PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli

Abstract

SummaryRecent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimuriumplasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu 82 and Ile 83 of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.

Demonstration of regulatory cross-talk between P fimbriae and type 1 fimbriae in uropathogenic Escherichia coli

“…Genomic DNA from each isolate and plasmid DNA were digested overnight with HindIII or BglII restriction endonucleases, resolved on agarose gels and blotted onto nitrocellulose (Hybond-N + ; Amersham) using the method described in Sambrook et al (1989). Two probes were used for hybridization to increase the probability of detecting all P-related clusters: the papF probe was amplified with primers 196P and 197P (Table 4), and the papB probe was amplified with papB1 and papB2, described in Holden et al (2001); both were labelled with fluorescein-dUTP using the ECF Random Prime Labelling kit (Amersham Biosciences). Following hybridization, the membrane was washed with high-stringency buffers and the signal was amplified using an ECF Signal Amplification System (Amersham Biosciences).…”

“…FimBpromoted recombination was measured using a minimal MOPS XGal plating assay, essentially as described previously (Gally et al, 1993;Holden et al, 2001). In brief, for off-to-on recombination frequencies, a white colony was selected, diluted in PBS to a suitable cell number, and plated onto minimal MOPS X-Gal agar.…”

“…As described, these Lrp-binding mutations do not completely lock P phase variation, and so a proportion of the bacterial population does still undergo phase transition. A completely locked on P regulatory region is likely to demonstrate an increased repression of FimB switching, perhaps achieving the complete repression observed for induced levels of the regulator cloned on a plasmid (Holden et al, 2001).A further demonstration of the repression of type 1 fimbriae by P/S clusters was provided by using the fim : : gfp + fusion in different E. coli 536 mutants that had had fim, prf, sfa clusters deleted in various combinations. This work showed that the proportion of bacteria expressing type 1 fimbriae increased by 2?3-fold in a strain that had had the Prf and S clusters deleted, and by 1?8-fold in a strain deleted for Prf.…”

“…Phase variation of type 1 fimbrial expression requires the activity of two recombinases; FimB promotes inversion in both directions, whereas FimE mediates predominantly on-to-off inversion (Gally et al, 1996;Klemm, 1986). Previous work has demonstrated that a regulator from the P-fimbrial gene cluster, PapB, when expressed from a plasmid, is able to prevent inversion of the fim switch (Holden et al, 2001;Xia et al, 2000). Cross-talk by PapB has been shown to occur by inhibition of FimBpromoted recombination together with increasing fimE expression (Xia et al, 2000).…”

“…The increase in FimE levels will turn the fim switch off and the inhibition of FimB-promoted recombination will maintain the fim switch in the off orientation. Subsequent work has shown that the closest homologues of PapB from other related fimbrial clusters have a similar activity (Holden et al, 2001). As E. coli clinical isolates often carry multiple adhesin gene clusters, such co-ordinate control between clusters would prevent co-expression of adhesins at the single-cell level.…”