Functional Analysis of Putative Thiol-Disulfide Oxidoreductases in Group A Streptococcus

Abstract

Group A Streptococcus (GAS) is a pathogenic bacterium that strictly infects humans, causing a diverse range of diseases from severe toxic shock syndrome to moderate strep throat infection, especially in children. GAS produces certain virulence factors that are structurally held together by complex molecular linkages, including covalent disulfide bonds formed between cysteine residues. Disulfide bonds are essential for protein stability and function, and are formed in vivo by enzymes called thiol-disulfide oxidoreductases (TDORs). Remarkably, disulfide bond formation pathways have not been studied in the context of GAS pathogenesis and could be an excellent approach to better understanding the structure and regulation of virulence factors in GAS. An in silico approach was used to identify five putative TDORs in GAS which were individually mutated and their subsequent biological functions characterized. Our results have identified 2037 as a novel TDOR enzyme in GAS. The Δ2037 mutant showed significantly increased sensitivity to oxidative stress-promoting compounds and was more susceptible to phagocytic death by murine macrophages, indicating a role for 2037 in maintaining thiol balance at the cell surface through an unknown mechanism. Most notably, 2037 is needed for proper disulfide bond formation in an important GAS exotoxin: streptococcal pyrogenic exotoxin A (SpeA). Redox state analysis of SpeA secreted by the parent culture showed the presence of a disulfide linkage (oxidized form) whereas this disulfide bond was broken (reduced form) in Δ2037 cultures. The 2037-complemented mutant restored SpeA to the oxidized state, implicating 2037 as a direct player in forming the disulfide bond of SpeA. Consistent with these data, 2037 exhibited functional oxidase activity in vitro and disulfide exchange reactions showed recombinant SpeA changed from its initial reduced form to an oxidized form following incubation with oxidized recombinant 2037 but not reduced 2037. Furthermore, preliminary point mutation data suggests that the active site 2037 cysteines 46 and 49 have different reactivites, with the N-terminal Cys46 possibly playing a distinct mechanistic role during protein complex formation with SpeA. This is the first report of an enzyme being directly involved in the proper folding of a superantigenic toxin. Our findings highlight the importance of studying disulfide pathways in modifying virulence factors secreted by pathogenic bacteria, and pave the way for new drug targets and vaccine development strategies that offer an alternative to antibiotics.