The fight between cells and bacteria: modulation of eukaryotic signalling by bacterial pathogens.
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Bacteria↗
Biomedical subjects
J Heesemann
Publications and source records attributed to J Heesemann.
Cloning and characterization of the gene encoding periplasmic 2',3'-cyclic phosphodiesterase of Yersinia enterocolitica O:8.
The gene encoding periplasmic 2',3'-cyclic phosphodiesterase in Yersinia enterocolitica O:8 (designated cpdB), was cloned and expressed in Escherichia coli. This enzyme enables Y. enterocolitica to grow on 2',3'-cAMP as a sole source of carbon and energy. Sequencing and analysis of a 3 kb ECO:RI fragment containing the cpdB gene revealed an open reading frame of 1179 bp, corresponding to a protein with a molecular mass of 71 kDa. The first 25 amino acid residues show features of a typical prokaryotic signal sequence. The predicted molecular mass of the mature peptide is therefore in agreement with the molecular mass estimated by SDS gel electrophoresis (68 kDa). The putative cpdB promoter region contains two possible -10 and -35 regions. Furthermore, the 5' untranslated region contains sequences with significant homology to the cyclic AMP-cyclic AMP receptor protein binding site and the sigma(28) consensus. This region is interrupted by an enterobacterial repetitive intergenic consensus (ERIC) sequence. Deletion of the ERIC element from the cpdB promoter region had no effect on cpdB expression. In the 3' untranslated region, a possible rho-independent transcriptional terminator was identified. The deduced amino acid sequence of the Y. enterocolitica CpdB protein shows 76% identity with CpdB of Salmonella typhimurium and E. coli. CpdB of Y. enterocolitica is exported to the periplasmic space. An isogenic Y. enterocolitica cpdB mutant strain, constructed by allelic exchange, was no longer able to grow on 2',3'-cAMP as sole source of carbon and energy. The CpdB mutant showed no significant change in virulence in an oral and intravenous mouse infection model.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Functional analysis of yersiniabactin transport genes of Yersinia enterocolitica.
Yersinia enterocolitica O:8, biogroup (BG) IB, strain WA-C carries a high-pathogenicity island (HPI) including iron-repressible genes (irp1-9, fyuA) for biosynthesis and uptake of the siderophore yersiniabactin (Ybt). The authors report the functional analysis of irp6,7,8, which show 98-99% similarity to the corresponding genes ybtP,Q,X on the HPI of Yersinia pestis. It was demonstrated that irp6,7 are involved in ferric (Fe)-Ybt utilization and mouse virulence of Y. enterocolitica, thus confirming corresponding results for Y. pestis. Additionally it was shown that inactivation of the ampG-like gene irp8 did not affect either Fe-Ybt utilization or mouse virulence. To determine whether irp6, irp7 and fyuA (encoding the outer-membrane Fe-Ybt/pesticin receptor FyuA) are sufficient to mediate Fe-Ybt transport/utilization, these genes were transferred into Escherichia coli entD,F and into non-pathogenic Y. enterocolitica, BG IA, strain NF-O. Surprisingly, E. coli entD,F but not Y. enterocolitica NF-O gained the capability to utilize exogenous Fe-Ybt as a result of this gene transfer, although both strains expressed functional FyuA (pesticin sensitivity). These results suggest that besides irp6, irp7 and fyuA, additional genes are required for sufficient Fe-Ybt transport/utilization. Finally, it was shown that irp6, irp7 and fyuA but not irp8 are involved in controlling Ybt biosynthesis and fyuA gene expression: irp6 and/or irp7 mutation leads to upregulation whereas fyuA mutation leads to downregulation. However, fyuA-dependent control of Ybt biosynthesis could be bypassed in a fyuA mutant by ingredients of chrome azurol S (CAS) siderophore indicator agar.
Animals↗
Arginine-143 of Yersinia enterocolitica YopP crucially determines isotype-related NF-kappaB suppression and apoptosis induction in macrophages.
Pathogenic Yersinia spp. counteract host defense mechanisms by modulating the cellular signal relay in response to infection. Subversion of the antiapoptotic NF-kappaB signaling pathway by the Yersinia enterocolitica virulence protein YopP crucially determines the induction of apoptosis in Yersinia-infected macrophages. Here, we analyzed a panel of pathogenic, phylogenetically distinct Y. enterocolitica serotypes for their abilities to trigger macrophage apoptosis. Y. enterocolitica from the highly pathogenic serogroup O8 was substantially more effective in apoptosis induction than Yersinia from the serogroups O3 and O9. Complementation of yopP-knockout mutants revealed that this effect was specifically conferred by the serogroup O8 YopP. The amino acid sequences of YopPO8 and YopPO9 share 94% identity, and both YopP isotypes were found to interact with the NF-kappaB-activating kinase IKKbeta in macrophages. However, selectively, YopPO8 mediated efficient inhibition of IKKbeta activities, which led to substantial suppression of NF-kappaB activation. To localize the YopPO8-related effector domain, we interchanged stretches of amino acids and single amino acid residues between YopPO8 and YopPO9. Functional characterization of the resulting mutants revealed a major role of the arginine-143 residue in determining the inhibitory impact of YopP on IKKbeta activity and survival of macrophages.
Amino Acid Sequence↗
Expression analysis of the yersiniabactin receptor gene fyuA and the heme receptor hemR of Yersinia enterocolitica in vitro and in vivo using the reporter genes for green fluorescent protein and luciferase.
The enteropathogenic Yersinia enterocolitica strains have several systems for scavenging iron from their environment. We have studied the expression of the fyuA gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the hemR gene, which encodes the receptor for heme, using the reporter genes gfp (encoding green fluorescent protein) and luc (encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of fyuA and hemR or expression of one gene by a gfp-luc tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that fyuA and hemR are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27 degrees C. In the in vivo BALB/C mouse infection model, tissue-specific expression of fyuA and hemR reporter fusions was observed. Surprisingly, fyuA and hemR reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying fyuA or hemR reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of fyuA and hemR under peritoneal growth conditions. Differential expression of the fyuA and hemR reporter fusions could not be observed, suggesting similar regulation of fyuA and hemR in the mouse infection model.
Animals↗
Infection of synovial fibroblasts in culture by Yersinia enterocolitica and Salmonella enterica serovar Enteritidis: ultrastructural investigation with respect to the pathogenesis of reactive arthritis.
Synovial fibroblasts were infected with Yersinia enterocolitica or Salmonella enterica serovar Enteritidis and analyzed by electron microscopy and fluorescence in situ hybridization. Intracellular bacterial replication was followed by degradation leading to "ghosts" possessing lipopolysaccharides but not DNA. However, single bacteria survived for more than 2 weeks. Therefore, transient intra-articular infection might be the missing link between initial intestinal infection and late synovial inflammation in the pathogenesis of reactive arthritis.
Arthritis, Reactive↗
Comparison of fluorescent in situ hybridization and conventional culturing for detection of Helicobacter pylori in gastric biopsy specimens.
In this study, we have investigated 201 gastric biopsy specimens obtained from dyspeptic patients for the presence of Helicobacter pylori. By means of fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescence-labeled oligonucleotide probes specific for H. pylori, this pathogen was detected in 63 biopsy specimens. By using conventional culturing, H. pylori was isolated from 49 of these 63 gastric biopsy specimens. In contrast, FISH failed to identify H. pylori in four samples from which the pathogen was cultured. The lowest sensitivity was obtained by using the urease test. H. pylori was detected indirectly by this method in 43 of 67 biopsy specimens, which were positive for the pathogen as determined by FISH and/or culturing. All 49 H. pylori isolates that were detected by FISH and culturing underwent antimicrobial susceptibility testing for clarithromycin, a macrolide drug that is a key component in the therapy of peptic ulcer disease caused by this pathogen. Clarithromycin susceptibility testing of cultured isolates was carried out by the E-test, whereas FISH was used on biopsy specimens to detect clarithromycin-resistant mutant strains. No discrepancies were found between these two methods. Thirty-seven strains were clarithromycin sensitive, and eight H. pylori isolates were resistant to the macrolide. From another four biopsy specimens, a mixture of clarithromycin-sensitive and -resistant strains was identified by both methods. Thus, FISH is a reliable technique for determining the clarithromycin susceptibility of this pathogen. Taken together, FISH is a more sensitive and rapid technique than culturing for detection of H. pylori in gastric biopsy specimens. However, in the microbiology routine diagnostic laboratory, the combination of both FISH and conventional culturing significantly increases the sensitivity in detection of H. pylori.
Adult↗
Evaluation of BACTEC MGIT 960 and BACTEC 460TB systems for recovery of mycobacteria from clinical specimens of a university hospital with low incidence of tuberculosis.
Clinical samples obtained over a period of 8 months (n = 2,624) were processed in parallel with the BACTEC 460TB system, with the MGIT 960 system, and in Löwenstein-Jensen (LJ) medium, resulting in the recovery of 127 mycobacteria. Recovery rates in combinations of the BACTEC 460TB or MGIT 960 system with LJ were, respectively, 94.7 and 94.7% for Mycobacterium tuberculosis complex (n = 57) and 91.4 and 70.0% for nontuberculous mycobacteria (n = 70). Contamination rates, elevated in the MGIT 960 system, were associated with patients (cystic fibrosis) and type of material but not with transport time. Detection time was reduced in the MGIT 960 system.
Bacteriological Techniques↗
Rapid and accurate determination of genotypic clarithromycin resistance in cultured Helicobacter pylori by fluorescent in situ hybridization.
Phenotypic susceptibility testing for clarithromycin by E-test and disk diffusion of 109 cultured Helicobacter pylori isolates was compared with the genotypic susceptibility determination by fluorescent in situ hybridization (FISH). No discrepancies were found between these three methods. However, FISH has the advantage of providing results after 3 h.
Anti-Bacterial Agents↗
Use of recombinant mitogillin for improved serodiagnosis of Aspergillus fumigatus-associated diseases.
During human infection, Aspergillus fumigatus secretes a 18-kDa protein that can be detected as an immunodominant antigen in the urine of infected patients. Recently, this protein was shown to be mitogillin, a ribotoxin that cleaves a single phosphodiester bond of the 29S rRNA of eukaryotic ribosomes. We proved the immunogenic capacity of mitogillin in a rabbit animal model, indicating its usefulness as an antigen for serological diagnosis of invasive aspergillosis. The mitogillin gene from A. fumigatus was transferred from plasmid pMIT+ to expression vector pQE30 and expressed in Escherichia coli as a fusion protein. Purified recombinant mitogillin was recognized by serum immunoglobulin G (IgG) of polyclonal rabbit sera that were obtained by immunization with purified native mitogillin. Consequently, we developed an enzyme-linked immunosorbent assay for detection of IgG, IgM, and IgA antibodies to recombinant mitogillin. In serum samples of patients suffering from aspergilloma (AO; n = 32), invasive pulmonary aspergillosis (IPA; n = 42), or invasive disseminated aspergillosis (IDA; n = 40), a good correlation of production of IgG antibody against mitogillin and clinical disease was observed (for patients with AO, 100% [32 of 32] were positive; for patients with IPA, 64% [31 of 42] were positive; for patients with IDA, 60% [24 of 40] were positive). In contrast, positive titers for serum IgG and IgM antibodies against mitogillin were found in only 1.3% of the serum samples of healthy volunteers and positive titers for IgA antibody were found in only 1.0% of the serum samples of healthy volunteers (n = 307; specificity = 95.4%). These results indicate that recombinant mitogillin expressed in E. coli can be used for improvement of the serodiagnosis of A. fumigatus-associated diseases.
Adolescent↗
Molecular diagnosis of an Enterocytozoon bieneusi human genotype C infection in a moderately immunosuppressed human immunodeficiency virus seronegative liver-transplant recipient with severe chronic diarrhea.
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Adult↗
Pneumocystis carinii carriage among cystic fibrosis patients, as detected by nested PCR.
A total of 137 sputa from 95 consecutive cystic fibrosis (CF) patients undergoing routine bacteriological surveillance were analyzed for Pneumocystis carinii colonization using nested PCR. Seven of 95 patients (7.4%) were PCR positive, suggesting that P. carinii carriage may exist among CF patients due to their underlying pulmonary disease.
Adult↗
Burkholderia/Stenotrophomonas.
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Burkholderia↗
Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins.
The non-fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.
Adhesins, Bacterial↗
The Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts with the small GTPases RhoA and Rac-1.
Pathogenic bacteria of the genus Yersinia counteract host defense by interfering with eukaryotic signal transduction pathways. YpkA of Yersinia pseudotuberculosis shares significant homology with eukaryotic Ser/Thr protein kinases, is translocated into the host cell and has been shown to be an essential virulence factor in a mouse infection model. In this study, we identify the small GTPases RhoA and Rac-1 as eukaryotic binding partners of YpkA and its homolog YopO of Yersinia enterocolitica. We demonstrate that the interaction is independent of phosphorylation of YpkA and nucleotide loading state of the GTPases. The interaction with RhoA and Rac-1 might provide an important clue to how YpkA interferes with eukaryotic signaling on a molecular level.
Animals↗
High-pathogenicity island of Yersinia pestis in enterobacteriaceae isolated from blood cultures and urine samples: prevalence and functional expression.
Production of the siderophore yersiniabactin is associated with virulence in Yersinia species. The genes for biosynthesis and uptake of yersiniabactin are located on a high-pathogenicity island (HPI). The distribution and functioning of the Yersinia HPI were assessed in different Enterobacteriaceae strains isolated from blood cultures and urine samples. In total, 550 clinical isolates from 10 Enterobacteriaceae species were investigated by polymerase chain reaction and DNA hybridization. The Yersinia HPI was most prevalent in Escherichia coli (overall prevalence, 72.3%) and, to a lesser extent, in Klebsiella oxytoca (58.3%), Citrobacter species (25%), Klebsiella pneumonia (17.7%), and Enterobacter species (12.2%). The production of the siderophore yersiniabactin was also demonstrated in these HPI-positive strains by use of a reporter gene bioassay. These findings indicate that the HPI of Yersinia is distributed and functions in clinical specimens of different Enterobacteriaceae species.
Blood↗
Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8.
The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.
AT Rich Sequence↗
Yersinia enterocolitica-mediated translocation of defined fusion proteins to the cytosol of mammalian cells results in peptide-specific MHC class I-restricted antigen presentation.
Yersinia enterocolitica delivers a set of effector proteins [Yersinia outer proteins (Yop)] into the cytosol of target cells to modulate host cell signal transduction pathways required for the extracellular survival of the bacterium. Secretion and subsequent translocation of Yop across the eukaryotic cell membrane are achieved via a type III secretion system. About 50 - 100 amino acids of the N terminus of Yop are required for chaperone-directed secretion and translocation. In this study, it is demonstrated by immunoblot analysis of Yersinia-infected cultured epithelial cells that one ot these proteins, YopE, can serve as a molecular carrier to deliver protein fragments of the heterologous p60 antigen of Listeria monocytogenes into the cytosol of target cells. T cell activation assays revealed that the observed type III-mediated antigen translocation led to a p60 peptide-specific MHC class I-restricted antigen presentation. Efficient translocation and antigen presentation were strictly dependent on the co-localized expression of hybrid YopE-p60 proteins and the YopE-specific chaperone SycE. These results suggest that the Yersinia type III secretion system may serve as an attractive tool for antigen delivery in Yersinia-based live vaccines to induce cellular immune responses.
Animals↗