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Studies of pore-forming bacterial protein toxins in Escherichia coli
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Group Bernt Eric Uhlin)
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Escherichia coli, a Gram-negative bacterium, which can be classified into three groups: the commensal, intestinal pathogenic (IPEC) and extra-intestinal pathogenic (ExPEC) E. coli. The cytolysin A (ClyA) protein, a 34-kDa pore-forming toxin, encoded by a gene found in both non-pathogenic and pathogenic E. coli and in Salmonella enterica serovars Typhi and Paratyphi. It mediates a cytotoxic effect on various mammalian cells. ClyA is released by E. coli via outer membrane vesicles (OMVs) after reaching the periplasm via an unknown mechanism through the inner membrane. The gene is silenced by mutations in some of the most studied ExPEC strains suggesting that the locus would be subject to patho-adaptive alterations.

To study if the mutations of the clyA gene in E. coli strains was particular to certain strains, the sequences of the clyA gene locus of a set of ExPEC isolates and of the E. coli collection of reference strains (ECOR) were compared. The ExPEC strains – uropathogenic and neonatal meningitis E. coli (UPEC and NMEC) strains contained various ΔclyA alleles. Next, a functional clyA gene locus was restored and tagged with luxAB in the chromosome of the UPEC strain 536. Luciferase activity of the bacteria carrying the restored gene showed that the clyA gene expression is highly increased at the late logarithmic growth phase when compared to the non-pathogenic E. coli K-12 strain. A higher transcriptional level of the clyA+ gene was observed when the SfaX regulatory protein was heterologously overproduced. It was concluded that the clyA+ gene is expressed at elevated levels in the UPEC strain and this is at least in part due to the SfaX/PapX transcriptional regulators.

Studies of clyA::phoA fusions obtained by transposon TnphoA insertion mutagenesis showed that the first 12 amino acid residues of ClyA was sufficient for translocation of the protein chimera into the periplasm and to the OMVs. The role of the two cysteine residues in ClyA for protein translocation was tested by introducing substitution mutations. The results indicated that the C-terminal Cys (ClyAC 285S) is important for localization and/or stability of the protein in the periplasm. Structural analysis of ClyAwt purified from the periplasm revealed that the protein forms dimeric complexes. Upon treatment with the reducing agent DTT the ClyA protein readily assembled into typical pore complexes as revealed by electron miscroscopic analysis. In conclusion, the ClyA protein is present in the periplasm in a conformation that prevents it from forming pores in the bacterial membranes.

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin which induces lysis of mammalian cells by forming transmembrane channels. Although the biophysical activities of VCC were well studied, there was no detailed analysis of VCC secretion. Our study demonstrated that a fraction of the VCC was secreted in association with OMVs. OMV-associated VCC from the wild type V. cholerae strain V:5/04 is biologically active as shown by toxic effects on mammalian cells, interestingly, OMV-associated VCC was more active than purified VCC. Both environmental and clinical V. cholerae isolates transport VCC via OMVs. In addition, when the vcc gene is heterologously expressed in E. coli, OMV-associated secretion of VCC was also observed. We suggest that OMV-mediated release of VCC is a feature shared with ClyA.

sted, utgiver, år, opplag, sider
Umeå: Umeå University , 2014. , s. 72
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1677
HSV kategori
Forskningsprogram
molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-93629ISBN: 978-91-7601-131-7 (tryckt)OAI: oai:DiVA.org:umu-93629DiVA, id: diva2:750670
Disputas
2014-10-24, Hörsal E 04, Unod R1, Norrlands universitetssjukhus, Umeå, 13:15 (engelsk)
Opponent
Veileder
Forskningsfinansiär
The Swedish Foundation for International Cooperation in Research and Higher Education (STINT), IG2008-2049Swedish Research Council, 2010-303Swedish Research Council, 349-2007-8673Swedish Research Council, 2006-4702Swedish Research Council, 2013-2392Tilgjengelig fra: 2014-10-03 Laget: 2014-09-29 Sist oppdatert: 2018-06-07bibliografisk kontrollert
Delarbeid
1. Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains
Åpne denne publikasjonen i ny fane eller vindu >>Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains
Vise andre…
2014 (engelsk)Inngår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, s. 216-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

There are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as “cryptic” since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA+ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA+ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA+ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA+ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium.

sted, utgiver, år, opplag, sider
BioMed Central, 2014
Emneord
ClyA cytolysin, Pathoadaptive mutations, clyA gene expression, Extraintestinal Escherichia coli, SfaX regulatory protein
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-93657 (URN)10.1186/s12866-014-0216-4 (DOI)000341665100001 ()
Tilgjengelig fra: 2014-09-29 Laget: 2014-09-29 Sist oppdatert: 2019-01-28bibliografisk kontrollert
2. Localization and structure of the ClyA protein in Escherichia coli before secretion and pore-formation
Åpne denne publikasjonen i ny fane eller vindu >>Localization and structure of the ClyA protein in Escherichia coli before secretion and pore-formation
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Publisher
s. 72
HSV kategori
Forskningsprogram
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-93656 (URN)
Forskningsfinansiär
Swedish Research Council
Tilgjengelig fra: 2014-09-29 Laget: 2014-09-29 Sist oppdatert: 2018-06-07bibliografisk kontrollert
3. Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains
Åpne denne publikasjonen i ny fane eller vindu >>Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains
Vise andre…
2014 (engelsk)Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 9, artikkel-id e106731Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.

Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.

Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

sted, utgiver, år, opplag, sider
Public library of science, 2014
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-93659 (URN)10.1371/journal.pone.0106731 (DOI)000341271500078 ()25187967 (PubMedID)
Forskningsfinansiär
Swedish Research Council, 2006-4702Swedish Research Council, 2013-2392Swedish Research Council, 353-2010-7074Swedish Research Council, 2010-3031Swedish Research Council, 2012-4638Swedish Research Council, 349-2007-8673The Swedish Foundation for International Cooperation in Research and Higher Education (STINT), IG2008-2049
Tilgjengelig fra: 2014-09-29 Laget: 2014-09-29 Sist oppdatert: 2018-10-10bibliografisk kontrollert

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