| 1. |
- Myint, Si Lhyam, et al.
(författare)
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Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae
- 2021
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier. - 0304-4165 .- 1872-8006. ; 1865:7
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Tidskriftsartikel (refereegranskat)abstract
- Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.
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| 2. |
- Seibt, Henrik, et al.
(författare)
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Elevated levels of VCA0117 in response to external signals activates type VI secretion in Vibrio cholerae A1552
- 2020
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Ingår i: Environmental Microbiology. - : John Wiley & Sons. - 1462-2912 .- 1462-2920. ; 22:10, s. 4409-4423
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co‐occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) – a σ54‐transcriptional activator – is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ54‐promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system.
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| 3. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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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
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:3, s. 150-158
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region ofthe main S fimbrial operon
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:3, s. 150-158
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Tidskriftsartikel (refereegranskat)abstract
- We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant inthe newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17-kDa 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 genefusions we found that different environmental conditions commonly affecting expression of fimbrialgenes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of themain 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 downregulationby the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did notreveal 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 caninfluence 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.
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| 5. |
- Sjöström, Annika E, 1963-, et al.
(författare)
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Growth phase regulated expression of sfaXII is negatively influenced by the RpoS sigma factor and the Hfq RNA chaperone
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Sjöström, Annika E., 1963-
(författare)
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Pathogenecity-associated genes modulate Escherichia coli adhesion and motility
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:5, s. 243-252
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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