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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, E-ISSN 1471-2180, Vol. 14, p. 216-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, p. 216-
Keywords [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)
Identifiers
URN: urn:nbn:se:umu:diva-93657DOI: 10.1186/s12866-014-0216-4ISI: 000341665100001Scopus ID: 2-s2.0-84907911169OAI: oai:DiVA.org:umu-93657DiVA, id: diva2:750624
Available from: 2014-09-29 Created: 2014-09-29 Last updated: 2024-01-17Bibliographically 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. p. 72
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1677
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
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)
Opponent
Supervisors
Funder
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: 2018-06-07Bibliographically approved
2. Regulatory mechanisms involved in pathoadaptation of extraintestinal pathogenic Escherichia coli
Open this publication in new window or tab >>Regulatory mechanisms involved in pathoadaptation of extraintestinal pathogenic Escherichia coli
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Establishment of commensal bacteria within a new niche of their host usually promotes the transition from commensalism to pathogenicity. Extraintestinal Pathogenic Escherichia coli (ExPEC) represent different pathovars with biphasic lifestyle – they can reside in the gut as commensals or they can escape and cause diseases elsewhere in the human body. Depending on the disease they are linked to, ExPEC can be divided into Uropathogenic E. coli (UPEC), Newborn Meningitis-causing E. coli (NMEC) and Sepsis-associated E. coli (SEPEC).

Pathoadaptive mutations linked to c-di-GMP signaling were investigated in the NMEC strain IHE3034 which lacks the main global stress regulator RpoS. We investigated the role of ycgG2 in the lifestyle of NMEC. Deletion of ycgG2, shown by us to encode an YcgG allozyme with c-di-GMP phosphodiesterase (PDE) activity, and the restored RpoS led to a decrease in the S-fimbriae, otherwise robustly produced in artificial urine, hinting that the urinary tract could serve as a habitat for NMEC. We showed that NMEC were capable of aerobic citrate utilization in the presence of a co-substrate - a property that normally does not exist in E. coli. Our data hint that this metabolic upgrade is enhanced by the lack, or reduced activity, of c-di-GMP PDEs. We also found that citrate utilization is a property of ExPEC, since we reconstituted it in E. coli UTI89 (a cystitis isolate) via inactivation of its RpoS, and since a set of pyelonephritis E. coli isolates use citrate aerobically in the presence of glucose. The main reason for this metabolic capability is the absence of RpoS which leads to the production of the citrate transporter CitT. Furthermore, we highlighted the deletion of the fec operon (required for the ferric citrate uptake) in a large group of different ExPEC strains and we showed that NMEC can use CitT for in vitro ferric citrate uptake dependent on YcgG2 as an alternative system.

Another pathoadaptive mutation which influences the fitness of ExPEC is the clyA (cytolysin A) gene inactivation, resulting from different deletions in different ExPEC genomes. When we restored the clyA+ locus, the UPEC strain 536 displayed increased susceptibility to antimicrobial peptides and urea. We also showed that the ClyA expression in 536 was increased by the presence of the DNA-binding regulator SfaX and another stand-alone PDE similar to YcgG2, called SfaY. The results were further confirmed by ClyA downregulation in NMEC deficient in SfaY and SfaX.

We also studied the role of sfaY - a gene coding for another stand-alone c-di-GMP PDE. The expression of sfaY is under the regulation of the main promoter of the horizontally acquired sfa gene cluster. The latter is responsible for the regulation and assembly of the virulence-associated S-fimbriae, via which ExPEC bacteria bind to sialylated receptors. We found that NMEC are competent for filamentation because of a c-di-GMP-dependent program under the control of a phase-variation event which selectively turns ‘ON’ the sfa promoter in a subpopulation of bacteria. When SfaY is present, c-di-GMP levels are reduced, thus inducing the SOS stress response via the canonical LexA-RecA pathway. The signaling resulted in an internal differentiation of the bacterial population into a subpopulation exhibiting a filamentous morphotype (bacteria with induced SOS stress response) and a subpopulation of small motile and non-motile bacteria. Hence, this molecular program could serve as a clue to explain the formation of the intracellular bacterial communities observed during urinary tract infection by UPEC.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2019. p. 74
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2008
Keywords
ExPEC, PAIs, pathoadaptation, citrate, metabolism, filamentation, toxins
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell and Molecular Biology
Research subject
Molecular Biology; Microbiology
Identifiers
urn:nbn:se:umu:diva-155816 (URN)978-91-7855-002-9 (ISBN)
Public defence
2019-02-20, Major Groove, NUS byggnad 6L, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2015-03007, 2015-06824, 2016-06598, 349-2007-8673, 829-2006-7431
Available from: 2019-01-30 Created: 2019-01-28 Last updated: 2019-01-29Bibliographically approved

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Enow Oben Ayuk, ConstanceOscarsson, JanZlatkov, NikolaDuperthuy, MaryliseWai, Sun NyuntUhlin, Bernt Eric

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