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Regulatory mechanisms involved in pathoadaptation of extraintestinal pathogenic Escherichia coli
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Prof. Bernt Eric Uhlin)
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 [en]
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: urn:nbn:se:umu:diva-155816ISBN: 978-91-7855-002-9 (print)OAI: oai:DiVA.org:umu-155816DiVA, id: diva2:1283211
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, 829-2006-7431Available from: 2019-01-30 Created: 2019-01-28 Last updated: 2019-01-29Bibliographically approved
List of papers
1. Absence of Global Stress Regulation in Escherichia coli Promotes Pathoadaptation and Novel c-di-GMP-dependent Metabolic Capability
Open this publication in new window or tab >>Absence of Global Stress Regulation in Escherichia coli Promotes Pathoadaptation and Novel c-di-GMP-dependent Metabolic Capability
2019 (English)In: Scientific Reports, ISSN 2045-2322, Vol. 9, article id 2600Article in journal (Refereed) Published
Abstract [en]

athoadaptive mutations linked to c-di-GMP signalling were investigated in neonatal meningitis-causing Escherichia coli (NMEC). The results indicated that NMEC strains deficient in RpoS (the global stress regulator) maintained remarkably low levels of c-di-GMP, a major bacterial sessility-motility switch. Deletion of ycgG2, shown here to encode a YcgG allozyme with c-di-GMP phosphodiesterase activity, and the restoration of RpoS led to a decrease in S-fimbriae, robustly produced in artificial urine, hinting that the urinary tract could serve as a habitat for NMEC. We showed that NMEC were skilled in aerobic citrate utilization in the presence of glucose, a property that normally does not exist in E. coli. Our data suggest that this metabolic novelty is a property of extraintestinal pathogenic E. coli since we reconstituted this ability in E. coli UTI89 (a cystitis isolate) via deactivation rpoS; additionally, a set of pyelonephritis E. coli isolates were shown here to aerobically use citrate in the presence of glucose. We found that the main reason for this metabolic capability is RpoS inactivation leading to the production of the citrate transporter CitT, exploited by NMEC for ferric citrate uptake dependent on YcgG2 (an allozyme with c-di-GMP phosphodiesterase activity).

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-155827 (URN)10.1038/s41598-019-39580-w (DOI)000459399400069 ()30796316 (PubMedID)
Note

Originally included in thesis in manuscript form.

Available from: 2019-01-29 Created: 2019-01-29 Last updated: 2019-05-22Bibliographically approved
2. 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
Open this publication in new window or tab >>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
Show others...
2014 (English)In: BMC Microbiology, ISSN 1471-2180, 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
Keywords
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:nbn:se:umu:diva-93657 (URN)10.1186/s12866-014-0216-4 (DOI)000341665100001 ()
Available from: 2014-09-29 Created: 2014-09-29 Last updated: 2019-01-28Bibliographically approved
3. C-di-GMP-mediated Morphotypic Pathoadaptability of Neonatal Meningitis Escherichia coli
Open this publication in new window or tab >>C-di-GMP-mediated Morphotypic Pathoadaptability of Neonatal Meningitis Escherichia coli
(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-155828 (URN)
Available from: 2019-01-29 Created: 2019-01-29 Last updated: 2019-01-29

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