fimB Expression Research Articles

Type 1 fimbrial phase variation in multidrug-resistant asymptomatic uropathogenic Escherichia coli clinical isolates upon adherence to HTB-4 cells.

The adherence of bladder uroepithelial cells, subsequent expression, and regulation of type 1 fimbrial genes (key mediator of attachment) in clinical multidrug-resistant uropathogenic Escherichia coli (MDR-UPECs) isolated from individuals with asymptomatic bacteriuria (ABU) remain unexplored till date. Therefore, this study aimed to investigate the underlying molecular mechanisms associated with the adherence of clinical MDR-ABU-UPECs to human a uroepithelial cell line (HTB-4), both in the absence and presence of D-Mannose. These investigations focused on phase variation, expression, and regulation of type 1 fimbriae and were compared to a prototype ABU-strain (E. coli 83972) and symptomatic MDR-UPECs. Discordant to the ABU prototype strain, MDR-ABU-UPECs exhibited remarkable adhesive capacity that was significantly reduced after D-mannose exposure, fairly like the MDR symptomatic UPECs. The type 1 fimbrial phase variation, determined by the fim switch analysis, asserted the statistically significant incidence of "both OFF and ON" orientation among the adherent MDR-ABU-UPECs with a significant reduction in phase-ON colonies post-D-mannose exposure, akin to the symptomatic ones. This was indicative of an operative and alternating type 1 fimbrial phase switch. The q-PCR assay revealed a coordinated action of the regulatory factors; H-NS, IHF, and Lrp on the expression of FimB and FimE recombinases, which further controlled the function of fimH and fimA genes in ABU-UPECs, similar to symptomatic strains. Therefore, this study is the first of its kind to provide an insight into the regulatory crosstalk of different cellular factors guiding the adhesion of ABU-UPECs to the host. Additionally, it also advocated for the need to accurately characterize ABU-UPECs.

RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12.

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE, and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.

Salicylate increases the expression of marA and reduces in vitro biofilm formation in uropathogenic Escherichia coli by decreasing type 1 fimbriae expression

Escherichia coli is one of the most frequent bacteria implicated in biofilm formation, which is a dynamic process whose first step consists in bacteria adhesion to surfaces through type 1 fimbriae. Salicylate induces a number of morphological and physiological alterations in bacteria including the activation of the transcriptional regulator MarA. In this report the effects of salicylate on biofilm formation and their relationship with MarA were studied. An inverse relationship was observed between in vitro biofilm formation and salicylate concentration added to the culture medium. Salicylate increases the expression of marA and decreases the expression of fimA and fimB genes in the wild-type strain. In addition, the fimA and fimB expression was decreased in a MarR mutant in which marA was also overexpressed. In conclusion, the expression of type 1 fimbriae in presence of salicylate may be regulated by the level of marA expression through fimB regulator, albeit through neither the ompX nor the tolC genes.

SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (OSA1 and OSA2), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap OSA1 and OSA2, respectively. Mutations that disrupt either OSA1 or OSA2 eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ∼1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

Inactivation of ibeA and ibeT results in decreased expression of type 1 fimbriae in extraintestinal pathogenic Escherichia coli strain BEN2908.

IbeA in extraintestinal pathogenic Escherichia coli (ExPEC) strains was previously described for its role in invasion. Here we investigated the role of IbeA and IbeT, encoded by a gene located downstream of ibeA, in the adhesion of the avian ExPEC strain BEN2908 to human brain microvascular endothelial cells (HBMEC). The DeltaibeA mutant was less adhesive to HBMEC than the wild-type strain BEN2908 was. Because strain BEN2908 also expresses type 1 fimbriae, we measured the adhesion specifically due to IbeA by comparing the adhesive properties of a Deltafim derivative of strain BEN2908 to those of a double Deltafim DeltaibeA mutant. No differences were observed, indicating that the reduction of adhesion in BEN2908 DeltaibeA could be due to a decrease in type 1 fimbria expression. We indeed showed that the decreased adhesion of BEN2908 DeltaibeA was correlated with a decrease in type 1 fimbria expression. Accordingly, more bacteria had a fim promoter orientated in the off position in a culture of BEN2908 DeltaibeA than in a culture of BEN2908. Expression of fimB and fimE, two genes encoding recombinases participating in controlling the orientation of the fim promoter, was decreased in BEN2908 DeltaibeA. A reduction of type 1 fimbria expression due to a preferential orientation of the fim promoter in the off position was also seen in an ibeT mutant of strain BEN2908. We finally suggest a role for IbeA and IbeT in modulating the expression of type 1 fimbriae through an as yet unknown mechanism.

Multiple co‐regulatory elements and IHF are necessary for the control of fimB expression in response to sialic acid and N‐acetylglucosamine in Escherichia coli K‐12

Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.

Distant cis-active sequences and sialic acid control the expression of fimB in Escherichia coli K-12.

The phase variation of type 1 fimbriation in Escherichia coli is controlled by the inversion of a 314 bp element of DNA, determined by FimB (switching in both directions) or FimE (switching from the ON-to-OFF orientation predominantly), and influenced by auxiliary factors IHF, Lrp and H-NS. The fimB gene is separated from the divergently transcribed yjhATS operon by a large (1.4 kbp) intergenic region of unknown function. Here, we show that fimB expression is regulated by multiple cis-active sequences that lie far upstream (>600 bp) of the transcription start sites for the recombinase gene. Two regions characterized further (regions 1 and 2) show sequence identity, and each coincides with a methylation-protected Dam (5'-GATC) site. Regions 1 and 2 apparently control fimB expression by an antirepression mechanism that involves additional sequences proximal to yjhA. Region 1 encompasses a 27 bp DNA sequence conserved upstream of genes known (nanAT ) or suspected (yjhBC) to be involved in sialic acid metabolism, and we show that FimB expression and recombination are suppressed by N-acetylneuraminic acid. We propose that E. coli recognizes the amino sugars as a harbinger of potential host defence activation, and suppresses the expression of type 1 fimbriae in response.

Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS.

The H-NS protein is a major component of the Escherichia coli nucleoid. Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter. We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping. In beta-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant. In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene. Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription. The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production. However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype. These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion.

Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS.

Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS.

The site-specific recombination system regulating expression of the type 1 fimbrial subunit gene of Escherichia coli is sensitive to changes in DNA supercoiling.

We have studied the effect of altering the in vivo level of DNA supercoiling on the phase-variable expression of the Escherichia coli fimA gene. Transcription from the fimA promoter was unaffected by changes in DNA supercoiling whether caused by the introduction of a topA::Tn10 mutation or by inhibition of DNA gyrase with the antibiotic novobiocin. However, inversion of the fimA promoter fragment was altered in response to perturbation of DNA supercoiling. Specifically, inactivation of topA reduced the rate of promoter fragment inversion in both the ON-to-OFF and the OFF-to-ON directions. This effect correlated with the loss of functional topA and not with the global level of DNA supercoiling. Inhibition of DNA gyrase introduced a bias in favour of the OFF-to-ON inversion; the ON-to-OFF inversion was affected only slightly. Changes in expression of fimB, the gene coding for the recombinase that catalyses fimA promoter fragment inversion in the strains used in this study, did not correlate with effects on fimA phase variation: we found that transcription of fimB was inhibited by loss of functional topA and was enhanced by inhibition of DNA gyrase in a manner that correlated well with the global level of in vivo DNA supercoiling. A model is presented to account for the effects of lost topoisomerase function on fimA gene expression.

Localization of promoters in the fim gene cluster and the effect of H-NS on the transcription of fimB and fimE.

The expression of type 1 fimbriae in Escherichia coli undergoes phase variation in which individual bacteria switch between a fimbriated and non-fimbriated state. The transition from one state to the other is caused by inversion of a DNA segment containing the promoter for the fimA gene. The orientation of the invertible segment is controlled by two proteins, FimB and FimE, which mediate an on/off and off only orientation of the segment, respectively. In this study we have mapped the 5' termini of the fimB, fimE and fimA transcripts. Furthermore, we show that expression of fimB and fimE is strongly influenced by the H-NS nucleoid protein.

Localization of promoters in the fim gene cluster and the effect of H-NS on the transcription of fimB and fimE

The expression of type 1 fimbriae in Escherichia coli undergoes phase variation in which individual bacteria switch between a fimbriated and non-fimbriated state. The transition from one state to the other is caused by inversion of a DNA segment containing the promoter for the fimA gene. The orientation of the invertible segment is controlled by two proteins, FimB and FimE, which mediate an on/off and off only orientation of the segment, respectively. In this study we have mapped the 5′ termini of the fimB, fimE and fimA transcripts. Furthermore, we show that expression of fimB and fimE is strongly influenced by the H-NS nucleoid protein.