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Pathogenecity-associated genes modulate Escherichia coli adhesion and motility
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli strains typical of UPEC (uropathogenic E. coli) and NBM (newborn meningitis) isolates carry chromosomally located PAIs (pathogenicity islands) that are absent in non-pathogenic E. coli strains. The PAIs include genes for virulence factors such as toxins and genes coding for specific adhesins and pili/fimbriae formation. Commonly, the gene clusters for such fimbriae in E. coli consist of a set of genes for biogenesis of the actual fimbriae organelles: a chaperone, an usher, the fimbrial subunits, and an adhesin, as well as some regulatory genes. Genetic studies of the fimbrial gene clusters in PAIs containing the pap genes, the prs genes, or the sfa genes led to the discovery of nearby open reading frames coding for putative cytoplasmic 17 kDa proteins — the X genes. Molecular genetic studies of the sfaXII locus in the clinical NMEC isolate IHE3034 have been performed. The results suggested that expression of the sfaXII gene had regulatory functions affecting both type 1 fimbriae expression and the flagella-mediated motility.

Type 1 fimbriae expression was found to be affected at the level of fim operon transcription and a major reason was SfaXII-mediated modulation of expression from the fimB and fimE recombinase genes. Quantification of SfaII-fimbriated bacteria in a comparison between wild type and SfaXII mutant strains gave no indication that the sfaXII gene product also would be affecting expression and/or biogenesis of SfaII fimbriae.

Biomechanical properties of the SfaII fimbriae produced by wild type and the sfaXII mutant IHE3034 were studied using force measuring optical tweezers (FMOT) and compared to other PAI-encoded fimbriae as well as to the type 1 fimbriae encoded on the core chromosome. The FMOT methodology assesses unfolding and refolding properties and we found that S fimbriae had weaker layer-to-layer interactions than both P and type 1 fimbriae, however the unfolding kinetics was slightly faster.

The expression profile and regulation of the sfaXII gene were determined by use of reporter gene fusions and it was found that expression was affected by environmental cues such as pH, osmolarity and temperature. It was also discovered that the nucleoid structuring protein H-NS and the sigma factor RpoS had strong direct or indirect repressive effects on sfaXII gene expression.

Further genomic analysis of the PAI fimbrial operons revealed that in some cases an additional ORF was found between the X genes and the fimbrial adhesion genes. Examination of the sfaII operon in IHE3034 indicated that this new gene, denoted sfaYII, coded for a protein that had the EAL domain motif and thereby could be considered a putative phosphodiesterase involved in controlling the level of cyclic-di-GMP in the bacterial cells.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2009. , 49 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1266
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-22302ISBN: 987-91-7264-789-3 OAI: oai:DiVA.org:umu-22302DiVA: diva2:214365
Distributor:
Molekylärbiologi (Medicinska fakulteten), 901 87, Umeå
Public defence
2009-05-28, Major Groove, Byggnad 6L, Institutionen för Molekylärbiologi, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-05-15 Created: 2009-05-05 Last updated: 2010-04-07Bibliographically approved
List of papers
1. The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression
Open this publication in new window or tab >>The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression
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2009 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 46, no 5, 243-252 p.Article in journal (Refereed) Published
Abstract [en]

The genomes of pathogenic E. coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaXII gene associated to the SfaII fimbrial determinant in the E. coli isolate IHE3034. sfaXII belongs to a subfamily of genes, the 17kDa genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaXII gene had an inhibitory effect on type 1 fimbriae expression. Down regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaXII on expression of the recombinase FimB that catalyzes OFF to ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaXII gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaXII gene, and presumably other members in the 17kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.

Place, publisher, year, edition, pages
Elsevier Ltd, 2009
Keyword
NMEC, Regulation, S fimbriae, Type 1 fimbriae, Motility
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-22286 (URN)10.1016/j.micpath.2009.01.007 (DOI)19486641 (PubMedID)
Available from: 2009-05-05 Created: 2009-05-04 Last updated: 2017-12-13Bibliographically approved
2. Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli
Open this publication in new window or tab >>Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli
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2010 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 39, no 8, 1105-1115 p.Article in journal (Refereed) Published
Abstract [en]

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S(II)) and believed to play a role in ascending urinary tract infections (S(I)). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S(II) and S(I) pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S(II) pili and 21 pN for S(I) pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX (II)) indicated that absence of the sfaX (II) gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S(I) are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.

Keyword
Fimbriae, uropathogenic escherichia coli, bond breaking, unfolding, optical tweezers
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32913 (URN)10.1007/s00249-009-0552-8 (DOI)000279194500001 ()19885656 (PubMedID)
Available from: 2010-03-30 Created: 2010-03-30 Last updated: 2017-12-12Bibliographically approved
3. Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon
Open this publication in new window or tab >>Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon
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2009 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 46, no 3, 150-158 p.Article in journal (Refereed) Published
Abstract [en]

We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17kDa genes, typically located downstream (300 – 3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaXII reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaXII gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did not reveal effects on SfaII fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaXII resulted in altered expression of other surface components. Moreover, we define a new gene, sfaYII, coding for a putative phosphodiesterase that is located in between the sfaXII gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaYII in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaYII can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.

Place, publisher, year, edition, pages
Elsevier Ltd, 2009
Keyword
NMEC, Fimbrial genes, Regulation, sfaXII, sfaYII
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-22279 (URN)10.1016/j.micpath.2008.12.001 (DOI)19103276 (PubMedID)
Available from: 2009-05-05 Created: 2009-05-04 Last updated: 2017-12-13Bibliographically approved
4. Growth phase regulated expression of sfaXII is negatively influenced by the RpoS sigma factor and the Hfq RNA chaperone
Open this publication in new window or tab >>Growth phase regulated expression of sfaXII is negatively influenced by the RpoS sigma factor and the Hfq RNA chaperone
(English)Manuscript (Other academic)
Abstract [en]

The regulatory gene sfaXII in the S fimbrial gene cluster of the newborn meningitis E. coli isolate IHE3034 is expressed in a growth phase dependent fashion with the highest levels at the onset of the stationary phase. It is then mainly transcribed from a promoter proximal to the gene. We have assessed the potential influence by the stationary phase sigma factor, σS, in the sfaXII regulation. In contrast to the stimulatory role commonly seen with stationary phase induced genes, we found that σS exerted a strong repressive effect on sfaXII transcription. Tests with an hfq mutant strain suggested that also the RNA chaperon Hfq caused negative regulation of sfaXII transcription. In both cases the effects were considered indirect via some other regulatory factor(s). Results from a transposon insertion mutagenesis experiment indicated that it may be possible to identify additional genes involved in sfaXII regulation.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-22281 (URN)
Available from: 2009-05-04 Created: 2009-05-04 Last updated: 2012-02-02Bibliographically approved

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