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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
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2014 (English)In: BMC Microbiology, ISSN 1471-2180, Vol. 14, 216- p.Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
BioMed Central, 2014. Vol. 14, 216- p.
Keyword [en]
ClyA cytolysin, Pathoadaptive mutations, clyA gene expression, Extraintestinal Escherichia coli, SfaX regulatory protein
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
URN: urn:nbn:se:umu:diva-93657DOI: 10.1186/s12866-014-0216-4ISI: 000341665100001OAI: diva2:750624
Available from: 2014-09-29 Created: 2014-09-29 Last updated: 2014-11-07Bibliographically approved
In thesis
1. Studies of pore-forming bacterial protein toxins in Escherichia coli
Open this publication in new window or tab >>Studies of pore-forming bacterial protein toxins in Escherichia coli
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2014. 72 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1677
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-93629 (URN)978-91-7601-131-7 (ISBN)
Public defence
2014-10-24, Hörsal E 04, Unod R1, Norrlands universitetssjukhus, Umeå, 13:15 (English)
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, 2013-2392
Available from: 2014-10-03 Created: 2014-09-29 Last updated: 2014-10-13Bibliographically approved

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Enow Oben Ayuk, ConstanceOscarsson, JanZlatkov, NikolaWestermark, MarieDuperthuy, MaryliseWai, Sun NyuntUhlin, Bernt Eric
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