The Complete Genome Sequence of Escherichia coli EC958: A High Quality Reference Sequence for the Globally Disseminated Multidrug Resistant E. coli O25b:H4-ST131 Clone

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum β-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.     

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.     

Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.Disulfide bonds bridging cysteine pairs impart structural stability and protease resistance to secreted and membrane-associated proteins. Most organisms contain specific mechanisms for the formation of disulfide bonds in proteins, a process called oxidative protein folding. In bacteria, this folding process is catalyzed by the disulfide bond family of proteins (18, 22). The best-characterized bacterial disulfide bond machinery is the Escherichia coli K-12 oxidative system, which consists of two enzymes, the periplasmic DsbA and the inner-membrane DsbB (25, 35). DsbA is a monomeric protein comprising a thioredoxin (TRX) domain with an embedded helical insertion and a redox-active CPHC motif (34). This highly oxidizing protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide (2, 4, 5), and as a result, the two cysteines contained in the CPHC catalytic motif become reduced. DsbB reoxidizes this cysteine pair and restores the oxidizing activity of DsbA, enabling it to assist the folding of a new substrate protein (21).The DsbAB oxidative protein folding system plays a well-documented part in bacterial virulence. Several studies have demonstrated a direct role for both enzymes, particularly DsbA, in the biogenesis of virulence factors utilized by bacterial pathogens in various stages of the infection process (19). The protein forming the P-ring of E. coli flagella, FlgI, was one of the first DsbA substrates identified (10) and flagellum-mediated motility was subsequently demonstrated to require the presence of functional DsbA in several gram-negative pathogens, including Salmonella enterica (1), Proteus mirabilis (8), Erwinia carotovora subsp. atroseptica (9), Burkholderia cepacia (17), and Campylobacter jejuni (42). In Yersinia pestis, S. enterica, Shigella flexneri, and enteropathogenic E. coli, deletion of dsbA results in defective type III secretion, a major virulence mechanism employed by these enteric pathogens to manipulate the host during infection. The defect was shown in each case to involve the outer membrane secretin (YscC, SpiA, Spa32, and EscC, respectively), which requires a single intramolecular disulfide bond to adopt a functional conformation (23, 36, 37, 49). Fimbria-mediated adhesion is a crucial first step of the infection process as it allows host colonization by mucosal pathogens. DsbA is required for functional assembly of several types of fimbriae, including P fimbriae of uropathogenic E. coli (UPEC) (24), bundle-forming pili (Bfp) of enteropathogenic E. coli (55), mannose-resistant Proteus-like (MR/P) fimbriae of Proteus mirabilis (8), plasmid-encoded fimbriae (Pef) of Salmonella enterica (6), type IV pili of Neisseria meningitidis (47), and toxin-coregulated pili (Tcp) of Vibrio cholerae (41). A number of studies have reported that dsbA and/or dsbB mutants are attenuated in infection models (9, 16, 41, 48, 52).The recent exponential increase in sequenced genomes has offered a first glimpse at the diversity of disulfide bond systems present in bacteria (13). In addition, it is now evident that several bacterial species encode multiple DsbA paralogues, often with demonstrated differences in substrate specificity. Neisseria meningitidis, for example, encodes three DsbA oxidoreductases: two inner membrane-associated lipoproteins (DsbA1 and DsbA2) and one periplasmic enzyme (DsbA3). While redundancy was observed in the oxidative folding of virulence-associated proteins by DsbA1 and DsbA2, DsbA3 alone was unable to restore important meningococcal virulence traits, such as type IV pilus-mediated adhesion to human endothelial cells (47). Recently, a second E. coli disulfide bond system (DsbLI) was identified in the genome-sequenced UPEC strain CFT073 and was demonstrated to be a functional paralogue of the prototypic DsbAB system (14). The oxidoreductase DsbL has the strongest oxidizing potential of all DsbA homologues characterized to date. Although the crystal structure of DsbL revealed a similar overall fold and domain architecture to DsbA, DsbL contains a longer helical insertion and deletions in the TRX domain that result in a truncated peptide binding groove. Moreover, DsbL shows different surface properties, including a distinct basic patch around the active site, which was suggested to allow stricter substrate specificity than the highly hydrophobic surface surrounding the active site of DsbA. Grimshaw and colleagues (14) demonstrated the specificity of the DsbLI system for the periplasmic enzyme arylsulfate sulfotransferase (ASST) encoded by assT, a gene found immediately upstream of dsbL and dsbI on the CFT073 chromosome. ASST belongs to a group of poorly characterized large bacterial ASSTs that are proposed to mediate detoxification of phenolic substances by catalyzing the transfer of sulfuryl groups from phenolic sulfates to phenol (26-28, 30). A reason for the specificity of DsbLI for ASST folding could be the presence of an allosteric disulfide bond, recently revealed by the enzyme''s crystal structure (33). This class of disulfide bond forms between C_α atoms of cysteines in unusually close proximity (3.8 Å in the case of ASST) and has higher steric strain energy than catalytic or structural disulfide bonds, thus explaining the requirement for the stronger DsbL oxidase for its formation (33). The activity of DsbL and DsbI was studied using plasmids introduced into E. coli K-12 strains with the native DsbAB system deleted. As yet, the role of the DsbLI system in UPEC virulence has not been investigated.E. coli CFT073 is a prototypic UPEC strain isolated from a female patient with acute pyelonephritis (38). UPEC strains are the causative agent of >80% of community-acquired urinary tract infections (UTIs) and >30% of nosocomial infections (7). The uropathogenic lifestyle of UPEC CFT073 is reflected in its genome, which contains several factors with an established role in urovirulence, including the well-studied type 1 and P fimbriae (50). Genomic comparison of CFT073—and other recently sequenced UPEC strains—with E. coli strains with distinct lifestyles (gut commensals, enteric pathogens, and avian pathogens) allows the discovery of genes unique to genomes of uropathogenic bacteria that are potentially novel urovirulence factors. One such UPEC-specific gene is assT, the gene located upstream of dsbL and dsbI in the chromosome of CFT073 (32).Here we characterize the DsbLI system in its native genetic background of UPEC CFT073 and compare and contrast the contribution of each of the two paralogous disulfide bond systems of CFT073 in the production of UPEC-associated virulence factors and in vivo uropathogenesis. Using isogenic dsbAB and dsbLI deletion mutants of CFT073, we demonstrate that the recently identified DsbLI oxidative protein folding machinery of UPEC CFT073 plays a secondary role in the production of urovirulence factors and does not appear to contribute to virulence in the mouse infection model used in this study. We also show that in the same infection model, an isogenic assT deletion mutant of CFT073 is not attenuated.     

Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that Bap_MS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bap_MS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.     

UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.     

Structural and Functional Characterization of Three DsbA Paralogues from Salmonella enterica Serovar Typhimurium

In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.