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.