Type IV pilus assembly in Pseudomonas aeruginosa over a broad range of cyclic di-GMP concentrations

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103ΔfimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the ΔfimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103ΔfimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle, type IV pili, over a broad range of c-di-GMP concentrations. Assembly of pili at low c-di-GMP concentrations requires a polarly localized c-di-GMP binding protein and phosphodiesterase, FimX; this requirement for FimX is bypassed at high c-di-GMP concentrations. Thus, P. aeruginosa can assemble the same surface organelle in distinct ways for motility or adhesion under very different environmental conditions.     

Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa

Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a DeltafimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP-FimXDeltaREC, unlike RFP-FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although DeltafimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia.     

The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.     

Lrp-DNA complex stability determines the level of ON cells in type P fimbriae phase variation

F165(1) and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors that play a key role in the establishment of disease caused by extraintestinal Escherichia coli (ExPEC) strains. They are both under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Our present study demonstrates that the high level of ON cells found during F165(1) phase variation is due to altered stability of the DNA complex formed by the leucine-responsive regulatory protein (Lrp) at its repressor binding sites 1-3; after each cell cycle, complex formation is also modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp towards its activator binding sites 4-6. Furthermore, we identified two nucleotides (T490, G508) surrounding the Lrp binding site 1 that are critical to maintaining a high OFF to ON switch rate during F165(1) phase variation, as well as switching Pap fimbriae towards the OFF state.     

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.     

Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.     

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.     

FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa

Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.     

肠外致病性大肠杆菌致病机制及公共卫生学意义

致病性大肠杆菌包括肠致病性大肠杆菌(intestinal pathogenic Escherichia coli, IPEC)和肠外致病性大肠杆菌(extraintestinalpathogenicE.coli,ExPEC),可引起人和动物多种感染性疾病。ExPEC主要在肠道外其他组织脏器定殖并导致感染,包括尿道致病性大肠杆菌(uropathogenicE.coli, UPEC)、新生儿脑膜炎大肠杆菌(newborn meningitis E. coli, NMEC)和禽致病性大肠杆菌(avian pathogenic E. coli, APEC)。人源ExPEC (UPEC和NMEC)主要引起人尿道感染、肾盂肾炎和新生儿脑膜炎,而APEC可导致禽类的大肠杆菌病,造成家禽业的巨大经济损失。另外,乳腺致病性大肠杆菌(mammary pathogenic E. coli, MPEC)和猪源ExPEC可导致奶牛乳房炎、猪的肺炎及急性败血症等病症。研究发现,ExPEC类菌株在基因组结构上很相似,与IPEC本质区别在于致病机制不同,ExPEC具有很多相同的毒力基因和耐药基因,而且动物源ExPEC...     

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.