Martina Bielaszewska
Research interests
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Interestsshiga toxin phages
Publications
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4.80Impact points
Epidemic Escherichia coli O104:H4: Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction.
Antimicrobial agents and chemotherapy. 03/2012;
The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicat... [more] The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated in some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased (P < 0.001 to P < 0.05), fosfomycin, gentamicin, and kanamycin insignificantly influenced (P > 0.1), and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased (P ≤ 0.05) stx(2)-phage induction and Stx2 production in outbreak isolates. Ciprofloxacin and chloramphenicol significantly upregulated and downregulated, respectively, stx(2) transcription (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which had been used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induce stx(2)-harboring phages nor increase stx(2) transcription and Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options in patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.
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4.16Impact points
Real-time multiplex polymerase chain reaction for detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools.
Journal of clinical microbiology. 02/2012;
A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for de... [more] A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and 132 patients' stool enrichment cultures. It is a rapid, sensitive and specific tool for detecting EHEC O104:H4 in human stools.
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3.93Impact points
Promiscuous Shiga toxin 2e and its intimate relationship to Forssman.
Glycobiology. 01/2012;
Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=... [more] Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders, and often fatal outcome. Stx2e-producing strains from the intestine of slaughtered pigs (n=3), feces of piglets with postweaning diarrhea or edema disease (n=12) and feces of humans with asymptomatic infections or mild diarrhea (n=13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.
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15.58Impact points
Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.
The Lancet infectious diseases. 06/2011; 11(9):671-6.
In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed viru... [more] In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain (rfb(O104), fliC(H4), stx(2), and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx(2), iha, lpf(O26), lpf(O113)) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. German Federal Ministry of Education and Research, Network Zoonoses.
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5.36Impact points
Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype.
Molecular microbiology. 02/2011; 79(4):1024-44.
Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five ... [more] Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (Tel(R) ) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml⁻¹) sustained large internal deletions within the Tel(R) OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10⁻³ and 1.97 × 10⁻⁴ cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10⁻¹ cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting Tel(R) OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness.
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5.50Impact points
Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.
Environmental microbiology. 02/2011; 13(5):1327-41.
The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified ... [more] The haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly) and the serine protease EspPα are putative virulence factors of EHEC. We investigated the interplay between these secreted factors and demonstrate that EspPα cleaves the 107 kDa large EHEC-Hly. Degradation was observed when purified EspPα was added to a growing culture of an EHEC-Hly-expressing strain, with isolated proteins and with coexpressing strains, and was independent of the EHEC serotype. EHEC-Hly breakdown occurred as a multistage process with the formation of characteristic fragments with relative molecular masses of ~82 kDa and/or ~84 kDa and ~34 kDa. The initial cleavage occurred in the N-terminal hydrophobic domain of EHEC-Hly between Leu(235) and Ser(236) and abolished its haemolytic activity. In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed. EHEC in contact with human intestinal epithelial cells simultaneously upregulated their EHEC-Hly and EspP indicating that both molecules might interact under physiological conditions. We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors. Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.
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30.76Impact points
Watch out for the even eviler cousin-sorbitol-fermenting E coli O157.
Lancet. 01/2011; 377(9762):298-9.
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4.92Impact points
Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains.
Journal of lipid research. 01/2011; 52(4):618-34.
Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a l... [more] Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.
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4.41Impact points
Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.
PloS one. 01/2011; 6(7):e22751.
An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with... [more] An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.
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4.45Impact points
Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells.
Thrombosis and haemostasis. 12/2010; 105(3):515-28.
Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by ... [more] Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by the two major Stx types, Stx1 and Stx2, in human brain microvascular endothelial cells (HBMECs) and EA.hy 926 macrovascular endothelial cells. Scanning electron microscopy of microcarrier-based cell cultures, digital holographic microscopy of living single cells, and quantitative apoptosis/necrosis assays demonstrate that Stx1 causes both necrosis and apoptosis, whereas Stx2 induces almost exclusively apoptosis in both cell lines. Moreover, microvascular and macrovascular endothelial cells have different susceptibilities to the toxins: EA.hy 926 cells are slightly, but significantly (∼ 10 times) more susceptible to Stx1, whereas HBMECs are strikingly (≥ 1,000 times) more susceptible to Stx2. These findings have implications in the pathogenesis of HUS, and suggest the existence of yet to be delineated Stx type-specific mechanisms of endothelial cell injury beyond inhibition of protein biosynthesis.
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3.21Impact points
Enterohemorrhagic Escherichia coli O26:H11-Associated Hemolytic Uremic Syndrome: Bacteriology and Clinical Presentation.
Seminars in thrombosis and hemostasis. 09/2010; 36(6):586-93.
Infection by enterohemorrhagic ESCHERICHIA COLI (EHEC) is the most frequent cause of hemolytic uremic syndrome (HUS) in childhood. During a 6-year period, all patients with the clinical diagnosis of HUS were registered in a prospective multicenter study in Austria and Germany. EHEC O26:H11 was the s... [more] Infection by enterohemorrhagic ESCHERICHIA COLI (EHEC) is the most frequent cause of hemolytic uremic syndrome (HUS) in childhood. During a 6-year period, all patients with the clinical diagnosis of HUS were registered in a prospective multicenter study in Austria and Germany. EHEC O26:H11 was the second most frequent detected serotype, accounting for 15.4% of all EHEC isolates. The presence of EHEC O26:H11 was significantly associated with young age at the disease onset ( P < 0.001). Patients infected with this serotype were not different in their clinical presentation than those infected with other serotypes. This study underlines the importance of EHEC serotypes other than O157 in the etiology of HUS and emphasizes the importance of implementation of appropriate diagnostic methods to identify the whole spectrum of EHEC associated with HUS.
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4.21Impact points
Distribution and phylogeny of immunoglobulin-binding protein G in Shiga toxin-producing Escherichia coli and its association with adherence phenotypes.
Infection and immunity. 08/2010; 78(8):3625-36.
eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variati... [more] eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H(-) and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-alpha, eibG-beta, and eibG-gamma). Strains expressing EibG-alpha and EibG-beta displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-gamma adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-alpha, eibG-beta, and eibG-gamma subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.
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6.79Impact points
Phylogenetic analysis of enterohemorrhagic Escherichia coli O157, Germany, 1987-2008.
Emerging infectious diseases. 04/2010; 16(4):610-6.
Multilocus variable number tandem repeat analysis (MLVA) is a subtyping technique for characterizing human pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157. We determined the phylogeny of 202 epidemiologically unrelated EHEC O157:H7/H- clinical isolates through 8 MLVA loci ... [more] Multilocus variable number tandem repeat analysis (MLVA) is a subtyping technique for characterizing human pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157. We determined the phylogeny of 202 epidemiologically unrelated EHEC O157:H7/H- clinical isolates through 8 MLVA loci obtained in Germany during 1987-2008. Biodiversity in the loci ranged from 0.66 to 0.90. Four of 8 loci showed null alleles and a frequency < or =44.1%. These loci were distributed among 48.5% of all strains. Overall, 141 MLVA profiles were identified. Phylogenetic analysis assigned 67.3% of the strains to 19 MLVA clusters. Specific MLVA profiles with an evolutionary persistence were identified, particularly within sorbitol-fermenting EHEC O157:H-.These pathogens belonged to the same MLVA cluster. Our findings indicate successful persistence of this clone.
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4.45Impact points
Vacuolisation of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli.
Thrombosis and haemostasis. 12/2009; 102(6):1080-92.
Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS), a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis ... [more] Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS), a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS. We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line. Because the vacuolating effect closely resembles those elicited on other cells by the vacuolating cytotoxin of Helicobacter pylori (VacA), we designated the factor responsible for this effect EHEC vacuolating cytotoxin (EHEC-Vac). EHEC-Vac (a secreted non-serine protease protein) binds to HBMECs rapidly and irreversibly, vacuolates within 30 min after exposure and the effect is maximally apparent at 48 h. Despite the vacuolisation, HBMECs survive for several days before they undergo necrosis. Electron and immunofluorescence microscopy demonstrate that the vacuoles induced by EHEC-Vac originate from lysosomes. Accordingly, they stain with neutral red indicating an acidic microenvironment. Similar to VacA, the EHEC-Vac-mediated vacuolisation is both prevented and reverted by the vacuolar proton pump inhibitor bafilomycin A1, suggesting a similar mechanism of vacuole formation by these toxins. Despite the similarity of phenotypes elicited by EHEC-Vac and VacA, genomic DNA from the EHEC-Vac-producing strain failed to hybridise to a vacA probe, as well as to probes derived from presently known E. coli vacuolating toxins. Through its microvascular endothelium-injuring potential combined with the ability to induce interleukin 6 release from these cells EHEC-Vac might contribute to the pathogenesis of HUS.
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6.79Impact points
Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91.
Emerging infectious diseases. 09/2009; 15(9):1474-7.
The diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.... [more] The diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.
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12.90Impact points
Intrahost genome alterations in enterohemorrhagic Escherichia coli.
Gastroenterology. 06/2009; 136(6):1925-38.
Bacterial chromosomes are not fixed molecules; they evolve over the course of infections in human beings. During infection, a variety of strong selective pressures are exerted on the pathogen. The resulting genetic changes that occur in intestinal pathogens might influence clinical outcome and have ... [more] Bacterial chromosomes are not fixed molecules; they evolve over the course of infections in human beings. During infection, a variety of strong selective pressures are exerted on the pathogen. The resulting genetic changes that occur in intestinal pathogens might influence clinical outcome and have an impact on diagnosis and epidemiology. Enterohemorrhagic Escherichia coli (EHEC) is a good example of this process. These zoonotic pathogens cause diarrhea, bloody diarrhea, and hemolytic uremic syndrome in human beings, whereas in their natural habitat they mostly are asymptomatic colonizers. Thus, EHEC must be able to quickly adapt from one milieu to another. The greatest challenge it might face is to infect human beings--profound chromosomal changes occur during the brief period that EHEC passes through the human gastrointestinal tract, leading to gains and losses of virulence determinants. The intensive study of human enteric factors that induce or modulate pathogen chromosome instability could provide important information about host-microbial interactions.
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4.16Impact points
Shiga Toxin, Cytolethal Distending Toxin and Hemolysin Repertoirs in Clinical Escherichia coli O91 Isolates.
Journal of clinical microbiology. 05/2009;
Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar ... [more] Shiga toxin (Stx)-producing Escherichia coli (STEC) of serogroup O91 are the most common human pathogenic eae-negative STEC. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (n = 1), H10 (n = 2), H14 (n = 52) or H21 (n = 20), or were H nontypeable (Hnt; n = 10); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the flagellin subunit-encoding gene fliCH14. Most STEC O91 possesed EHEC-hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx2d-activatable encoding mucus- and elastase-activatable Stx2d was solely present in STEC O91:H21, whereas most strains in the other serotypes possessed stx1. Moreover, only STEC O91:H21 possessed the cdt-V cluster encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes mostly originated in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients stools on enterohemolysin agar and their rapid initial subtyping using fliC genotyping facilitates identification of these emerging pathogens in clinical and epidemiological studies and enables prediction of the risk of a severe clinical outcome.
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1.90Impact points
Phenotypic and Genotypic Traits of Shiga Toxin-Negative E. coli O157:H7/H(-) Bovine and Porcine Strains.
Foodborne pathogens and disease. 04/2009; 6(2):235-43.
Abstract Enterohemorrhagic E. coli (EHEC) O157:H7/H(-) (nonmotile) exist as Shiga toxin gene (stx)-positive and stx-negative variants in patients and the environment. We analyzed molecular characteristics, phenotypes, and the phylogenetic background of three stx-negative E. coli O157:H7/H(-) strains... [more] Abstract Enterohemorrhagic E. coli (EHEC) O157:H7/H(-) (nonmotile) exist as Shiga toxin gene (stx)-positive and stx-negative variants in patients and the environment. We analyzed molecular characteristics, phenotypes, and the phylogenetic background of three stx-negative E. coli O157:H7/H(-) strains isolated from cattle and a pig and compared them with those of human EHEC and stx-negative E. coli O157:H7/H(-). All three animal strains contained fliCH7 and two contained eae. One eae-positive strain (O157:H(-)) was sorbitol-fermenting (SF) and the other (O157:H7) was non-sorbitol-fermenting (nSF). These two strains shared a spectrum of non-stx putative virulence and fitness genes with human nSF and SF EHEC and stx-negative E. coli O157:H7/H(-) and belonged, similar to the vast majority of human isolates, to sequence type (ST) 11 in multilocus sequence typing. In contrast, the eae-negative O157:H7 animal isolate, which was SF, differed in spectrum of virulence genes and also differed phylogenetically (ST717) from the two eae-positive strains and the human EHEC and stx-negative E. coli O157:H7/H(-). In contrast to efforts with human stx-negative E. coli O157:H(-), attempts to transduce the two stx-negative/eae-positive animal O157:H7/H(-) strains with stx(2)-encoding phages from human SF and nSF EHEC O157:H7/H(-) failed, despite the animal strains having intact loci where such phages integrate in human EHEC O157 (wrbA and yecE). The role of animal stx-negative/eae-positive and stx-negative/eae-negative E. coli O157:H7/H(-) in their natural source and in human infections needs further investigation.
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5.36Impact points
Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin.
Molecular microbiology. 03/2009;
Summary Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin ... [more] Summary Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h vs. 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.
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4.41Impact points
Structure and function relationship of the autotransport and proteolytic activity of EspP from Shiga toxin-producing Escherichia coli.
PloS one. 02/2009; 4(7):e6100.
BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities ... [more] BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta.
Following (28)
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Jan Thiele
Westfälische Wilhelms-Universität Münster -
Karl Cammann
Westfälische Wilhelms-Universität Münster -
Christian Mück-Lichtenfeld
Westfälische Wilhelms-Universität Münster -
Benedikt Sundermann
University Hospital Münster, Germany -
Dag Harmsen
Westfälische Wilhelms-Universität Münster
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