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

Abstract

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 welldocumented 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 carotovo...

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Our results, together with previous reports 13,23,24,48 , suggest a straightforward model for the FimC-catalyzed subunit folding of FimA and other pilus subunits. As the FimA fold has a high topological complexity (contact order) 28 , this might be the dominant factor determining the high kinetic barrier of spontaneous FimA folding.…”

“…The deletion of the dsbA gene on the E. coli genome leads to the complete absence of type 1 pili 22 . Only when the entire type 1 pilus gene cluster was overexpressed via a plasmid in a dsbA deletion strain could type 1 pilus biogenesis still be observed, but at reduced levels compared to wild-type cells 23,24 . The latter finding does not contradict the dependence on DsbA of type 1 pilus biogenesis in wild-type E. coli strains and may be explained by partial oxidation of overexpressed pilus subunits by air oxygen, which is possibly catalyzed by traces of transition metals such as copper 45 .…”

“…The periplasmic disulfide oxidoreductase DsbA is the principal oxidant of periplasmic proteins with structural disulfide bonds 20,21 . DsbA is essential for the in vivo assembly of pili and other disulfide-bonded virulence factors, as deletion of the dsbA gene from E. coli abolishes pilus biogenesis [22][23][24] .…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…The pilus subunits, when released into the periplasm, form a disulfide bond between their β-strands A and B (figure 2), a process catalysed by the periplasmic oxidoreductase DsbA [28,29]. It has been shown that the chaperone is able to discriminate subunits with a correct disulfide bond, offering an early stage of subunit quality control [30].…”

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA fromSalmonella entericaserovar Typhimurium

“…Two of these proteins, NmDsbA1 and NmDsbA3, have been structurally characterized and the disulfide-oxidation mechanism in this pathogen has been investigated (Vivian et al, 2008(Vivian et al, , 2009Lafaye et al, 2009). Multiple Dsb proteins are also found in uropathogenic E. coli (UPEC), which contains a DsbL/DsbI pair in addition to the oxidase and isomerase pathways found in the prototypic K-12 (Grimshaw et al, 2008;Totsika et al, 2009).…”