Pap Pili Research Articles

English

The aim of this study was to gain further insight into the interplay between both extended spectrum β-lactamase (ESBL) and AmpC enzymes and different virulence factors (VFs) among Escherichia coli (E. coli) isolated from Mansoura University Hospitals, Egypt. For this purpose 100 E. coli isolates were collected from different clinical sources. All isolates were investigated for production of ESBL and AmpC enzymes. The prevalence of VF encoding genes including: KPsMII (group2 capsule synthesis), FyuA (ferric yersiniabactin uptake), Afa/dra (Dr-binding a fimbrial adhesins), PapA (P fimbriae), PapC (formation of digalactoside-binding Pap pili), iutA (aerobactin receptor) and BssS (biofilm formation) and associations of these genes with both enzyme types were analyzed by polymerase chain reaction. ESBL was produced by 37% of the isolates while AmpC enzyme was produced by 29%. Virulence genes prevalence among ESBL phenotypes were 43.2% KPsMII, 51.35% FyuA, 62.1% Afa/dra, 43.2% PapA, 16.2% PapC, 67.56% iutA and 78.4% BssS. Regarding AmpC phenotypes, the prevalence of  virulence genes were: 51.7% KPsMII, 86.2% FyuA, 68.96% Afa/dra, 55.17% PapA, 24.1% PapC, 82.7% iutA and 100% BssS. Of the tested virulence factor encoding genes, BssS, FyuA and iutA were significantly more prevalent among AmpC producers. In addition, AmpC producers exhibited a statistically significant higher prevalence of multivirulence (MV) (MV≥4) than ESBL producers. Furthermore, AmpC phenotypes showed very significantly higher expression (P>0.01) of BssS gene than ESBLs phenotypes. Our results suggest a correlation between AmpC phenotypes and production of some factors that are reported to be involved in the virulence of E. coli. Key words: extended spectrum β-lactamase, AmpC enzymes, virulence factors, Escherichia coli.

Capture of uropathogenic E. coli by using synthetic glycan ligands specific for the pap-pilus.

Biotinylated mono- and biantennary di-/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di-/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galβ(1→4)Glc and GalNHAcα(1→4)Galβ(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin-coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galβ(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.

Solution Structure of Escherichia coli PapI, a Key Regulator of the Pap Pili Phase Variation

Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K d in the 10 μM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings.

Mechanism of fibre assembly through the chaperone–usher pathway

The chaperone-usher pathway directs the formation of adhesive surface fibres in numerous pathogenic Gram-negative bacteria. The fibres or pili consist exclusively of protein subunits that, before assembly, form transient complexes with a chaperone in the periplasm. In these chaperone:subunit complexes, the chaperone donates one beta-strand to complete the imperfect immunoglobulin-like fold of the subunit. During pilus assembly, the chaperone is replaced by a polypeptide extension of another subunit in a process termed 'donor strand exchange' (DSE). Here we show that DSE occurs in a concerted reaction in which a chaperone-bound acceptor subunit is attacked by another chaperone-bound donor subunit. We provide evidence that efficient DSE requires interactions between the reacting subunits in addition to those involving the attacking donor strand. Our results indicate that the pilus assembly platforms in the outer membrane, referred to as ushers, catalyse fibre formation by increasing the effective concentrations of donor and acceptor subunits.

Regulation of the Pap Epigenetic Switch by CpxAR: Phosphorylated CpxR Inhibits Transition to the Phase ON State by Competition with Lrp

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

The intricate workings of a bacterial epigenetic switch.

Bacteria have developed epigenetic mechanisms to control the reversible Off-to-On switching of cell surface structures such as pyelonephritis-associated pili (PAP). The pap pili switch is primarily controlled by the global regulator leucine-responsive regulatory protein (Lrp), the local regulator PapI, and DNA adenine methylase (Dam). There are two sets of binding sites for Lrp in the pap regulatory region: promoter proximal sites 1,2,3 and promoter distal sites 4,5,6. The pilin promoter proximal (GATCprox) and distal (GATCdist) targets for Dam are located within Lrp binding sites 2 and 5, respectively. In the Off state, Lrp binds cooperatively to sites 1,2,3 overlapping the papBA pilin promoter, shutting off pilin transcription, and blocking methylation of GATCprox. Binding of Lrp at sites 1,2,3, together with methylation of GATCdist, reduces the affinity of Lrp for sites 4,5,6, preventing simultaneous binding of Lrp at sites 4,5,6 upstream. Switching to the phase. On state requires the environmentally regulated PapI co-regulator, which increases the affinity of Lrp for sites 5 and 2. PapI binds specifically to Lrp-pap DNA complexes via binding with Lrp as well as contact with DNA sequences within pap sites 5 and 2. Directionality in switching from Off to On appears to be due to methylation of GATCprox, which prevents formation of the PapI-Lrp-pap site 2 ternary complex. A switch model is presented in which DNA replication is proposed to play a critical role by generating a hemimethylated GATCdist site and displacing Lrp from sites 1,2,3. This facilitates methylation of GATCprox and binding of PapI-Lrp to sites 4,5,6, with subsequent activation of pap transcription. The first gene product of the pap operon, PapB, positively regulates papI transcription, resulting in a positive feedback loop that helps maintain the On state. The pap switch is environmentally regulated by a number of factors including the CpxAR two-component regulatory system, the Histone-like nucleoid structuring protein H-NS, and cAMP-Catabolite Gene Activator Protein (CAP), which all involve binding of regulatory binding proteins to pap DNA sequences with subsequent alteration of PapI and Lrp binding. The Pap switch mechanism, with interesting variations, is conserved among a number of enteric bacteria, controlling expression of many unrelated pili-adhesin complexes.

The Pix pilus adhesin of the uropathogenic Escherichia coli strain X2194 (O2 : K(-): H6) is related to Pap pili but exhibits a truncated regulatory region.

Adhesins provide a major advantage for uropathogenic Escherichia coli in establishing urinary tract infections (UTIs). A novel gene cluster responsible for the expression of a filamentous adhesin of the pyelonephritogenic E. coli strain X2194 has been identified, molecularly cloned, and characterized. The 'pix operon' contains eight open reading frames which exhibit significant sequence homology to corresponding genes in the pap operon encoding P pili, the prevalent E. coli adhesins in non-obstructive acute pyelonephritis in humans. Although a pixB gene corresponding to the PapB regulator was identified, a papI homologue could not be found in the pix operon. Instead, a fragment of the R6 gene of the highly uropathogenic E. coli strain CFT073 was identified upstream of pixB. The R6 gene is located in a pathogenicity island containing several pilus-encoding sequences and shows homology to a transposase of Chelatobacter heintzii. In a pixA-lacZ fusion system it was demonstrated that the expression of Pix pili is regulated at the transcriptional level by the R6 gene sequence. A significantly reduced transcription was observed by deleting this fragment and by lowering the growth temperature from 37 to 26 degrees C. In contrast to other filamentous adhesin systems, Pix pili are mainly expressed in the steady state growth phase and were not repressed by the addition of glucose.

Escherichia coli DegP protease cleaves between paired hydrophobic residues in a natural substrate: the PapA pilin.

The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report here the identification of the major pilin subunit of the Pap pilus, PapA, as a natural DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. Using overlapping peptide arrays, we identified three regions in PapA that are preferentially cleaved by DegP. A 7-mer peptide was found to be a suitable substrate for cleavage by DegP in vitro. In vitro proteolysis of model peptide substrates revealed that cleavage is dependent upon the presence of paired hydrophobic amino acids; moreover, cleavage was found to occur between the hydrophobic residues. Finally, we demonstrate that the conserved carboxyl-terminal sequence in pilin subunits, although not a cleavage substrate for DegP, activates the protease and we propose that the activating peptide is recognized by DegP's PDZ domains.

DNA methylation affects the cell cycle transcription of the CtrA global regulator in Caulobacter.

The Caulobacter chromosome changes progressively from the fully methylated to the hemimethylated state during DNA replication. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. Using analysis of CcrM mutant strains, transcriptional reporters integrated at different sites on the chromosome, and a ctrA P1 mutant, we demonstrate that transcription of the P1 promoter is repressed by DNA methylation. Moreover moving the native ctrA gene to a position near the chromosomal terminus, which delays the conversion of the ctrA promoter from the fully to the hemimethylated state until late in the cell cycle, inhibited ctrA P1 transcription, and altered the time of accumulation of the CtrA protein and the size distribution of swarmer cells. Together, these results show that CcrM-catalyzed methylation adds another layer of control to the regulation of ctrA expression.

The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215.

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP-CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP-dependent activation of papBA requires binding of the leucine-responsive regulatory protein (Lrp) at binding sites that extend from -180 to -149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP-dependent promoters. Also, like class I promoters, the C-terminal domain of the alpha-subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an 'oriented heterodimer' approach with wild-type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter-distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H-NS. The results presented here show that AR1 of the promoter-proximal CAP subunit was required for papBA transcription even in the absence of the histone-like protein H-NS. These results show that the promoter-proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C-terminal domain of the alpha-subunit of RNA polymerase.

Characterization of the Escherichia coli AF/R1 pilus operon: novel genes necessary for transcriptional regulation and for pilus-mediated adherence.

We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E. coli strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the E. coli strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA. AF/R1 pili mediate the adherence to Peyer's patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E. coli. Our observations will facilitate the further study of the phenomena of M-cell adherence.

Formation of DNA methylation patterns: nonmethylated GATC sequences in gut and pap operons.

Most of the adenine residues in GATC sequences in the Escherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44. 5) centered 44.5 bp upstream of the transcription start site of the gutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44. 5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of the pap GATC-I and GATC-II sites.

Regulation of UropathogenicEscherichia coliAdhesin Expression by DNA Methylation

Pap pili play an important role in the pathogenesis of upper urinary tract infections by enabling uropathogenicEscherichia colito adhere to host epithelial cells. Pap pili are coded for by the pyelonephritis-associated pili (pap) operon, which consists of 11 genes required for the expression and assembly of Pap pili. Expression of Pap pili is regulated by a phase variation mechanism in which the pili expression state of the bacterial population is skewed between phase-on (expression positive) and phase-off (expression negative) states. Pap phase variation is controlled by the cooperative binding of leucine-responsive regulatory protein (Lrp) to two sets of Lrp binding sites in the upstream regulatory region of thepapoperon. A single GATC sequence, which is the target site for deoxyadenosine methylase (Dam), is centrally located within each Lrp binding region. Dam plays a critical role in the expression of Pap pili via the formation of DNA methylation patterns. Methylation of GATC-I reduced the affinity of Lrp forpapDNA by about twofold. Conversely, Lrp specifically blocked methylation ofpapGATC-Iin vitro.These data support the hypothesis that Lrp and Dam compete for binding to GATC-I, and are consistent with previous results indicating that methylation of GATC-I is important for stability of the phase-off state.

New tools in an old trade: CS1 pilus morphogenesis.

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.

Renal Scarring by Mannose-Sensitive Adhesin of Escherichia Coli Type 1 Pili

Most Escherichia coli isolates from patients with pyelonephritis possess both pap (mannose-resistant) pili and type 1 (mannose-sensitive) pili. In the experimental pyelonephritis model of rats, the mannose-sensitive-piliated strain caused severe renal scarring, whereas the mannose-resistant or nonpiliated strain did not. Type 1 pili consist of several subunits; one major subunit and other minor subunits. One of the minor subunits, adhesin, is responsible for mannose-sensitive adhesion to eukaryotic cells. The role of adhesin was examined in scar formation after infection with a newly constructed adhesin-deficient mutant which has pilus structure but cannot agglutinate guinea pig erythrocytes. A mutant plasmid, pYMZ84, containing a deletion in the adhesin gene of type 1 pili, failed to agglutinate guinea pig erythrocytes even though the bacteria expressed pili morphologically indistinguishable from those produced by plasmid pSH2, carrying the intact genes for the type 1 pili. E. coli harboring pYMZ84 caused negligible or minimal renal scarring, whereas E. coli harboring pSH2 caused severe renal scarring in rats. These data suggest that the mannose-sensitive adhesin of type 1 pili stimulates renal scarring.

Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor.

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26 degrees C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23 degrees C. The histone-like nucleoid structuring protein H-NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H-NS or a factor regulated by H-NS bound within the pap regulatory region at 23 degrees C but not at 37 degrees C, as evidenced by H-NS-dependent inhibition of methylation of the pap GATC sites designated GATC-I and GATC-II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment containing the GATC-I and GATC-II sites. Moreover, H-NS blocked methylation of these pap GATC sites in vitro: H-NS blocked pap GATC methylation at 1.4 microM but was unable to do so at higher concentrations at which non-specific binding occurred. Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H-NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H-NS with pap regulatory DNA. We hypothesize that a function of H-NS such as oligomerization was altered at 23 degrees C, which enabled H-NS to repress pap gene expression through the formation of a specific nucleoprotein complex.

Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.

Renal scarring by mannose-sensitive adhesin of Escherichia coli type 1 pili.

Most Escherichia coli isolates from patients with pyelonephritis possess both pap (mannose-resistant) pili and type 1 (mannose-sensitive) pili. In the experimental pyelonephritis model of rats, the mannose-sensitive-piliated strain caused severe renal scarring, whereas the mannose-resistant or nonpiliated strain did not. Type 1 pili consist of several subunits; one major subunit and other minor subunits. One of the minor subunits, adhesin, is responsible for mannose-sensitive adhesion to eukaryotic cells. The role of adhesin was examined in scar formation after infection with a newly constructed adhesin-deficient mutant which has pilus structure but cannot agglutinate guinea pig erythrocytes. A mutant plasmid, pYMZ84, containing a deletion in the adhesin gene of type 1 pili, failed to agglutinate guinea pig erythrocytes even though the bacteria expressed pili morphologically indistinguishable from those produced by plasmid pSH2, carrying the intact genes for the type 1 pili. E. coli harboring pYMZ84 caused negligible or minimal renal scarring, whereas E. coli harboring pSH2 caused severe renal scarring in rats. These data suggest that the mannose-sensitive adhesin of type 1 pili stimulates renal scarring.

Mutations affecting mRNA processing and fimbrial biogenesis in the Escherichia coli pap operon

The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in "crew-cut" bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.

Leucine‐responsive regulatory protein plays dual roles as both an activator and a repressor of the Escherichia coli pap fimbrial operon

The expression of the pap pilus operon of Escherichia coli is under a phase-variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states. In this study, we explore the roles of leucine-responsive regulatory protein (Lrp) and the histone-like protein H-NS in the regulation of pap phase variation. Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H-NS, each of which can act independently as transcriptional repressors. Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H-NS does not. In contrast, in the ON state, Lrp, in conjunction with PapI, activates pap transcription. This activation is not merely a result of alleviating the H-NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H-NS and Lrp. Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus. Together, our results show that Lrp functions as a transcriptional activator in phase-ON cells and as a repressor of basal transcription in phase-OFF cells. Because pap phase variation occurs in the absence of H-NS, it is not clear what role this regulatory protein plays in pap gene regulation.