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J Heesemann

Publications and source records attributed to J Heesemann.

At least 73 records · Page 4Linked to original sources
PubMed1999

Common and specific characteristics of the high-pathogenicity island of Yersinia enterocolitica.

Yersinia pestis, Y. pseudotuberculosis O:1, and Y. enterocolitica biogroup 1B strains carry a high-pathogenicity island (HPI), which mediates biosynthesis and uptake of the siderophore yersiniabactin and a mouse-lethal phenotype. The HPI of Y. pestis and Y. pseudotuberculosis (Yps HPI) are highly conserved in sequence and organization, while the HPI of Y. enterocolitica (Yen HPI) differs significantly. The 43,393-bp Yen HPI sequence of Y. enterocolitica WA-C, serotype O:8, was completed and compared to that of the Yps HPI of Y. pseudotuberculosis PB1, serotype O:1A. A common GC-rich region (G+C content, 57.5 mol%) of 30.5 kb is conserved between yersinia strains. This region carries genes for yersiniabactin biosynthesis, regulation, and uptake and thus can be considered the functional "core" of the HPI. In contrast, the second part of the HPI is AT rich and completely different in two evolutionary lineages of the HPI, being 12.8 kb in the Yen HPI and 5.6 kb in the Yps HPI. The variable part acquired one IS100 element in the Yps HPI and accumulated four insertion elements, IS1328, IS1329, IS1400, and IS1222, in the Yen HPI. The insertion of a 125-bp ERIC sequence modifies the structure of the promoter of the ybtA yersiniabactin regulator in the Yen HPI. In contrast to the precise excision of the Yps HPI in Y. pseudotuberculosis, the Yen HPI suffers imprecise deletions. The Yen HPI is stably integrated in one of the three asn tRNA copies in Y. enterocolitica biogroup 1B (serotypes O:8, O:13, O:20, and O:21), probably due to inactivation of the putative integrase. The 17-bp duplications of the 3' end of the asnT RNA are present in both Yersinia spp. The HPI attachment site is unoccupied in nonpathogenic Y. enterocolitica NF-O, biogroup 1A, serotype O:5. The HPI of Yersinia is a composite and widely spread genomic element with a highly conserved yersiniabactin functional "core" and a divergently evolved variable part.

Base Sequence↗
PubMed1999

Rational live oral carrier vaccine design by mutating virulence-associated genes of Yersinia enterocolitica.

Three different Yersinia enterocolitica serotype O8 strains harboring mutations in virulence-associated genes coding for Yersinia adhesin A (YadA), Mn-cofactored superoxide dismutase (SodA), and high-molecular-weight protein 1 were analyzed for their ability to colonize and persist in tissues after orogastric immunization of C57BL/6 mice. We demonstrated that all three Yersinia mutant strains were markedly impaired in their ability to disseminate into the spleens and livers of immunized mice but were able to colonize the Peyer's patches for at least 12 days, resulting in the induction of significant antibody titers against Yersinia outer proteins (Yops) and in the priming of Yersinia antigen-specific CD4+ Th1 cells isolated from spleens. The high level of attenuation did not diminish the immunogenic properties of the mutant strains. In fact, mice immunized with a single oral dose of any of the mutant strains were protected against a lethal oral-challenge infection with wild-type Y. enterocolitica. Moreover, adoptive transfer of Yersinia-specific antibodies from sera of mice immunized with the mutant WAP-314 sodA revealed that this protection could be mediated by Yersinia-specific immunoglobulins.

Administration, Oral↗
PubMed1999

Ferric enterochelin transport in Yersinia enterocolitica: molecular and evolutionary aspects.

Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae.

Base Sequence↗
PubMed1999

Pneumocystis carinii carriage in immunocompetent patients with primary pulmonary disorders as detected by single or nested PCR.

Ninety-five bronchoalveolar lavage specimens from 63 immunocompetent adult patients with primary pulmonary disease were analyzed for Pneumocystis carinii colonization by primary and nested PCR. Twelve of 63 patients (19%) were PCR positive. None of them developed P. carinii pneumonia. These results suggest that P. carinii carriage may exist in immunocompetent patients with underlying pulmonary disease.

Adult↗
PubMed1999

Comparison of MB/BacT and BACTEC 460 TB systems for recovery of mycobacteria in a routine diagnostic laboratory.

MB/BacT, BACTEC 460 TB, and Löwenstein-Jensen (LJ) medium were evaluated in parallel for recovery of mycobacteria from 3,700 continuous clinical specimens in a routine laboratory. Mycobacteria were identified from 123 (3.3%) specimens. The recovery rates for all mycobacteria by the different systems were 91.0, 73.0, and 53.6% for BACTEC 460 TB, MB/BacT, and LJ medium, respectively. The recovery rates for Mycobacterium tuberculosis complex were 97.1, 80. 2, and 67.6%, respectively. The lack of sensitivity of the MB/BacT system was more pronounced with smear-negative specimens and resulted in a failure to detect three patients with infectious tuberculosis.

Bacterial Typing Techniques↗
PubMed1999

First-glance diagnosis of Strongyloides stercoralis autoinfection by stool microscopy.

We report a case of autoinfection due to Strongyloides stercoralis in a 27-year-old Ethiopian AIDS patient living in Germany for nearly 3 years. This case was diagnosed on the basis of a single-view field in microscopy of a freshly obtained formalin-fixed stool specimen showing both rhabditiform and filariform larvae. The diagnosis of autoinfection by microscopy is discussed in detail.

Adult↗
PubMed1998

Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase.

Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.

Bacterial Proteins↗
PubMed1998

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

Animals↗
PubMed1998

IL-18 (IFN-gamma-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice.

IL-12-induced IFN-gamma production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-gamma-inducing factor) is produced by macrophages and induces IFN-gamma production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-gamma production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-gamma production was not IL-18 dependent. IL-18-induced IFN-gamma production was to a higher degree dependent on IFN-gammaR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-gamma levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-gamma production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.

Animals↗
PubMed1998

In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene.

The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE-GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co-expressed in Y. enterocolitica (i) on a low-copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co-integration by homologous recombination of a yopE-gfp-carrying suicide plasmid into pYV08 (co-integrate configuration). After 30min of infection of HEp-2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE-GFP into HEp-2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp-2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE-GFP from selectively solubilized HEp-2 cells. The co-translocation of other Yops was not significantly impaired by YopE-GFP as shown by YopH/YopE-mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE-gfp expression (in trans as well as in the co-integrate configuration) in the HEp-2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE-LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.

Animals↗
PubMed1998

Transverse myelitis associated with Mycoplasma pneumoniae infection.

A 14-year-old boy developed acute transverse myelitis with severe abdominal pain, bladder dysfunction, weakness, and sensory loss of the lower extremities. Magnetic resonance imaging revealed a segmental expanded central edema affecting parts of the spinal cord, including the caudal medulla oblongata. Antibody response to Mycoplasma pneumoniae was negative in microparticle agglutination assays (1:40 in the acute serum and 1:160 in the convalescent serum) and complement fixation tests (1:20 and 1:10). However, analysis of acute-phase serum revealed a specific IgA and IgG response but no IgM response. Detection of M. pneumoniae in the cerebrospinal fluid by nested polymerase chain reaction and in nasopharyngeal aspirate by culture confirmed an M. pneumoniae infection. Treatment with doxycycline (100 mg daily) was started on the second day after admission to the hospital and continued for 14 days; the patient recovered completely and was discharged 20 days after onset of the disease, with no signs of neurological deficits.

Adolescent↗
PubMed1998

Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans.

The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this "high pathogenicity island" (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregative Escherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella enterica strains investigated. Polypeptides encoded by the fyuA, irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess the hms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the juA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.

Bacterial Outer Membrane Proteins↗
PubMed1998

The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation.

The ability to synthesize and uptake the Yersinia siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3, irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica 08, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 or irp2 abrogates yersiniabactin synthesis. Mutations in irp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1 mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.

Amino Acid Sequence↗
PubMed1998

Abiotrophia elegans sp. nov., a possible pathogen in patients with culture-negative endocarditis.

We isolated a hitherto undescribed microorganism from a patient with endocarditis. The microscopic appearance, a negative catalase reaction, and growth as satellite colonies next to Staphylococcus epidermidis suggested that this microorganism is a member of the genus Abiotrophia, formerly known as nutritionally variant streptococci. However, the clinical isolate described herein differed markedly from the known Abiotrophia spp., A. adiacens and A. defectiva, in terms of its (i) biochemical properties, (ii) restricted growth temperature range, (iii) whole-cell lysate polypeptide profile, and (iv) unique nutritional requirements. In contrast to the type strains of A. adiacens and A. defectiva, which used L-cysteine and pyridoxal hydrochloride as growth factors, the growth of the clinical isolate was only supported by L-cysteine hydrochloride and not by pyridoxal hydrochloride when the organism was tested in Todd-Hewitt or casein-soy peptone broth. Comparative 16S rRNA gene sequence analysis revealed that the microorganism was a member of the genus Abiotrophia and was most closely related to A. adiacens (96.9% homology). Phenotypic and phylogenetic data are consistent with the assumption of a new species within the genus Abiotrophia, for which we propose the name Abiotrophia elegans sp. nov. The unique nutritional requirements of this strain are of importance for diagnostic laboratories. The media of blood culture systems supplemented only with pyridoxal hydrochloride as a growth factor may fail to promote the growth of A. elegans sp. nov., and thus, these systems might not detect this microorganism as a possible cause of endocarditis.

Adult↗
PubMed1998

PCR for detection and identification of Abiotrophia spp.

Members of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of Abiotrophia spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of Abiotrophia spp. were developed. The first PCR assay was positive for all Abiotrophia spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for A. elegans, the most fastidious species of Abiotrophia. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of Abiotrophia spp.

Bacteremia↗
PubMed1998

Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae.

In this prospective study, the use of a culture-enhanced PCR assay for the detection of Mycoplasma pneumoniae, followed by hybridization with a specific probe (MP-HPCR) or without hybridization (MP-PCR), and the use of a nested PCR (MP-NPCR) were evaluated. Clinical samples (190 specimens) from 190 patients with respiratory complaints were incubated in culture broth overnight and then subjected to PCR. The results of the PCR were compared to those obtained by culture, the direct antigen test, and serologic testing by microparticle agglutination and by immunoblotting in unclear cases. The sensitivities were 19 CFU for MP-PCR, 1.9 CFU for MP-HPCR, and 0.019 CFU for MP-NPCR. PCR amplification of the beta-globin gene was possible in 98% of cases: after dilution of the beta-globin-negative samples, all samples were reactive. Correlation between negative MP-NPCR results and negative serology results was found in 89% of cases; a positive correlation was found with 10% of the patients. Samples from three immunocompromised patients were MP-NPCR positive but serologically negative. High respiratory colonization by M. pneumoniae (>10(5) CFU/ml) in patients with acute respiratory disease could be detected by culture, MP-PCR, and MP-NPCR. These results indicate that MP-PCR and MP-NPCR are reliable methods for the detection of M. pneumoniae in respiratory tract samples of patients with respiratory complaints.

Adult↗