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J Heesemann

Publications and source records attributed to J Heesemann.

At least 163 records · Page 9Linked to original sources
PubMed1993

Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples.

By hybridizing colonies grown from 928 individual stool samples of patients suffering from diarrhea with oligonucleotide probes 772 and 849 complementary to Shiga-like toxin I (SLT-I) and SLT-II gene sequences, respectively, we identified two strains that hybridized with probe 849, which biochemical identification revealed as Citrobacter freundii. An additional five slt-II probe-positive isolates were screened from 81 beef samples. Polymerase chain reaction analysis and restriction of amplified products provided evidence for slt-II-related genes in all seven strains. From C. freundii LM 76, the genes encoding the A and B subunits were cloned in pUC 18 vectors and sequenced. The gene encoding the A subunit differed from that of Escherichia coli slt-IIvhc in 4 bases, resulting in two amino acid residue differences. In 11, 13, and 11 nucleotides, differentiation of slt-IIA, slt-IIcA, and vtx2haA, respectively, was found. These differences affected the predicted amino acid sequence as follows: there were six amino acid differences with SLT-IIA, five with SLT-IIcA, and four with VTx2haA. The nucleotide sequence of the gene encoding the B subunit is identical to slt-IIvhcB and differed from slt-IIcB and vtx2haB by only a single nucleotide base, but this resulted in a predicted amino acid sequence identical to that reported for these toxins. We therefore termed the toxin genes C. freundii slt-IIcA and slt-IIcB. Culture filtrates inoculated with material from the colonies from primary cultures were cytotoxic to Vero cells. Neutralization assays with antisera to E. coli SLT-I, SLT-II, and SLT-IIvhc revealed that antibodies against SLT-IIvhc reduced the C. freundii cytotoxic activity specifically and to the same degree as with the E. coli SLT-IIvhc control strain. In five of the seven strains tested, subcultivation on both a liquid or solid medium resulted in loss of cytotoxic activity. With polymerase chain reaction, we demonstrated that loss of cytotoxic activity ran parallel with the loss of slt genes. These data demonstrate the intergeneric occurrence of SLT-II-related toxins, which may well be a new marker of enteropathogenicity in C. freundii. Our findings that the toxin genes belong to the slt-II family plus their evident instability in the majority of strains should help pave the way to a better understanding of their role in diarrhea or food poisoning.

Animals↗
PubMed1993

Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages.

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.

Animals↗
PubMed1993

Outer membrane protein YadA of enteropathogenic yersiniae mediates specific binding to cellular but not plasma fibronectin.

The binding of bacteria or bacterial products to host proteins of tissue extracellular matrix may be a mechanism of tissue adherence. We investigated interactions of the plasmid-encoded outer membrane protein YadA, which confers pathogenic functions on enteropathogenic yersiniae, with fibronectin. Attachment of YadA-positive and YadA-negative recombinant Yersinia enterocolitica strains to cartilage-derived human cellular fibronectin and human plasma fibronectin in the solid phase revealed that YadA mediates binding of yersiniae to cellular fibronectin in a saturable, concentration-dependent manner. The interaction could be inhibited by an anti-YadA-specific anti-serum. An anti-beta 1-integrin antibody and the synthetic peptide G-R-G-D-S-P, representing the binding site for alpha 5 beta 1-integrin on fibronectin, did not block attachment of YadA-positive yersiniae to cellular fibronectin, indicating a binding site for YadA on cellular fibronectin independent of the R-G-D-S-containing site. By contrast, YadA failed to mediate binding to plasma fibronectin immobilized on nitrocellulose or plastic surfaces. These observations provide evidence for the hypothesis that the binding region for YadA in cellular fibronectin is not present in plasma fibronectin. This study is the first report on differential binding of bacteria to splicing variants of fibronectin. Further experiments might answer the question whether binding of YadA to cellular fibronectin contributes to the pathogenesis of yersiniae, both to the initial adhesion of the bacteria to the matrices of the host and to the arthritogenic potential of enteropathogenic yersiniae.

Adhesins, Bacterial↗
PubMed1993

Experimental Yersinia enterocolitica infection in euthymic and T-cell-deficient athymic nude C57BL/6 mice: comparison of time course, histomorphology, and immune response.

To elucidate the role of T lymphocytes in primary infection with Yersinia enterocolitica, we investigated the elimination rate of this pathogen, the histomorphology of tissue lesions, and the immune responses of athymic T-cell-deficient C57BL/6 nude mice and their euthymic littermates after parenteral infection with Y. enterocolitica of serotype O:8. While a low inoculum of 3 x 10(2) Y. enterocolitica cells (about 0.01 times the median lethal dose for normal C57BL/6 mice) was cleared by normal C57BL/6 mice within 7 to 10 days, athymic nude C57BL/6 mice developed progressive infections after this inoculum, leading to death on days 20 to 25 postinfection (p.i.). While normal C57BL/6 mice experienced short-term transient infections, nude mice exhibited a biphasic, progressive infectious process. Thus, in the early phase (days 1 to 7 p.i.), a rapid influx of CD11b/18-positive cells (Mac-1 antigen) and natural killer cells was evident in the spleens and livers of the nude mice. The late phase (from day 8 p.i. onward) was characterized by a rapid progression of the infection and a further influx of CD11b/18-positive cells into the liver accompanied by an increase in bacterial counts and development of tissue lesions particularly in the liver and spleen. In normal mice, granuloma-like lesions composed of CD11b/18-, CD4-, and CD8-positive cells could be observed. However, granulomata were not found in nude mice. Yersinia-specific immunoglobulin G antibodies appeared on day 15 p.i. in the sera of normal mice, while nude mice failed to develop significant antibody titers. Adoptive transfer of Yersinia-specific T cells into athymic nude mice mediated resistance to Y. enterocolitica infection and restored both the ability of granuloma formation and the production of specific antibodies. In summary, the data presented herein strongly suggest that T lymphocytes play an essential role in the defense of C57BL/6 mice against Y. enterocolitica.

Animals↗
PubMed1993

Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-.

We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-like toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics.

Bacterial Adhesion↗
PubMed1993

Differentiation between mouse-virulent and -avirulent strains of Toxoplasma gondii by a monoclonal antibody recognizing a 27-kilodalton antigen.

Using a murine monoclonal antibody, we were able to differentiate between mouse-virulent and -avirulent strains of Toxoplasma gondii. Monoclonal antibody TB6G5 was reactive with eight clinical mouse-avirulent isolates but not with mouse-virulent laboratory strains RH and BK. The antibody-reactive antigen was identified by indirect immunofluorescence and immunoblot as a 27-kDa cytoplasmic protein expressed by tachyzoites as well as by bradyzoites.

Animals↗
PubMed1993

Phenotypical and genotypical characterization of epidemic clumping factor-negative, oxacillin-resistant Staphylococcus aureus.

A total of 50 oxacillin-resistant Staphylococcus aureus (ORSA) strains that were clumping factor negative (CFN) and protein A negative by latex agglutination were collected from patients in six different hospitals at different locations in Germany during 1991 and 1992. Antibiograms, bacteriophage typing, and plasmid analysis were performed. The antibiograms showed that, besides oxacillin, all CFN ORSA strains were resistant to gentamicin, clindamycin, erythromycin, ciprofloxacin, and fosfomycin. All these isolates were nontypeable with an international set of phages, and an additional experimental phage set indicated that the strains were phage type 16, 192. Moreover, all isolates possessed a single plasmid of 30 kb, and restriction analysis of those plasmids revealed identical patterns. For genotyping, these 50 isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR) of the coagulase and protein A genes and then by restriction enzyme digestion and analysis of restriction fragment length polymorphisms (RFLPs). With 49 strains, electrophoresis of SmaI-digested chromosomal DNA revealed identical PFGE patterns regarding the number and size of the DNA fragments, which could be differentiated from those of clumping factor-positive ORSA strains. Typing for the coagulase gene by PCR revealed PCR products of identical sizes. The AluI restriction digestion patterns of the PCR products were identical. PCR with primers derived from the region of that part of the protein A gene that encodes the immunoglobulin G-binding domains showed a PCR product that was about 170 bp smaller than that of the protein A gene from strains that were positive in the protein A latex agglutination test. Since it is precisely this size that is required in order to encode one immunoglobulin G-binding region, we assume that this is not present in the CFN ORSA strains. The phenotypical and genotypical features identify these very unusual CFN ORSA stains as being of clonal origin.

Base Sequence↗
PubMed1993

Experimental Yersinia enterocolitica infection in rodents: a model for human yersiniosis.

Yersinia enterocolitica infection in humans causes a broad spectrum of diseases ranging from acute bowel disease to extraintestinal manifestations such as reactive arthritis, erythema nodosum and uveitis. During the last decade a fascinating part of the molecular biology of the pathogenicity of human pathogenic Yersinia species has been unraveled. Pathogenicity factors such as protein tyrosine phosphatase, protein kinase, thrombin- and collagen-binding factors have been identified and characterized on the molecular level. In contrast to many animal models for human enteropathogenic microorganisms, experimental Y. enterocolitica infection in rodents resembles yersiniosis in humans and thus offers extraordinary opportunities to study the sequential steps of the infectious process. Rabbits are suitable animals in which to study Yersinia-induced enteritis (enterotoxin-mediated) and the humoral immune response after oral infection. The role of Peyer's patches (PP) in the entry of enteropathogenic Yersinia species has been elucidated in mice and rabbits. M cells are probably the primary target cells of invading Yersiniae. Surprisingly, after penetration of the mucosal epithelial cell layer Yersinia bacilli were visualized to be exclusively extracellular in PP tissue. Obviously neutrophils within PP were unable to phagocytize the invading microorganisms. Presently, it is not clear how the microorganisms disseminate from PP into lymph nodes, spleen, liver and lung of mice where they form abscesses and granuloma-like lesions. Immunohistologically the involvement of macrophages and T cells could be demonstrated in Yersinia-induced lesions of mice. Direct evidence for the role of T cells and cytokine-activated macrophages in the host defense reaction against a primary Yersinia infection in mice could be obtained from experiments including adoptive transfer of Yersinia-specific T cells and in vivo neutralization of TNF-alpha and IFN-gamma. The experimental rat model turned out to be a suitable model for studying Yersinia-induced aseptic arthritis. Lewis- and SHR rats proved to be arthritis-susceptible. Arthritogenicity of Yersinia for rats appeared to be restricted to Y. enterocolitica of serotype 08 and correlated with the virulence potential of this serotype. Surprisingly, expression of YadA, the collagen-binding factor, was not necessary for arthritis induction. A close association between both susceptibility to arthritis induction and Yersinia infection could be demonstrated in various rat strains. Depletion of alpha/beta T-cell receptor (alpha beta-TCR)-positive T cells by treatment with alpha beta-TCR-specific antibody revealed that T cells were required for clearance of the pathogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice.

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF-alpha or anti-IFN-gamma antibodies ("serotherapy") prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF-alpha treatment. The later treatment with anti-TNF-alpha or anti-IFN-gamma antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF-alpha and IFN-gamma is required for the restriction of a primary Y. enterocolitica infection in mice.

Animals↗
PubMed1992

Experimental Yersinia enterocolitica infection in rats: analysis of the immune response to plasmid-encoded antigens of arthritis-susceptible Lewis rats and arthritis-resistant Fischer rats.

Recently, experimentally Yersinia-induced arthritis in Lewis and SHR rats has been described as a potential animal model for analyzing the pathomechanism of reactive arthritis (Toivanen et al. 1986; Hill and Yu 1987). We could confirm that Lewis but not Fischer rats develop aseptic arthritis about 2 weeks after an intravenous inoculation of Yersinia enterocolitica, serotype 0:8 (Hill and Yu 1987). Moreover, we compared the antibody response to virulence-associated Yersinia antigens (Yops) of experimentally infected Lewis rats with that of Fischer rats using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The ELISA results revealed a more rapid and vigorous specific IgG, IgA and IgM response for Lewis rats than for Fischer rats in the early state of infection. As demonstrated by immunoblotting, the IgG response was directed predominantly against the plasmid-encoded YopM, YopH, YopD and the V-antigen during the acute phase followed by antibodies against YopE and YadA. Although both rat strains seroconverted against this set of antigens, IgG antibodies to YadA were more prevalent and of higher titer in arthritis-susceptible rats compared to arthritis-resistant rats.

Animals↗
PubMed1992

Improved sensitivity of the polymerase chain reaction for detection of Toxoplasma gondii in biological and human clinical specimens.

The aim of the present study was to improve the sensitivity of the polymerase chain reaction for detection of Toxoplasma gondii in biological and clinical specimens. Using a pair of primers amplifying a 634 bp fragment of the B1 gene of this parasite, it was possible to detect ten parasites in 100 microliters of sample suspensions containing a high concentration of concomitant host cells. A comparison of different DNA purification methods indicated that cell-rich clinical specimens intended for use as samples for the polymerase chain reaction should be digested with proteinase K prior to DNA amplification. By using the described sample preparation methods and the polymerase chain reaction, Toxoplasma gondii DNA was demonstrated in ten of 52 clinical specimens of patients with clinical or serological indications of toxoplasmosis.

Animals↗
PubMed1992

Genetics of Escherichia coli uropathogenicity: analysis of the O6:K15:H31 isolate 536.

E. coli strain 536 (O6:K15:H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12 linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.

Chromosome Mapping↗
PubMed1992

Cloning and sequencing of a Shiga-like toxin II-related gene from Escherichia coli O157:H7 strain 7279.

Escherichia coli O157:H7 strains are newly recognized pathogens associated with haemorrhagic colitis and haemolytic-uremic syndrome. In addition to Shiga-like toxin types I and II known to be produced by E. coli O157 isolates, we have identified in E. coli O157:H7 strain 7279 a third toxin, designated SLT-IIvhc, that is neutralized neither by anti-SLT-I nor by anti-SLT-II antibodies. The genes for this toxin were isolated by using a PCR-mediated cell-free cloning technique. DNA sequence analysis revealed a high degree of homologies to SLT-II and several SLT-II variants. The predicted amino acid sequence of the A subunit of SLT-IIvhc differed from that of the O91:H7 toxin VT2ha and SLT-IIc from E. coli O157:H- strain E2511 by 2 and 3 amino acids, respectively. The amino acid sequence of its B subunit was identical to VT2ha and SLT-IIc but different from SLT-II and SLT-IIv. Immunological differences of SLT-IIvhc and SLT-II as well as their different toxicity to HeLa cells presumably resulted from the small deviations within the primary structure of the B subunit.

Amino Acid Sequence↗
PubMed1992

Mechanism of YadA-mediated serum resistance of Yersinia enterocolitica serotype O3.

Complement activation via the alternative pathway was analyzed with isogenic strains of Yersinia enterocolitica serotype O3 differing in plasmid content (p- or p+ strains) or selective lack of YadA expression (YadA- strain). The p+ strain was serum resistant, even after antibody-enhanced complement activation. Serum sensitivity was observed with the p- and YadA- strains but was more pronounced in the p- strain. The p+ strain deposited less C5b-9(m) complexes on its surface than the p- and YadA- strains. No size difference, however, was detected with solubilized C5b-9(m) complexes obtained from resistant and sensitive strains. At the C3 level, it became evident that surface-bound C3b was degraded faster into iC3b on the p+ strain than on the p- and YadA- strains. Our results demonstrate that YadA inhibits complement activation at the C3 and C9 level. As a result, reduced amounts of C5b-9(m) are generated on the surface of YadA-bearing bacteria. In addition, YadA seems to protect against the lytic action of those C5b-9(m) complexes whose deposition could not be prevented.

Adhesins, Bacterial↗
PubMed1992

T lymphocytes mediate protection against Yersinia enterocolitica in mice: characterization of murine T-cell clones specific for Y. enterocolitica.

Yersinia enterocolitica is enteropathogenic for humans and rodents, causing intestinal and extraintestinal diseases. The cellular immune response of the infected host has not yet been analyzed in detail. Therefore, we used a parenteral mouse infection model to determine the role of T lymphocytes in immunity against Y. enterocolitica. We report the generation and characterization of Y. enterocolitica-specific T-cell clones isolated from spleens of intravenously infected C57BL/6 mice. The T-cell clones obtained showed the phenotype of helper T cells (L3T4) or cytotoxic T cells (Lyt2). All T-cell clones were positive for the interleukin-2 (IL-2) receptor (Tac antigen, p55 subunit) and were negative for the gamma delta T-cell receptor. L3T4+ clones produced small quantities of IL-2 (less than 1 U/ml) when stimulated with heat-killed Y. enterocolitica, whereas Lyt2+ clones produced no or extremely low levels of IL-2. In contrast to IL-2 production, both L3T4+ and Lyt2+ T-cell clones produced considerable quantities of gamma interferon (500 U/ml). When transferred into nonimmune mice, some of the L3T4+, as well as the Lyt2+, T-cell clones could mediate at least partial protection against a challenge of a lethal dose of Y. enterocolitica. These data demonstrate for the first time the generation and characterization of Y. enterocolitica-specific T-cell clones and provide evidence that T cells may be involved in protection against enteropathogenic Y. enterocolitica.

Animals↗
PubMed1992

Plasmid-encoded outer membrane protein YadA mediates specific binding of enteropathogenic yersiniae to various types of collagen.

The plasmid-encoded outer membrane protein YadA of enteropathogenic yersiniae is associated with pathogenicity. Recently, collagen binding of YadA-positive yersiniae was reported without detailed characterization (L. Emödy, J. Heesemann, H. Wolf-Watz, M. Skurnik, G. Kapperud, P. O'Toole, and T. Wadström, J. Bacteriol. 171:6674-6679, 1989). To elucidate the nature of collagen binding to YadA, we used a recombinant Yersinia strain expressing the cloned YadA gene. Direct binding of YadA-positive yersiniae to collagens was demonstrated in affinity blot experiments on nitrocellulose filters. A spectrum of collagen types in a wide concentration range were tested for their ability to block binding of 125I-labeled collagen type II to YadA-positive yersiniae. The results indicate a specific binding site(s) for YadA in collagen types I, II, III, IV, V, and XI. In contrast, collagen type VI did not bind to YadA. To characterize the binding site(s) more precisely, isolated collagen chains and cyanogen bromide fragments were investigated. These studies revealed that binding of YadA to collagen type I is confined to the alpha 1(I) chain, whereas the binding site within collagen type XI is localized in the alpha 3(XI) chain. alpha 2(I), alpha 1(XI), and alpha 2(XI) did not bind to YadA. Most interestingly, in the alpha 1(II) chain the specific binding site for YadA resides in the cyanogen bromide fragment CB10. The latter might indicate a binding site that does not depend on conformation. Based on these findings, further fragmentation and the synthesis of peptides may allow definition of the peptide sequence(s) relevant for YadA binding.

Bacterial Adhesion↗
PubMed1992

Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation.

Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections. This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E. coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences. When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs. The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes.

Bacterial Toxins↗