Thioredoxin Profiling of Multiple Thioredoxin-Like Proteins in Staphylococcus aureus

Abstract

Hydrogen sulfide (H2S) is thought to signal through protein S-sulfuration (persulfidation; S-sulfhydration) in both mammalian systems and bacteria. We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (kcat = 0.13 s-1) and TrxP (kcat = 0.088 s-1) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (kcat = 0.015 s-1). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress.

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases

“…Peng et al studied the reactivity of TrxA, TrxP, and TrxQ from Staphylococcus aureus with persulphidated pyruvate kinase as a model substrate. The three redoxins displayed small differences in substrate specificity that were also discussed to be the result of electrostatic differences in the area surrounding the N-terminal active site thiol [79]. We have previously proposed and provided evidence that the binding of Trx family proteins to their interaction partners and likely all protein-protein interactions in aqueous solution require geometrically compatible surfaces and, that given, are controlled by complementary electrostatic surfaces [14,39,80].…”

A Fluorescent Probe to Detect Quick Disulfide Reductase Activity in Bacteria

“…In this study, B. cereus showed much higher disulfide reducibility among E. coli , P. aeruginosa and B. subtilis . Besides, a previous study identified two thioredoxin-like proteins TrxP and TrxQ, which could reduce protein disulfide as a potential regulatory mechanism [ 47 ]. This may account for the highest disulfide reducibility of S. aureus among other strains.…”

H2S and reactive sulfur signaling at the host-bacterial pathogen interface

“…The characterization of two thioredoxins in S. aureus that have significant activities on protein persulfides relative to disulfides versus the canonical TrxA certainly suggests a role for Trx in this process, as well as the possibility that bacteria encode specific thioredoxin-like proteins for this purpose (96). Furthermore, a thioredoxin-based proteomic profiling strategy was used to identify potential cellular targets for these persulfide-reducing thioredoxins in S. aureus (193). Analogous strategies might be applied to STRs in an effort to identify protein targets that could function as donors or acceptors in transsulfuration reactions.…”