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.Pathogenic bacteria often interact with their host through surface proteins referred to as adhesins. Two major classes of adhesins have been described, and both utilize vastly different mechanisms for their assembly at the cell surface. Fimbrial adhesins such as the prototypical type 1 fimbriae of Escherichia coli are composed primarily of a major repeating subunit protein and often contain minor-subunit proteins (including the adhesin) at the tip of the organelle. The biogenesis of these fimbriae is dependent on a highly conserved chaperone-usher system (20,29). Nonfimbrial adhesins are a second class of adherence factors and encompass a diverse range of adhesins that includes the autotransporter (AT) family of proteins. AT proteins are particularly unique in that the information required for receptor recognition and routing and anchorage to the outer membrane is provided by the protein itself (25). Recent studies have identified a new group of nonfimbrial adhesins that have the capacity to form stable trimeric structures on the bacterial cell surface. These adhesins are characterized by a membrane-anchored C-terminal domain that forms a trimeric ␤-barrel pore and facilitates the translocation of a passenger domain (consisting of an extended stalk and an N-terminal head) to the bacterial cell surface via the type V secretion pathway (54, 67).Trimeric AT proteins have been identified from a range of different gram-negative bacterial pathogens. Where characterized, the function of trimeric AT proteins is universally associated with bacterial adherence, and thus they constitute an important group of virulence factors (14). The YadA adhesin from Yersinia enterocolitica represents the best-characterized trimeric AT protein. YadA mediates adherence to host epithelial cells an...

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined irondeficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a "cheater" and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the UnitedStates. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens.Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States, where it accounts for approximately 40% of all nosocomial infections (49). Although CAUTI is usually asymptomatic, it is a frequent cause of bacteremia and infected patients can also experience fever, acute pyelonephritis, and death (59). The risk of bacteriuria following urethral catheterization is estimated to be 5 to 10% per day (60). Most patients with indwelling urinary catheters for 30 days or longer develop bacteriuria (49).Nosocomial CAUTI is caused by a range of gram-negative and gram-positive organisms, including Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Providencia stuartii, Staphylococcus epidermidis, and Enterococcus faecalis (60). These infections are often polymicrobial and can last from several days to months (29). E. coli is one of the most common organisms isolated from the urine of CAUTI patients. Like uropathogenic E. coli (UPEC) strains that cause cystitis and pyelonephritis, CAUTI E. coli strains possess a range of virulence factors, including adhesins (e.g., P and type 1 fimbriae) and toxins (e.g., hemolysin), and express certain O antigen and capsule (K) types (29). Adherence is important for the colonization of the urinary tra...

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Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest ␤-helix as the predominant structure in the large N-terminal passenger (␣) domain and a 12-strand ␤-barrel for the C-terminal ␤-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.