococcus pyogenes (group A streptococcus) can result in the recently described streptococcal toxic shock syndrome (STSS), which is characterized by rashes, hypotension, multiorgan failure, and a high mortality rate. S. pyogenes isolates associated with STSS usually produce streptococcal pyrogenic exotoxin A (SpeA), a bacterial superantigen capable of stimulating host immune cells. Most of the symptoms of STSS are believed to result from cytokine release by the stimulated cells. To better understand the pathogenesis of STSS, we began studies on the SpeA-immune cell interaction. We generated 20 mutant forms of SpeA1 (SpeA encoded by allele 1), and the mutant toxins were analyzed for mitogenic stimulation of human peripheral blood mononuclear cells, affinity for class II major histocompatibility complex molecules (DQ), and disulfide bond formation. Residues necessary for each of these functions were identified. There are four alleles of speA, and STSS strains usually contain either allele 2 or allele 3. The product of allele 2, SpeA2, had slightly higher affinity for the class II MHC molecule compared with SpeA1 but not significantly greater mitogenic activity. SpeA3, however, was significantly increased in mitogenic activity and affinity for class II MHC compared with SpeA1. Thus, we have evidence that the toxin encoded by some of the highly virulent S. pyogenes STSS-associated isolates is a more active form of SpeA.
Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis
Authors: Phadnis S H , Parlow M H , Levy M , Ilver D , Caulkins C M , Connors J B , Dunn B E
Published: January 2020

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and is associated with peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The bacterium is characterized by potent urease activity, thought to be located on the outer membrane, which is essential for survival at low pH. The purpose of the present study was to investigate mechanisms whereby urease and HspB, a GroEL homolog, become surface associated in vitro. Urease, HspB, and catalase were located almost exclusively within the cytoplasm in fresh log-phase cultures assessed by cryo- immunoelectron microscopy. In contrast, significant amounts of surface-associated antigen were observed in older or subcultured preparations concomitantly with the appearance of significant amounts of extracellular antigen, amorphous debris, and membrane fragments. By use of a variety of biochemical methods, a significant fraction of urease and HspB was associated with the outer membrane in subcultured preparations of H. pylori. Taken together, these results strongly suggest that H. pylori cells undergo spontaneous autolysis during culture and that urease and HspB become surface associated only concomitant with bacterial autolysis. By comparing enzyme sensitivity to flurofamide (a potent, poorly diffusible urease inhibitor) in whole cells with that in deliberately lysed cells, we show that both extracellular and intracellular urease molecules are active enzymatically. Autolysis of H. pylori is an important phenomenon to recognize since it likely exerts significant effects on the behavior of H. pylori. Furthermore, the surface properties of H. pylori must be unique in promoting adsorption of cytoplasmic proteins.
Population structure of Streptococcus agalactiae reveals an association between specific evolutionary lineages and putative virulence factors but not disease
Authors: Hauge M , Jespersgaard C , Poulsen K , Kilian M
Published: January 2020

To evaluate the genetic diversity and relationships in a collection of 85 Danish strains of Streptococcus agalactiae (group B streptococcus) we have performed restriction fragment length polymorphism analysis on EcoRI- and MspI-digested whole-cell DNA using as probes rRNA, DNA fragments representing the genes encoding hyaluronidase, C5a-peptidase, alpha-antigen, and beta-antigen as well as two randomly selected genomic DNA fragments for which the coding potential is unknown. In addition, we have assayed for expression of hyaluronidase activity and beta-antigen. Combined analyses of our data and those previously obtained by multilocus enzyme electrophoresis and serotyping revealed a population separating into six major lineages that correlate with individual serotypes. The significant linkage disequilibrium of alleles indicates that the S. agalactiae population examined is predominantly clonal. Notably, strains expressing the serotype III capsule divide into two distant evolutionary lineages, of which one lacks expression of hyaluronidase activity. Six North American isolates of serotype III clustered together with multiple Danish serotype III strains, showing that the combinations of characters on which the phylogenetic tree was based are conserved worldwide. Occurrence of beta-antigen correlated with a specific version of the alpha-antigen gene and was exclusively associated with a single major phylogenetic lineage. Comparisons with the clinical history of the strains revealed no evidence of differences in pathogenic potential among the six major genetic divisions.
Transfer of a CD4+ Th1 cell line to nude mice effects clearance of Rhodococcus equi from the lung
Authors: Kanaly S T , Hines S A , Palmer G H
Published: January 2020

Rhodococcus equi, and intracellular respiratory pathogen, causes sever e granulomatous pneumonia in humans with AIDS and in young horses. Pulmonary clearance of R. equi requires functional CD4+ T cells and gamma interferon (IFN-gamma) expression from bronchial lymph node cells. The purpose of this study was to investigate whether R. equi-specific CD4+ Th1 cells could effect clearance of R. equi from the lung. Adoptive transfer of a clearance of R. equi from the lungs. In contrast, mice transfused with a R. equi-specific CD4+ Th2 cell line expressed interleukin-4 but not IFN-gamma mRNA, failed to clear pulmonary infection, and developed granulomas in the lung. Control mice, which did not receive cells, did not produce IFN-gamma or interleukin-4 and developed small pulmonary granulomas. These results clearly show that a Th1 response is sufficient to effect pulmonary clearance of R. equi.
Delineation by use of specific monoclonal antibodies of the T-cell receptor and major histocompatibility complex interaction sites on the superantigen toxic shock syndrome toxin 1
Authors: Shimonkevitz R , Boen E , Malmstrom S , Brown E , Hurley J M , Kotzin B L , Matsumura M
Published: January 2020

Murine monoclonal antibodies (MAbs) specific for toxic shock syndrome toxin 1 (TSST-1), a bacterial superantigen, showed the ability either to detect TSST-1 bound to histocompatibility locus antigen (HLA)-DR molecules or to inhibit TSST-1 binding to HLA-DR. A MAb capable of detecting DR-bound TSST-1 could also inhibit the toxin-induced activation of a T-cell receptor Vbeta15-expressing murine T-cell hybridoma. Alternatively, MAbs with specificity for the HLA-DR association site could present TSST-1 in vitro, stimulating CD4+ human T cells to proliferate. These functional activities correlated directly with with MAb specificity for HLA-DR versus T-cell receptor Vbeta interaction sites on TSST-1 as determined by reactivity with a panel of recombinant TSST-1 mutant molecules. Therefore, these MAbs discriminate the superantigen functional sites on the TSST-1 molecule and constitute reagents with the property of being potent modulators of the toxic activity of TSST-1.
Susceptibility of Neisseria gonorrhoeae to protegrins
Authors: Qu X D , Harwig S S , Oren A M , Shafer W M , Lehrer R I
Published: January 2020

We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility of six strains of Neisseria gonorrhoeae to antimicrobial peptides derived from mammalian leukocytes. The test organisms included the well-characterized serum-resistant FA19 and serum-sensitive F62 strains plus four antibiotic-resistant clinical isolates. Although each N. gonorrhoeae strain was resistant to human neutrophil defensins, all six were exquisitely sensitive to protegrins, a family of small beta-sheet antimicrobial peptides recently identified in porcine leukocytes. Protegrin-treated N. gonorrhoeae became vacuolated and had striking membrane changes when viewed by transmission and scanning electron microscopy. Because low concentrations of protegrins can also inactivate Chlamydia trachomatis and human immunodeficiency virus, they show promise for development as topical agents to avert sexually transmitted diseases.
Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the Cholera toxin B subunit
Authors: Russell M W , Moldoveanu Z , White P L , Sibert G J , Mestecky J , Michalek S M
Published: January 2020

Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma. In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens. Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system. IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization. Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period. IgA antibodies were found in nasal and vaginal washes. Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation. Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions. Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes. Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.
Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni
Authors: Dalton J P , Clough K A , Jones M K , Brindley P J
Published: January 2020

Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into the culture medium. The specific activity of cathepsin L was about double that of cathepsin B when each was assayed at its pH optimum, and moreover, the specific activities of cathepsins L and B in extracts of female schistosomes were 50 to 100% higher than in extracts of male schistosomes. Analysis of the primary structure of two cloned S. mansoni cathepsins L, here termed cathepsin L1 and cathepsin L2, revealed that they are only 44% similar and that cathepsin L2 showed more identity (52%) with human cathepsin L than with schistosome cathepsin L1. Moreover, differences in their active site, propeptide region, and potential for glycosylation suggest separate functions for schistosome cathepsin L1 and cathepsin L2.
Changes in the peripheral blood T-Cell receptor V beta repertoire in vivo and in vitro during shigellosis
Authors: Islam D , Wretlind B , Lindberg A A , Christensson B
Published: January 2020

A sequential activation of T cells in peripheral blood during shigello sis has been observer (D. Islam, P.K. Bradham, A. A. Lindberg, and B. Christensson, Infect. Immun 63:2941-2949, 1995). To further investigate the cellular response during the course of Shigella infection, changes in the T-cell receptor (TCR) repertoire in the subsets in blood in patients during shigellosis was that Shigella antigens may modulate the function of T cells carrying TCRs capable of recognizing Shigella-specific epitopes or superantigens. Such a selective preference for T cells expressing certain TCR Vbeta types could lead to the expansion or deletion of these T cells. In the present study of 27 adult male Bangladeshi patients with dysentery (14 cases caused by Shigella Dysenteriae 1 and 13 cases caused by Shigella flexneri), the changes in the TCR Vbeta repertoire of peripheral blood CD4+ and CD8+ T-cell subsets have been analyzed with a panel of nine anti-Vbeta monoclonal antibodies by flow cytometry. Twenty healthy males from Bangladesh and 20 healthy males from Sweden served as controls. Compared with the Bangladeshi controls, the patients had an increased frequency of CD4+T cells expression Vbeta2, Vbeta3, and Vbeta17, with a maximum at day 7 after the onset of disease. The frequency of CD4+T cells expressing Vbeta5.1 was increased only in patients with S. flexneri infection. Peripheral blood T cells from Shigella-infected patients also responded to in vitro stimulation in a TCR Vbeta-specific manner. Stimulation with heat-killed S. dysenteriae 1 and Shiga toxin enhanced the frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, Vbeta13.6, and Vbeta17, especially in samples obtained at day 7. The enhanced frequency of cells expressing Vbeta2, Vbeta3, Vbeta5.1, and Vbeta17 found both in in vivo and in vitro could suggest that in shigellosis antigens or superantigens are presented to the immune system and preferentially activate certain TCR Vbeta types in T-cell subsets. The kinetics of the change in the TCR Vbeta repertoire in blood during shigellosis may indicate that following local activation, the antigen activated T cells can be retrieved in the blood and restimulated in vitro. If confirmed by parallel analysis of T cells in the gut and blood by TCR sequence analysis, the possibility suggested by our findings would facilitate further analysis of the role of cell-mediated immune responses in the pathogenesis of and protection against Shigella infection.
Immunoglobulin E elevation in Plasmodium chabaudi malaria
Authors: Helmby H , Perlmann H , Troye-Blomberg M , Perlmann P
Published: January 2020

In order to investigate the mechanism of immunoglobulin E (IgE) elevation in malaria we studies mice infected with asexual blood stages of the rodent malaria parasite Plasmodium chabaudi chabaudi for total IgE and IgE antimalarial antibodies. Multiply infected mice had elevated levels of total as well as malaria-specific IgE in their sera. Sera taken from mice 3 weeks after one infection with P. chabaudi showed no IgE elevation, indicated that prolonged or repeated exposure to the parasite is necessary for the induction of an IgE response, which also is induced independently of previous or simultaneous infection with other pathogens such as helminths.
Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene sequences in enterotoxigenic E. coli strains pathogenic for humans
Authors: Yamamoto T , Echeverria P
Published: January 2020

The sequence of the enteroaggregative Escherichia coli enterotoxin 1 ( EAST1) gene was present in most (or all) strains of human-colonizing enterotoxigenic E. coli (ETEC) with colonization factor antigen II (CFA/II) or CFA/IV (CS6). The EAST1 gene was also strongly associated with PCF09+ ETEC or CFA/I+ ETEC elaborating heat-liable enterotoxin (and heat-stable enterotoxin I). In contrast, CFA/I+ ETEC elaborating heat-stable enterotoxin I, CFA/III+ ETEC, or CS17+ ETEC exhibited very weak or no associated. E. coli from healthy volunteers had no EAST1 gene sequence. A CFA/I+ ETEC strain (H10407) possessed multiple copies of the EAST1 gene on the CFA/I-encoding plasmid and chromosome. In one CFA/II+ ETEC strain, the EAST1 gene was present on the CFA/II-encoding plasmid. The EAST1 gene sequences of the CFA/I+ and CFA/II+ ETEC strains were identical to each other and 99.1% homologous to the reported gene sequence of enteroaggregative E. coli. The data indicate that the EAST1 gene is distributed among ETEC strains with a case of the presence of multiple copies in a single cell and that this distribution is associated with the adherence factor type.
Randomized, double-blind placebo-controlled trial to evaluate the safety and immunogenicity of combined Salmonella typhi Ty21a and Vibrio cholerae CVD 103-HgR live oral vaccines
Authors: Kollaritsch H , Furer E , Herzog C , Wiedermann G , Que J U , Cryz S J
Published: January 2020

Healthy adults (n=330) were randomized to receive either a bivalent vaccine composed of Vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a or a placebo. The combined vaccine was well tolerated. Approximately 80% of vaccines manifested a significant rise in anti-S. typhi immunoglobulin G or immunoglobulin A lipopolysaccharide antibody levels. Significant (fourfold or greater) rises in anti-Inaba or anti-Ogawa vibriocidal antibody titer were achieved by 94 and 80% of vaccine recipients, respectively. Elevated baseline vibriocidal antibody titers showed a modest suppressive effect on the rate of seroconversion.
Construction and preliminary characterization of three hemagglutinin mutants of Porphyromonas gingivalis
Authors: Lépine G , Ellen R P , Progulske-Fox A
Published: January 2020

Targeted insertional mutagenesis was used to construct hagA, hagB, and hagC hemagglutinin mutants of Porphyromonas gingivalis. pJRD215-derived plasmids containing tetA(Q)2 and portions of the targeted genes were conjugated into P. gingivalis. Interruption of the three loci was confirmed by Southern hybridization, sequencing, reverse transcription-PCR, and microtiter hemagglutination assays. No significant differences in hydrophobicity or coadherence to Actinomyces viscosus were detected between the mutants and the wild-type strain.
Reactivation of Chlamydia pneumoniae infection in mice by cortisone treatment
Authors: Laitinen K , Laurila A L , Leinonen M , Saikku P
Published: January 2020

Reactivation of Chlamydia pneumoniae infection was studied by inducing immunosuppression by cortisone acetate treatment given every other day for 14 days in intranasally infected NIH/s mice. The treatment started 2 or 4 weeks after primary infection, when no C. pneumoniae was detected. C. pneumoniae could be recovered from the lung cultures on days 7 and 9 in 10 and 60% of the mice, respectively, when cortisone treatment was begun 30 days after infection. These results confirm the persistent nature of C. pneumoniae infection.
An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies
Authors: Levine M , Miller F C
Published: January 2020

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.
A factor from Trypanosoma cruzi induces repetitive cytosolic free Ca2+ transients in isolated primary canine cardiac myocytes
Authors: Barr S C , Han W , Andrews N W , Lopez J W , Ball B A , Pannabecker T L , Gilmour R F
Published: January 2020

An unusual 120-kDa alkaline peptidase contained in a trypomastigote soluble fraction (TSF) of Trypanosoma cruzi is associated with the induction of repetitive Ca2+ transients and subsequent invasion by the parasite of a number of mammalian cell lines, including tissue culture L6E2 myoblasts (B. A. Burleigh and N. W. Andrews, J. Biol. Chem. 270:5172-5180, 1995; S. N. J. Moreno, J. Silva, A. E. Vercesi, and R. Docampo, J. Exp. Med. 180:1535-1540, 1994; A. Rodríguez, M. G. Rioult, A. Ora, and N. W. Andrews, J. Cell Biol. 129:1263-1273, 1995; I. Tardieux, M. H. Nathanson, and N. W. Andrews, J. Exp. Med. 179:1017-1022, 1994). Using single cell spectrofluorometry and whole-cell patch clamping, we show that TSF produces rapid repetitive cytosolic Ca2+ transients (each associated with cell contraction) in primary cardiac myocytes isolated from dogs. The response of myocytes to TSF was dose dependent in that increasing numbers of cells responded to increasing concentrations of TSF. The TSF-induced Ca2+ transients could be obliterated when TSF was heated or treated with trypsin or the protease inhibitor leupeptin. Aprotinin, pepstatin A, and E-64 did not affect TSF activity. The TSF-induced Ca2+ transients and trypomastigote cell invasion could not be inhibited by alpha (prazosin)- or beta (propanolol)-adrenergic blockers or L-type Ca2+ channel blockers (verapamil, nisoldipine, or cadmium) or by removal of extracellular Ca2+. However, inhibition of pertussis toxin-sensitive G proteins and Ca2+ release from the sarcoplasmic reticulum (with thapsigargin or ryanodine) prevented the TSF-induced Ca2+ transients and cell invasion by trypomastigotes. These data suggested that cardiac myocyte pertussis toxin-sensitive G proteins are associated with the regulation of TSF-induced Ca2+ transients and myocyte invasion by trypomastigotes but are independent of Ca2+ entry into the cytosol via L-type Ca2+ channels. The Ca2+ transients are dependent on release of Ca2+ from sarcoplasmic reticulum Ca2+ stores, but this release is not dependent on extracellular Ca2+ or on the classic model of Ca2+ -induced Ca2+ release in cardiac myocytes. Further, subthreshold depolarizations, together with cell contraction as demonstrated by whole-cell patch clamping, occurred with each Ca2+ transient. However, the depolarizations were of magnitude insufficient to generate an action potential, providing further evidence for a lack of dependence on L-type Ca2+ channels and other voltage-dependent channels (Na+ and K+ channels) in the generation of TSF-induced Ca2+ transients. Our findings suggest that primary canine cardiac myocytes respond to TSF and parasite invasion in ways similar to those of the in vitro cell lines studied to date. Since cardiac myocytes are primary targets for T. cruzi in the vertebrate host, our study indicates that TSF may play a role in the pathogenesis of Chagas' disease in humans.
Cryptosporidium parvum infection in T-cell receptor (TCR)-alpha- and TCR-delta-deficient mice
Authors: Waters W R , Harp J A
Published: January 2020

Mice deficient in either alpha beta or gamma delta T cells were more susceptible to infection with the protozoan parasite Cryptosporidium parvum than were control mice. Both neonatal and adult alpha beta-T-cell-deficient mice developed chronic infection. Gamma delta-T-cell-deficient neonatal mice were more susceptible than control mice but were able to clear the infection. Adult gamma delta-T-cell-deficient mice were not susceptible to infection. These data indicate that alpha beta T cells are important for resistance to C. parvum while gamma delta T cells have a less critical role.
Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium
Authors: Bäumler A J , Tsolis R M , Heffron F
Published: January 2020

The role of the Salmonella typhimurium fimbrial operons, lpf, fim, and pef, in adhesion to and invasion of epithelial cell lines was investigated. An S. typhimurium lpfC mutant was unable to adhere to or to invade HEp-2 cells, while an S. typhimurium fim deletion mutant did not attach to or enter HeLa cells. These results suggest that adhesion is a prerequisite for invasion and that distinct fimbrial adhesins select different target cells for invasion by S. typhimurium.
Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load
Authors: Ashman R B , Fulurija A , Papadimitriou J M
Published: January 2020

After systemic infection with the yeast Candida albicans, inbred mice show substantial differences in mortality, organ colonization, and severity of tissue damage. To examine the relationships between these variables, which are not directly correlated with each other, fungal colonization of the kidneys and brain was enumerated in six inbred strains that exhibit different patterns of tissue damage and mortality. Mice lacking the fifth component of complement (C5) are highly susceptible to lethal challenge, and A/J and DBA/2 mice, both C5 deficient, showed the highest colony counts in the kidneys after challenge with 10(5) blastoconidia. In contrast, colony counts in the brain of all six strains were equivalent at this challenge dose. A/J and DBA/2 mice died after challenge with 3 x 10(5) blastoconidia, but other strains showed an increase in kidney colonization, and strain-dependent differences in clearance from the brain became evident. The data suggest that mortality in A/J and DBA/2 mice is related to an unusual susceptibility of the kidneys to colonization by C. albicans and that there may be tissue-specific differences in host protective mechanisms.
Peptide epitopes from noncytosolic Listeria monocytogenes can be presented by major histocompatibility complex class I molecules
Authors: Zwickey H L , Potter T A
Published: January 2020

Listeria monocytogenes is an intracellular pathogen which escapes the phagosome and resides in the cytosol of the host cell. Using Listeria innocua and a mutant strain of L. monocytogenes (listeriolysin O negative), which do not enter the cytosol of the host cell, we demonstrate class I presentation of an epitope of p60, a protein secreted by L. monocytogenes, to a class I-restricted CD8+ cytotoxic T lymphocyte clone.
The second capsule gene of cryptococcus neoformans, CAP64, is essential for virulence
Authors: Chang Y C , Penoyer L A , Kwon-Chung K J
Published: January 2020

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.
Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity
Authors: Appelmelk B J , Simoons-Smit I , Negrini R , Moran A P , Aspinall G O , Forte J G , De Vries T , Quan H , Verboom T , Maaskant J J , Ghiara P , Kuipers E J , Bloemena E , Tadema T M , Townsend R R , Tyagarajan K , Crothers J M , Monteiro M A , Savio A , De Graaff J
Published: January 2020

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.
Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene
Authors: Cloeckaert A , Verger J M , Grayon M , Zygmunt M S , Grépinet O
Published: January 2020

The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.
CD4+ T-helper lymphocyte responses against Babesia bigemina rhoptry-associated protein I
Authors: Rodríguez S D , Palmer G H , McElwain T F , McGuire T C , Ruef B J , Chitko-McKown M G , Brown W C
Published: January 2020

A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.
Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine
Authors: Lebens M , Shahabi V , Bäckström M , Houze T , Lindblad N , Holmgren J
Published: January 2020

Three variants of the cholera toxin B subunit (CTB) were generated by site-specific mutagenesis in which regions of the mature protein were altered to the composition found at the corresponding positions of the closely related B subunit of the heat-labile enterotoxin of enterotoxigenic Escherichia coli (LTB). The mutant proteins were expressed in Vibrio cholerae and purified from the growth medium. In the first of the mutant proteins, the first 25 amino acids corresponded to the sequence found in LTB, and in the second, changes were made at positions 94 and 95 of the mature protein. The third mutant protein combined the changes made in the first two. Analysis of the immunological properties of these novel proteins by using monoclonal antibodies and absorbed polyclonal antiserum demonstrated that they had acquired LTB-specific epitopes. Immunizations with the mutant proteins resulted in antisera containing LTB-specific as well as CTB-specific and cross-reactive antibodies. The sera were also found to be more strongly cross-reactive in the in vitro neutralization of both cholera toxin and heat-labile enterotoxin than were antisera raised against either CTB or LTB. The results suggest that such hybrid CTB-LTB proteins may be useful in a broad-spectrum vaccine against enterotoxin-induced diarrhea.
Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin
Authors: Meng T C , Hetsko M L , Gillin F D
Published: January 2020

Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II). Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ agglutinin (WGA) inhibited excystation by > 90%. Adsorption of either ligand with PCWs eliminated inhibition, demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid, which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA- and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall glycoproteins during stage II.
Preclinical immunoprophylactic and immunotherapeutic efficacy of antisera to capsular polysaccharide-tetanus toxoid conjugate vaccines of Vibrio vulnificus
Authors: Devi S J , Hayat U , Powell J L , Morris J G
Published: January 2020

Vibrio vulnificus is an oyster-associated bacterial pathogen that causes life-threatening fulminating septicemia and necrotizing wound infections in humans. The capsular polysaccharide of V. vulnificus (VvPS) is critical for virulence. Previously we showed that active immunization of mice with a VvPS-tetanus toxoid (VvPS-TTa) conjugate vaccine conferred significantly higher protection against subsequent lethal challenge than immunization with VvPS alone. In the current study, we examined the utility of immunoprophylaxis or immunotherapy with hyperimmune antisera elicited by VvPS-TTa and VvPS-TTb conjugate vaccines prepared by different synthetic schemes. First we demonstrated that the Ribi adjuvant significantly enhanced the murine antibody response (P < or = 0.02) to both conjugates. Subsequently, high-titered polyclonal antisera were raised to VvPS-TTa and VvPS-TTb conjugate vaccines by using Ribi adjuvant or Freund's adjuvants. Antisera were observed to have protective effects when administered before and after acute lethal infection. All animals receiving prophylactic antisera intraperitoneally 24 h before lethal challenge with homologous carbotype 1 were protected, while 73 to 100% of control mice succumbed. Immunotherapy was also effective, with survival rates of 60 to 73% seen among mice when antisera were administered 2 h after bacterial challenge, at a time when symptoms of infection were already apparent. The protective effect of capsular antiserum appeared to be serotype specific. Antisera to the, carbotype 1 VvPS-TTa vaccine did not confer cross-protection against lethal challenge with carbotype 2 V. vulnificus despite partial structural similarity and a weak serological cross-reaction between the two carbotypes. Immune globulins induced by a potential multivalent VvPS conjugate vaccine composed of clinically prevalent carbotypes may have utility in the management of V. vulnificus infections and deserve further evaluation.
Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells
Authors: McKee M L , O'Brien A D
Published: January 2020

Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12.
Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to the coccoid form
Authors: Benaissa M , Babin P , Quellard N , Pezennec L , Cenatiempo Y , Fauchère J L
Published: January 2020

In vitro, Helicobacter pylori converts from a bacillary to a full coccoid form via an intermediate U-shaped form. Organisms with a full coccoid form keep a double membrane system, a polar membrane, and invagination structures. Western blots (immunoblots) of sera from colonized patients show that some high-molecular-mass antigenic fractions are expressed only in coccoids. Conversely, fractions of 30 and 94 kDa were more intensively detected in the bacillary forms. These results suggest that (i) coccoid conversion is not a degenerative transformation and (ii) antigens specific to the coccoid forms are expressed in vivo.
Influence of opsonization conditions on C3 deposition and phagocyte binding of large- and small-capsule Cryptococcus neoformans cells
Authors: Kozel T R , Tabuni A , Young B J , Levitz S M
Published: January 2020

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per microl of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.
Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes
Authors: Eaton K A , Suerbaum S , Josenhans C , Krakowka S
Published: January 2020

Helicobacterpylori possesses two flagellin molecules, MA, the major species, and FlaB, which is expressed in minor amounts. This study sought to determine if one or both flagellin species are necessary for colonization or persistence by H. pylon. Thirty-six gnotobiotic piglets from six litters were given one of four isogenic strains of H. pylon orally. The bacterial strains used were strain N6, the wild type, which produced both FlaA and FlaB and was fully motile; N6flaB::km, which produced FlaA but not FlaB and was weakly motile; N6flaA::km, which expressed FlaB but not FlaA and was nonmotile; and N6flaA::cat/flaB::km, which produced neither flagellin and was nonmotile. Strain N6 colonized all piglets and persisted for 2, 4, and 10 days after inoculation. Both N6flaA::km and N6flaB::km colonized for 2 and 4 but not 10 days, and colonization was weak. N6flaA::cat/flaB:: km colonized for 2 days but did not persist for 4 or 10 days after inoculation. These findings demonstrate that both flagellin species are necessary for full colonization by H. pylon. Colonization for up to 4 days is possible in the absence of either flagellin species but not both.
The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells
Authors: Yang Y , Merriam J J , Mueller J P , Isberg R R
Published: January 2020

Yersinia pseudotuberculosis inv mutant strains cured of the virulence plasmid exhibit thermoinducible adhesion to cultured mammalian cells. To identify the genes responsible for this phenotype, Y. pseudotuberculosis homologs of the Y. enterocolitica ail and the Y. pestis psa loci were identified. Mutations in the Y. pseudotuberculosis ail and psa loci were constructed and tested for thermoinducible binding. Results of cellular binding assays indicated that only mutations in psa, not in ail, resulted in defects for thermoinducible binding, with inv yadA psa strains showing no detectable cell adhesion. In addition, an inv psa strain was defective for hemagglutination of sheep erythrocytes, in contrast to an inv psa+ strain which was fully competent for hemagglutination. The introduction of a plasmid containing a 6.7-kb KpnI-ClaI fragment of Y. pseudotuberculosis encompassing the psa locus was sufficient to complement both the cell adhesion and hemagglutination defects of the psa mutant. Results from subcloning and transposon mutagenesis indicated that the complete 6.7-kb region was required for thermoinducible binding and hemagglutination.