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Molecular analysis of transcription factors in uropathogenic E. coli adhesin operons
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Bernt Eric Uhlin)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Molekylär analys av transkriptionsfaktorer i adhesin operon hos uropatogena E. coli (Swedish)
Abstract [en]

The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2009. , 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1304
Keyword [en]
UPEC, E. coli, F1C, P fimbriae, cross-talk, FocB, PapB, crystal structure, OMVs
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-27757ISBN: 978-91-7264-879-1 (print)OAI: oai:DiVA.org:umu-27757DiVA: diva2:277507
Public defence
2009-12-16, Major Groove, Institutionen för Molekylärbiologi, Byggn. 6L, Norrlands Universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-11-26 Created: 2009-11-19 Last updated: 2011-04-28Bibliographically approved
List of papers
1. Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli
Open this publication in new window or tab >>Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli
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2008 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, no 2, 771-780 p.Article in journal (Refereed) Published
Abstract [en]

Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

Keyword
Uropathogenic E. coli, PapB, FocB, F1C, fimbriae, pili, cross-talk
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-19587 (URN)10.1128/IAI.01010-07 (DOI)18039830 (PubMedID)
Available from: 2009-03-06 Created: 2009-03-06 Last updated: 2017-12-13Bibliographically approved
2. Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
Open this publication in new window or tab >>Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
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2010 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, no Pt 3, 337-341 p.Article in journal (Refereed) Published
Abstract [en]

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.

Keyword
fimbriae, FocB, transcription factors
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-27753 (URN)10.1107/S1744309110002204 (DOI)000275057700031 ()20208176 (PubMedID)
Available from: 2009-11-19 Created: 2009-11-19 Last updated: 2017-12-12Bibliographically approved
3. Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
Open this publication in new window or tab >>Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli
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2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 16, 3368-3381 p.Article in journal (Refereed) Published
Abstract [en]

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form different protein-protein complexes and that they may exert both positive and negative effects on transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all alpha helical structure with a helix-turn-helix (HTH) motif. Interestingly, conserved residues important for DNA-binding are not located in the recognition helix of the HTH-motif, but in the preceding helix. Results from protein-DNA binding studies indicated that FocB interacts with minor groove of its cognate DNA, which also points to a DNA-interaction unusual for this motif. Packing interactions in the crystals gave two plausible dimerization interfaces. Conserved residues known to be important for protein oligomerization are present at both interfaces, suggesting that both sites play a role in a functional FocB protein.

Place, publisher, year, edition, pages
Wiley, 2010
Keyword
fimbriae, FocB, repressor protein, uropathogenic Escherichia coli, X-ray crystallography
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-27755 (URN)10.1111/j.1742-4658.2010.07742.x (DOI)000280631300010 ()
Available from: 2009-11-19 Created: 2009-11-19 Last updated: 2017-12-12Bibliographically approved
4. Interaction between the PapI protein and the alpha subunit of Escherichia coli RNA polymerase
Open this publication in new window or tab >>Interaction between the PapI protein and the alpha subunit of Escherichia coli RNA polymerase
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Uropathogenic Escherichia coli (UPEC) expressing P fimbriae (also denoted Pap or P pili) have the ability to adhere in a specific manner to uroepithelial cells in the cause of urinary tract infections. P fimbriae expression is subject to an intricate regulatory mechanism playing an important role in pathogenesis, allowing bacteria to sense its environment and express fimbriae when needed, and turn off the expression when it may be unnecessary or detrimental to the bacteria. The expression of P fimbriae in the uropathogenic E. coli depends on transcription of the major pap operon in a gene cluster that consists of the polycistronic papB operon and the divergently orientated monocistronic papI operon. The transcription of pap is positively controlled by the local transcription factors PapI (8 kDa) and PapB (12 kDa) in concert with the global regulators leucine-responsive regulatory protein (Lrp) and the cAMP receptor protein (CRP). DNA adenine methylase (Dam) activity is also required for pap transcription. PapI is known to interact with Lrp and its role in relation to the transcriptional activation of pap is thought to be indirect without contact with the RNA polymerase. The CRP-cAMP complex is known to activate the expression of pap via direct contact with the alpha (α) subunit of E. coli RNA polymerase. Methodology/Principal findings: Here, we show that an excess of α protein inhibits the transcription of pap as revealed by using a collection of strains which carry plasmids expressing α subunits with different point mutations in the α subunit of E. coli RNA polymerase. The inhibition was partially restored by the introduction of a papI clone expressing higher level of PapI. Experiments with purified proteins verified that PapI physically interacts with the α subunit of E. coli RNA polymerase in vitro. Conclusion/Significance: Our findings indicate that the PapI protein may interact directly with the RNA polymerase complex and that such interactions need to be considered in its role as a transcription factor in the activation of pap transcription.

Keyword
Escherichia coli, PapI, alpha subunit, RNAP
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-27738 (URN)
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2009-11-26Bibliographically approved
5. Release of the type I secreted α-haemolysin via outer membrane vesicles from Escherichia coli
Open this publication in new window or tab >>Release of the type I secreted α-haemolysin via outer membrane vesicles from Escherichia coli
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2006 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, no 1, 99-112 p.Article in journal (Refereed) Published
Abstract [en]

The α-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the α-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The α-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The α-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the α-haemolysin were distinct from the OMVs not containing α-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of α-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted α-haemolysin from the bacteria to the host cells during bacterial infections.

Place, publisher, year, edition, pages
Hoboken, NJ, United States: Wiley-Blackwell, 2006
Keyword
Escherichia coli, OMVs, outer membrane vesicles, haemolysin, UPEC
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-27736 (URN)10.1111/j.1365-2958.2005.04938.x (DOI)
Note
Stina Berglund nu Stina LindbergAvailable from: 2009-12-08 Created: 2009-11-18 Last updated: 2017-12-12Bibliographically approved
6. Detection of functional P fimbriae proteins in E. coli outer membrane vesicles
Open this publication in new window or tab >>Detection of functional P fimbriae proteins in E. coli outer membrane vesicles
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Outer membrane vesicles (OMVs) are continuously released from the surface of Gram negative bacteria. The composition of the OMVs is similar to that of the outer membrane and may include periplasmic constituents. Studies have shown that OMVs also may have a role in the delivery of bacterial toxins to the extracellular space. Results: In this study we investigate the association of P fimbriae proteins to OMVs from both genetically manipulated E. coli laboratory strains and a uropathogenic isolate. We found that the OMVs could carry functionally active P fimbriae proteins on their surface and we observed specific attachment of OMVs to beads coated with the P fimbrial galabiose receptor. Conclusions: Our results suggest that OMVs can carry functionally active fimbrial proteins on their surface and that they may have a role in specific delivery of bacterial components to target cells.

Keyword
Eshcerichia coli, outer membrane vesicles, OMVs, P fimbriae, adhesion
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-27737 (URN)
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2009-11-26Bibliographically approved

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