Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the most common etiologic agent of uncomplicated urinary tract infection (UTI). An important mechanism of gene regulation in UPEC is phase variation that involves inversion of a promoter-containing DNA element via enzymatic activity of tyrosine recombinases, resulting in biphasic, ON or OFF expression of target genes. The UPEC reference strain CFT073 has five tyrosine site-specific recombinases that function at two previously characterized promoter inversion systems, fimS and hyxS Three of the five recombinases are located proximally to their cognate target elements, which is typical of promoter inversion systems. The genes for the other two recombinases, IpuA and IpuB, are located distal from these sites. Here, we identified and characterized a third phase-variable invertible element in CFT073, ipuS, located proximal to ipuA and ipuB The inversion of ipuS is catalyzed by four of the five CFT073 recombinases. Orientation of the element drives transcription of a two-gene operon containing ipuR, a predicted LuxR-type regulator, and upaE, a predicted autotransporter. We show that the predicted autotransporter UpaE is surface located and facilitates biofilm formation as well as adhesion to extracellular matrix proteins in a K-12 recombinant background. Consistent with this phenotype, the ipuS ON condition in CFT073 results in defective swimming motility, increased adherence to human kidney epithelial cells, and a positive competitive kidney colonization advantage in experimental mouse UTIs. Overall, the identification of a third phase switch in UPEC that is regulated by a shared set of recombinases describes a complex phase-variable virulence network in UPEC.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). ON versus OFF phase switching by inversion of small DNA elements at two chromosome sites in UPEC regulates the expression of important virulence factors, including the type 1 fimbria adhesion organelle. In this report, we describe a third invertible element, ipuS, in the UPEC reference strain CFT073. The inversion of ipuS controls the phase-variable expression of upaE, an autotransporter gene that encodes a surface protein involved in adherence to extracellular matrix proteins and colonization of the kidneys in a murine model of UTI.

Strain- and host species-specific inflammasome activation, IL-1ß release, and cell death in macrophages infected with uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1ß (IL-1ß) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1ß secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1ß secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15.

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and impact of the study: A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium.

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 A and belonged to space groups P2(1), P2(1)2(1)2 and C2, respectively.

Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa.

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.

Capacitation-like changes in equine spermatozoa throughout the cryopreservation process.

Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P<0.001), with a corresponding decrease in the proportion of spermatozoa displaying the F staining pattern (2.5%; P<0.001). Further analysis of CTC fluorescence staining patterns showed that there was a major decrease in the proportion of F pattern spermatozoa corresponding to an increase in B pattern spermatozoa following removal of seminal plasma after centrifugation and resuspension in freezing medium. There was a further decline in the proportion of F pattern spermatozoa, corresponding to increases in B and AR pattern spermatozoa, after the freezing and thawing steps. Resuspension of centrifuged spermatozoa in homologous seminal plasma did not induce capacitation-like changes. These data indicate that the process of freezing and thawing stallion semen induces capacitation-like changes in spermatozoa and that most of the change is brought about by removal of seminal plasma, with further changes induced by the actual freezing and thawing step.

DNA microarray analysis of fim mutations in Escherichia coli.

Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type 1 fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated with virulence in the urinary tract. A typical type 1 fimbriated bacterium has up to 500 fimbriae on its surface, with each fimbria consisting of approximately 1000 individual subunits. This equates to approximately 8% of the total cellular protein and is potentially a significant resource drain for the cell. Here we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type 1 fimbriae. Changes in fim expression correlated directly with alterations in colony morphology. Deletion of the entire fim gene cluster resulted in the converse expression of another surface protein Antigen 43 (Ag43). Specific deletion of the fimH gene did not affect expression of other fim genes or Ag43, but did dramatically reduce the number of fimbriae expressed on the cell surface. The use of high-resolution oligonucleotide arrays for defining points of transcription initiation and termination is also demonstrated.

Novel Zn(2+)-chelating peptides selected from a fimbria-displayed random peptide library.

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of approximately 40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn(2+). By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn(2+) were enriched. None of the isolated sequences showed similarity to known Zn(2+)-binding proteins, indicating that completely novel Zn(2+)-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn(2+)-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.

FimH-mediated autoaggregation of Escherichia coli.

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N-terminal receptor-binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30 degrees C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild-type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYP020B were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii.

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli.

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.

Coordinate gene regulation by fimbriae-induced signal transduction.

Fimbriae are thread-like polymers displayed in large amounts on the bacterial surface and used by many pathogens to attach to receptors on host tissue surfaces. Fimbriae contain disulfide bridges, contrary to many Escherichia coli surface proteins produced in bulk amounts. Here we investigate whether fimbriae expression can affect expression of other genes. Analysis of gene expression in two E.coli strains, differing in the fim locus, indicated the flu gene to be affected. The flu gene encodes the antigen 43 (Ag43) surface protein, specifically involved in bacterial aggregation, and microcolony and biofilm formation. Ag43 production is repressed by the global regulator OxyR, which monitors the cell's thiol-disulfide status. Only the thiol form of OxyR represses Ag43 production. We demonstrate that production of several different disulfide-containing fimbriae results in the abolition of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated with the expression of other disease- and colonization-related genes.

Biofilm formation in a hydrodynamic environment by novel fimh variants and ramifications for virulence.

Type 1 fimbriae are surface-located adhesion organelles of Escherichia coli that are directly associated with virulence of the urinary tract. They mediate D-mannose-sensitive binding to different host surfaces by way of the minor fimbrial component FimH. Naturally occurring variants of FimH that bind strongly to terminally exposed monomannose residues have been associated with a pathogenicity-adaptive phenotype that enhances E. coli colonization of extraintestinal locations such as the urinary tract. The FimH adhesin also promotes biofilm formation in a mannose-inhibitable manner on abiotic surfaces under static growth conditions. In this study, we used random mutagenesis combined with a novel selection-enrichment technique to specifically identify mutations in the FimH adhesin that confer on E. coli the ability to form biofilms under hydrodynamic flow (HDF) conditions. We identified three FimH variants from our mutant library that could mediate an HDF biofilm formation phenotype to various degrees. This phenotype was induced by the cumulative effect of multiple changes throughout the receptor-binding region of the protein. Two of the HDF biofilm-forming FimH variants were insensitive to mannose inhibition and represent novel phenotypes not previously identified in naturally occurring isolates. Characterization of our enriched clones revealed some similarities to amino acid alterations that occur in urinary tract infection (UTI) strains. Subsequent screening of a selection of UTI FimH variants demonstrated that they too could promote biofilm formation on abiotic surfaces under HDF conditions. Interestingly, the same correlation was not observed for commensal FimH variants. FimH is a multifaceted protein prone to rapid microevolution. In addition to its previously documented roles in adherence and invasion, we have now demonstrated its function in biofilm formation on abiotic surfaces subjected to HDF conditions. The study indicates that UTI FimH variants possess adaptations that enhance biofilm formation and suggests a novel role for FimH in UTIs associated with medical implants such as catheters.

Bacterial adhesins: function and structure.

Specific adhesion to host tissue cells is an essential virulence factor of most bacterial pathogens. The fundamental processes that determine bacterial attachment to host tissue surfaces are mediated by microbial adhesins. Host specificity and tissue tropism are characteristics exhibited by different bacteria and are determined (at least in part) by the interaction between adhesins and their complementary receptors on host cell surfaces. A detailed picture of how bacteria are able to target to various receptors is emerging. A large number of bacterial adhesins with individual receptor specificities have been identified. Furthermore, recent research has shown that individual adhesins are prone to rapid microevolution that results in changes in the receptor specificity of individual adhesins. Microbial adhesins are often assembled into complex polymeric organelle structures, however non-organelle adhesins linked to the cell surface as monomers or simple oligomers also exist. This review gives an overview of bacterial adhesins and focuses on some general aspects of their biogenesis and role in bacterial colonization of host cell surfaces and as virulence factors.

Fimbriae-assisted bacterial surface display of heterologous peptides.

The display of peptide segments on the surface of bacteria offers many new and exciting applications in biotechnology and medical research. Fimbria-assisted display of heterologous sequences is a paradigm for chimeric organelle display on bacteria. Fimbriae are particularly attractive candidates for epitope display for several reasons: (1) they are present in extremely high numbers at the cell surface, (2) they are strong immunogens, (3) they possess inherent adhesive properties, and (4) they can be easily purified. The majority of work dealing with fimbria-assisted peptide display has been focused on the development of recombinant vaccines. A number of different fimbrial types have been used to display immune-relevant sectors of various foreign proteins. Chimeric fimbrial vaccines can be used in the context of purified proteins, however the potential also exists to exploit this technology for the development of live recombinant vaccines. Work has also been performed demonstrating the amenability of fimbriae towards the powerful technology of random peptide display. This review summarises the current state of research in this field.

Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens.

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.

Expression and purification of the mannose recognition domain of the FimH adhesin.

Type 1 fimbriae have been shown to be specifically required for Escherichia coli colonisation and pathogenesis of the urinary tract. These structural organelles mediate specific adhesion to alpha-D-mannosides by virtue of the FimH adhesin. FimH is a two-domain protein in which the N-terminal domain contains the receptor-binding site and the C-terminal domain is required for organelle integration. To date, FimH has only been isolated as a complex with the system-specific chaperone FimC. Here we report that a functional form of the FimH receptor-binding domain can be readily isolated and characterised by replacing the C-terminal domain with a histidine tag.

Functional flexibility of the FimH adhesin: insights from a random mutant library.

Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. Naturally occurring variants of the FimH protein have been selected in nature for their ability to recognize specific receptor targets. In particular, variants that bind strongly to terminally exposed monomannose residues have been associated with a pathogenicity-adaptive phenotype that enhances E. coli colonization of extraintestinal locations such as the urinary bladder. In this study we have used random mutagenesis to specifically identify nonselective mutations in the FimH adhesin which modify its binding phenotype. Isogenic E. coli clones expressing FimH variants were tested for their ability to bind yeast cells and model glycoproteins that contain oligosaccharide moieties rich in either terminal monomannose, oligomannose, or nonmannose residues. Both the monomannose- and the oligomannose-binding capacity of type 1 fimbriae could be altered by minor amino acid changes in the FimH protein. The monomannose-binding phenotype was particularly sensitive to changes, with extensive differences in binding being observed in comparison to wild-type FimH levels. Different structural alterations were able to cause similar functional changes in FimH, suggesting a high degree of flexibility to target recognition by this adhesin. Alteration of residue P49 of the mature FimH protein, which occurs within the recently elucidated carbohydrate-binding pocket of FimH, completely abolished its function. Amino acid changes that increased the binding capacity of FimH were located outside receptor-interacting residues, indicating that functional changes relevant to pathogenicity are likely to be due to conformational changes of the adhesin.

Sequestration of zinc oxide by fimbrial designer chelators.

Type 1 fimbriae are surface organelles of Escherichia coli. By engineering a structural component of the fimbriae, FimH, to display a random peptide library, we were able to isolate metal-chelating bacteria. A library consisting of 4 x 10(7) independent clones was screened for binding to ZnO. Sequences responsible for ZnO adherence were identified, and distinct binding motifs were characterized. The sequences selected exhibited various degrees of affinity and specificity towards ZnO. Competitive binding experiments revealed that the sequences recognized only the oxide form of Zn. Interestingly, one of the inserts exhibited significant homology to a specific sequence in a putative zinc-containing helicase, which suggests that searches such as this one may aid in identifying binding motifs in nature. The zinc-binding bacteria might have a use in detoxification of metal-polluted water.