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Biomedical subjects

J Heesemann

Publications and source records attributed to J Heesemann.

At least 127 records · Page 7Linked to original sources
PubMed1995

[Infections in diabetes mellitus].

Diabetes mellitus is one of the most frequent metabolic distortions predisposing for infectious diseases. Characteristic features of patients with diabetes mellitus are dysfunctions of professional phagocytes, in particular of polymorphonuclear leucocytes. Infections of skin and soft tissue in particular of the lower extremities, rhinocerebral mucormycosis, invasive external otitis and urinary tract infections are typically associated with diabetes mellitus. Frequently, a rapidly progressive infection requires urgent surgical intervention and parenteral antimicrobial therapy. Short-interval metabolic controls, improvement of metabolic functions, extended vaccination and strict hygiene measures are supportive to prevent infections or to reduce a complicated outcome of infections.

Bacterial Infections↗
PubMed1994

Interaction of enteropathogenic Yersinia enterocolitica with complex basement membranes and the extracellular matrix proteins collagen type IV, laminin-1 and -2, and nidogen/entactin.

The plasmid-encoded virulence factor yersinia adhesin A (YadA) contributes to pathogenicity of Yersinia enterocolitica which might be related to its adhesive potential. Therefore, we have investigated the interaction of Y. enterocolitica with basement membrane (BM) and with the major BM proteins collagen type IV, laminin, and nidogen/entactin. Recombinant YadA-positive but not YadA-negative yersiniae bound specifically to lens capsule BM tissue, as well as purified collagen type IV and the laminin-1 and -2 (formally known as merosin) isoforms. Binding sites are located on the alpha 1 chain of the 58-nm amino-terminal 7sL fragment of collagen type IV and on the elastase-fragment E1 of laminin-1. YadA-mediated binding of yersiniae to collagen type IV was rapid and saturable, it was independent of divalent cations, stable over a wide pH range, and not influenced by higher salt concentrations. D-Glucose and D-galactose did not interfere with binding, indicating a protein-protein interaction. In contrast, adhesion of yersiniae to the laminin-2 isoform occurred also independent of YadA expression and no binding was observed to nidogen/entactin. The results support the hypothesis that adhesion of Y. enterocolitica could contribute to pathogenicity of enteropathogenic yersiniae. Further definition of binding sites for YadA on BM proteins might allow determination of the relevance of Yersinia-BM interactions to infection.

Adhesins, Bacterial↗
PubMed1994

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100.

Insertion sequence IS100 was localized on a 9.5-kb plasmid of Yersinia pestis and was shown to be specific for Y. pestis and serotype I strains of Y. pseudotuberculosis. The nucleotide sequence of IS100 isolated from this plasmid was determined. The element, which was flanked by 5-bp direct repeats, contained 1953 bp including imperfect inverted terminal repeats of 52 and 61 bp long (43 bp were identical). Two open reading frames encoding potential polypeptides of 340 and 252 amino acids were identified on one DNA strand. Nucleotide sequence as well as deduced polypeptide sequences of IS100 were homologous to those of IS21, IS232 and IS640.

Base Sequence↗
PubMed1994

Differentiation in virulence patterns of Escherichia coli possessing eae genes.

In this study 98 Escherichia coli strains which belonged to traditional enteropathogenic (EPEC) serotypes and 82 enterohemorrhagic E. coli (EHEC) strains were screened by polymerase chain reaction (PCR) for the presence of E. coli-attaching and -effacing (eae) genes. These strains were also hybridized with the enteropathogenic adherence factor (EAF) probe and examined in the fluorescence actin staining (FAS) test. The results obtained from the individual strains demonstrated that all 26 class I EPEC with localized adherence to HEp-2 cells carried EAF and eae genes. In contrast, of 72 EPEC strains with no or diffuse adherence only 1 strain was EAF positive and 6 strains had eae. Of 82 EHEC strains a total of 75 carried eae sequences. Of considerable interest, 15 of 21 E. coli strains that lost their slt genes during subcultivation were found to be eae positive. As controls a total of 53 enterotoxigenic and enteroinvasive E. coli, and 125 E. coli strains from the normal flora were investigated and all displayed negative results in the eae-PCR. From the 201 strains comprising classical EPEC serotypes, EHEC and E. coli with lost slt genes, a total of 126 displayed a positive FAS test and 122 reacted in the eae-PCR. Only 4 strains were FAS test positive but eae-PCR negative. Our data indicate that E. coli strains possessing the eae genes are heterogenous with respect to their virulence determinants. Loss of virulence plasmids and phage-encoded slt genes either in the host or during storage may contribute to this heterogeneity. The eae-PCR detected all class I EPEC and 91.5% of the EHEC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗
PubMed1994

Characterization of different oligomeric species of the Yersinia enterocolitica outer membrane protein YadA.

The oligomeric structure of the plasmid-encoded outer membrane protein YadA of Yersinia enterocolitica was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sucrose gradient sedimentation, respectively. The apparent molecular weight (M(r)) of the oligomeric 200-kDa YadA species detected by SDS-PAGE varied from 152,000 to 240,000 depending on the respective acrylamide concentration. The atypical electrophoretic behavior of the 200-kDa YadA species results form an exceptionally high relative free mobility as revealed by the Ferguson plot. In contrast, the apparent M(r) of 53,000 of the YadA monomer was independent of the acrylamide concentration. An additional oligomeric 116-kDa YadA species was detected by SDS-PAGE when membrane preparations of Y. enterocolitica were solubilized in SDS at 37 degrees C. The gel-purified 116-kDa YadA species was completely converted to the 200-kDa species by heating at 100 degrees C and to the monomeric form (M(r) 53,000) by heating in the presence of 10 M urea without reducing agents, respectively. This suggests that the 116-kDa YadA species represents the native oligomeric form of YadA, whereas the 200-kDa species is only generated from native YadA during denaturation in SDS. The significance of the 116-kDa YadA species is also supported by the rather slow sedimentation at about 6 S of detergent-solubilized YadA in sucrose gradients, which probably contains only two or three monomers.

Adhesins, Bacterial↗
PubMed1994

Prevalence of attaching and effacing Escherichia coli in stool samples from patients and controls.

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) have the ability to cause 'attaching and effacing' (AE) lesions; the genes necessary to cause AE in both of these pathogroups have been identified and termed eae. Using colony hybridization, we screened 237 stool samples from patients with diarrhea, and 237 stool samples from age-matched controls for the presence of E. coli carrying eae. Individual colonies harbouring eae could be recovered from 7 (2.9%) of the patient stools, as well as from 6 (2.5%) of the control stools. All these E. coli isolates were positive in the fluorescence actin staining (FAS) test. In addition, all the samples were also probed for Shiga-like toxin (slt) genes and the EPEC adherence factor (EAF) to evaluate whether testing for eae identified all EHEC and class I EPEC. Of the 7 patient samples harbouring E. coli with eae, 4 had E. coli with eae and slt genes, and 2 had E. coli with eae and EAF sequences. In 2 of the 237 patient stools, E. coli which were eae and EAF negative but slt probe positive could be recovered. These 2 E. coli strains were non-reactive in the FAS test. Of the control samples, none of the E. coli strains, including the 6 samples containing eae positive strains, possessed EAF or slt-sequences. In concrete terms, the similar eae incidence found in both E. coli isolates from patients and controls is currently of limited clinical diagnostic value and more importantly, the eae probe could not identify all slt-harbouring E. coli. On the basis of these results, the use of the eae-probe cannot be recommended in preference to the slt probes for the detection of EHEC.

Adhesins, Bacterial↗
PubMed1994

Desferrioxamine-promoted virulence of Yersinia enterocolitica in mice depends on both desferrioxamine type and mouse strain.

The effects of desferrioxamine B (DFOB) and G (DFOG) on growth promotion and virulence of Yersinia enterocolitica as well as on T cell activation and proliferation were investigated. Both desferrioxamines promoted growth of Y. enterocolitica O:8 (WA-314) and O:3 (Y-108). DFOB had a greater immunosuppressive effect on T cells than did DFOG. These results suggest a dual role of DFOB in yersiniosis: growth promotion of the pathogen and immunosuppression of the host. The LD50 of both Yersinia strains for mice was reduced by DFOB but not by DFOG. However, the LD50 of yersiniae was reduced by DFOB to a greater extent in Yersinia-resistant C57BL/6 than in Yersinia-susceptible BALB/c mice. The different impact of DFOB on the LD50 of Y. enterocolitica in C57BL/6 and BALB/c mice might be due to an immunomodulating effect of DFOB.

Animals↗
PubMed1994

Interaction of polymorphonuclear leukocytes with Yersinia enterocolitica: role of the Yersinia virulence plasmid and modulation by the iron-chelator desferrioxamine B.

The impact of desferrioxamine B (DFOB) on intracellular calcium mobilization, chemoluminescence (CL) signaling, phagocytosis, and bacterial killing of Yersinia enterocolitica by polymorphonuclear leukocytes (PMNL) was investigated. DFOB did not alter calcium signaling in PMNL but transiently increased phagocytosis. Likewise, higher counts of viable bacteria in PMNL and increased CL signals after stimulation with Y. enterocolitica were observed in the presence of DFOB. However, DFOB reduced the CL signal elicited individually by zymosan, PMA, and FMLP. Comparison of plasmid-cured with plasmid-harboring Y. enterocolitica revealed that plasmid-encoded determinants modulate phagocytosis, CL signal, and bactericidal properties of PMNL. These data demonstrate that DFOB alters microbicidal properties of PMNL and modulates their interaction with Y. enterocolitica and provide further evidence for a putative dual role of DFOB in the pathogenesis of Yersinia infection, namely growth support of the pathogen and modulation of the antimicrobial host response.

Calcium↗
PubMed1994

Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome.

The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli (O)157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs--one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.

Animals↗
PubMed1994

Characterisation of pathogenic Yersinia enterocolitica serogroups by pulsed-field gel electrophoresis of genomic NotI restriction fragments.

Enteropathogenic Yersinia enterocolitica is an important cause of human and animal disease. Phenotypic and genotypic characteristics currently used to identify Y. enterocolitica are not necessarily sufficient to differentiate pathogenic from non-pathogenic strains or to analyse the epidemiology of yersiniae at a molecular level. To improve the characterisation of Yersinia isolates, NotI restriction fragment length polymorphisms (RFLPs) of chromosomal DNA of more than 100 clinical, animal and environmental isolates were analysed in pulsed-field gel electrophoresis. Highly conserved RFLP patterns with fragments ranging from 15 to 400 kb were detected within each of 10 Y. enterocolitica serogroups tested. Determination of RFLP types makes it possible to discriminate between isolates of different Y. enterocolitica serogroups and other Yersinia spp. Moreover, NotI restriction endonuclease analysis allows even subtyping of strains belonging to a unique serogroup-biotype. Identification of NotI fragments hybridising with inv- or ail-homologous sequences was used as an additional discriminating marker. The results indicate that NotI RFLP typing can provide a powerful new tool for the differentiation of clinical Y. enterocolitica isolates.

Animals↗
PubMed1994

The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function.

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacteriocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-virulent Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-resistant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse virulence). The reisolated transconjugants which survived in mice for 3 d harboured a unique cosmid and phenotypically were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcloned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73,677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71,368 Da. The open reading frame is preceded by a sequence which shares homology with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, IutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cloned fyuA gene, yersiniabactin uptake and mouse virulence were restored. These studies demonstrate that the cloned pesticin/yersiniabactin receptor FyuA of Y. enterocolitica has the typical features of iron-regulated TonB-dependent outer membrane receptors for siderophores and bacteriocins and is required for mouse virulence.

Amino Acid Sequence↗
PubMed1994

Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.

Stage conversion between tachyzoites and bradyzoites of Toxoplasma gondii was investigated in vitro by using murine bone marrow-derived macrophages (BMMs) as host cells. Following infection of untreated BMMs with tachyzoites, spontaneous expression of bradyzoite-specific antigens (Bsa) occurred at low frequency with Toxoplasma strain-dependent ratios from 0.03 to 2%. As previously described for peritoneal macrophages, activation of tachyzoite-infected BMMs with gamma interferon (IFN-gamma) or lipopolysaccharide resulted in the induction of Bsa. When bradyzoites were used for infection, prolonged expression of Bsa could be observed in IFN-gamma-activated BMMs. The induction of Bsa expression seemed to be closely linked to parasite multiplication and increased to maximal values of 50 to 70% in intermediately activated macrophages with nitric oxide (NO) levels that allowed reduced parasite replication. Identical results in stage conversion were obtained when sodium nitroprusside was used as a source of exogenous NO, indicating that NO might be a molecular trigger of stage conversion. NO is reactive with iron-sulfur centers in proteins, thereby inhibiting proteins involved in the mitochondrial respiratory chain. Using oligomycin and antimycin A as inhibitors of mitochondrial function, growth inhibition of parasites and induction of Bsa were obtained. Since microglia are the functional correlates of macrophages in the central nervous system and inhibit T. gondii upon activation with IFN-gamma, a similar mechanism might be involved during cyst development in the brain.

Animals↗
PubMed1994

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon.

Susceptibility of mice to infection with Yersinia enterocolitica has been shown to be related to neither the Ity locus encoding for resistance to Salmonella typhimurium and other pathogens nor the H-2 locus. Recent studies in our laboratory have demonstrated that T-cell-mediated immune responses are required for overcoming primary Yersinia infection. In the present study, we investigated the course of infection with Y. enterocolitica and the resulting immune responses in Yersinia-susceptible BALB/c and Yersinia-resistant C57BL/6 mice. In the early phase of infection, the clearance of the pathogen was comparable in both strains of mice, suggesting similar mechanisms of innate resistance. Splenic T cells from Yersinia-infected C57BL/6 mice exhibited marked proliferative responses and produced gamma interferon (IFN-gamma) upon exposure to heat-killed yersiniae. By contrast, the Yersinia-specific T-cell response in BALB/c mice was weak, and IFN-gamma production could not be detected before day 21 postinfection. T cells isolated from C57BL/6 mice 7 days after infection mediated immunity to Y. enterocolitica but those from BALB/c mice did not, while at 21 days postinfection T cells from both strains mediated protection. Neutralization of IFN-gamma abrogated resistance to yersiniae in C57BL/6 mice but to a far smaller extent in BALB/c mice. Administration of recombinant IFN-gamma or anti-interleukin-4 antibodies rendered BALB/c mice resistant to yersiniae, whereas this treatment did not significantly affect the course of the infection in C57BL/6 mice. These results indicate that the cellular immune response, in particular the production of IFN-gamma by Yersinia-specific T cells, is associated with resistance of mice to Y. enterocolitica.

Animals↗
PubMed1994

Protective role for heat shock protein-reactive alpha beta T cells in murine yersiniosis.

To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype O8. A fragment of Y. enterocolitica O8 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90% homology with HSP60 of Y. enterocolitica O3 and GroEL of Escherichia coli and 59% homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65 (amino acid residues 180 to 188) was not found on Yersinia HSP60. To determine whether Yersinia HSP60 is an immunodominant antigen, the immune responses of Yersinia-infected C57BL/6 mice were analyzed. Yersinia-infected mice evolved a significant serum antibody and splenic T-cell response against Yersinia HSP60. CD4+ alpha beta T-cell clones which were generated from splenic T cells isolated from either Yersinia-infected or Yersinia HSP60-immunized mice, recognized both heat-killed Yersinia serotypes O3 and O8 as well as recombinant Yersinia HSP60 but not heat-killed Yersinia pseudotuberculosis, Salmonella typhimurium, or recombinant HSP65 of Mycobacterium bovis. The adoptive transfer of HSP60-reactive T-cell clones mediated significant protection against a lethal infection with Y. enterocolitica O8. These results indicate that HSP60 of Y. enterocolitica is an immunodominant antigen which is recognized by both antibodies and CD4+ alpha beta T cells. Moreover, this is the first report providing direct evidence that microbial HSP may elicit a protective immune response which is not associated with autoimmunity.

Amino Acid Sequence↗