DisarmingBurkholderia pseudomallei: Structural and Functional Characterization of a Disulfide Oxidoreductase (DsbA) Required for VirulenceIn Vivo

Abstract

Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei. Conclusion: These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism. Antioxid. Redox Signal. 20,[606][607][608][609][610][611][612][613][614][615][616][617]

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria

“…These findings suggest that the bacterial thiol-disulfide oxidoreductases would provide excellent targets for the development of antimicrobials. Since dsbA mutants are attenuated in rodent models of infection (86,87), efforts have recently been made to identify inhibitors of DsbA/DsbB as antivirulence agents. By screening a library of 1,123 fragments for compounds that bind to oxidized DsbA, Adams and colleagues (88) identified a small set of molecules that exhibit high binding affinity to DsbA and inhibit its activity in vitro.…”

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

“…pseudomallei possesses a large genome (ϳ7 Mb) that encodes numerous virulence factors, including capsular polysaccharide, lipopolysaccharide, type III secretion system 1 (T3SS-1), T3SS-3, T6SS-1, TssM, type IV pili, MviN, PlcN3, type III O-PS, type IV O-PS, LfpA, ecotin, BPSL0918, SodC, BbfA, DsbA, RelA, SpoT, and BLF1 (4)(5)(6)(7)(8)(9)(10)(11)(12). Many of these virulence determinants are secretion systems or secreted proteins that promote survival in vivo by disarming elements of the host immune system.…”

Targeting virulence not viability in the search for future antibacterials

“…Quinones (labelled Q) generate disulfides in DsbB (orange), which are transferred to DsbA (green), which catalyzes oxidative protein folding in substrate virulence factors (blue; and indicated above). In concert, the disulfide in DsbA (labelled S-S) is reduced to two thiols (labelled SH) to complete the catalytic cycle Antivirulence strategies for bacterial infection an animal model of melioidosis in which mice infected with the causative agent of melioidosis, Burkholderia pseudomallei, all died within 42 days whereas mice infected with B. pseudomallei lacking the gene for DsbA all survived [64]. Similarly, animal infection models have demonstrated that deletion of dsbA or dsbB in uropathogenic E. coli (UPEC) severely attenuated its ability to colonize the bladder [65], and that dsbA mutants in Salmonella enterica serovar Typhimirium were avirulent [66].…”