The primary function of thioredoxin 1 (TrxA) is to maintain the redox balance in living cells, including eukaryotes and prokaryotes. It is maintained in a reduced state by thioredoxin reductase in a reaction that consumes NADPH. By reducing the disulfide bonds, thioredoxin helps biomolecules maintain their native structure. The genome of Salmonella encodes two independent thioredoxin proteins. TrxA promotes antioxidant defenses of Salmonella against NADPH phagocyte oxidase-mediated oxidative stress in vivo. Previous work from our research identified that TrxA promotes antioxidant defenses of Salmonella independently of the canonical oxidoreductase activity that maps to the catalytic cysteine residues. We demonstrated that TrxA helps maintain the solubility of SsrB response regulator, increasing the expression of the Salmonella pathogenicity island-2 (SPI-2) genes and thus enhancing the Salmonella virulence in mice and macrophages.
The SsrA-SsrB two-component system regulates the type III secretion system encoded by SPI2, reducing contact between Salmonella-containing vacuoles and phagocytic NADPH oxidase-containing vesicles. The ssrA-ssrB operon is regulated by several two-component systems such as EnvZ-OmpR and PhoP-PhoQ in response to changes in osmolality, pH, and the presence of anti-microbial peptides. The structure of response regulator such as SsrB, OmpR and PhoP is remarkably conserved, raising the possibility that TrxA could regulate intracellular growth of Salmonella by binding not only to SsrB but also to other response regulators.
In this study, using an in vivo pull-down assay, our team discovered that TrxA binds promiscuously to several response regulators including OmpR and PhoP. Our biochemical assays proved that TrxA interacts with these response regulators in a redox-independent manner. Protein structure analysis was desperately needed to reveal which part of TrxA directly binds to them. To address this important question, we collaborated with Dr. Beat Vogeli at the Department of Biochemistry and Molecular Genetics at the University of Colorado School of Medicine. Using NMR, Alexandra Born, at the time a graduate student in the Vogeli lab, determined the binding residues of TrxA that make contact with OmpR and PhoP. This NMR approach showed that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using a similar approach, Dr. Vogeli’s group identified residues in the flexible linker and the C-terminal β-hairpin of OmpR that make specific associations of this archetypical response regulator with thioredoxin. Importantly, our biochemical and functional analyses demonstrated that the interfacial residues in OmpR that make contact with TrxA, do not mediate interactions of this response regulator to its cognate sensor kinase EnvZ, DNA or RNA polymerase. We also showed that interfacial residues in both TrxA and OmpR enable the intracellular growth of Salmonella in mice and macrophages, while activating transcription of SPI-2 genes. Our collaborative effort has uncovered the interfacial residues by which TrxA plays critical roles in Salmonella pathogenesis, which are independent of its well-characterized thio-disulfide exchange function. Our investigations also have uncovered the critical role those promiscuous interactions of several response regulators with a common area in thioredoxin play in the transmission of signals by otherwise highly discriminatory two-component signaling pairs.