The multiplicity of Thioredoxin systems meets the specific needs of Clostridia

Oxidative stress is a highly common stress for cells, which targets proteins with oxidation of cysteine residues. The thioredoxin (Trx) system, which is a ubiquitous system for thiol- and protein-repair, is composed of a thioredoxin (TrxA) and a thioredoxin-reductase (TrxB). TrxAs reduce disulfide bonds of oxidized proteins and are then usually recycled by a single pleiotropic NAD(P)H-dependent TrxB (NTR). However, some Clostridia have also ferredoxin-dependent TrxBs. In this work, we first analyzed the composition of Trx systems across Bacteria. Most of bacteria have only one NTR, but organisms in some Phyla including Firmicutes have several TrxBs. In Firmicutes, this multiplicity of TrxBs is observed only in Clostridia. We thus used Clostridioides difficile as a model to investigate the biological relevance of TrxB multiplicity by studying the physiological roles of the Trx systems in this gut pathogen. Three TrxAs and three TrxBs are present in the 630{Delta}erm strain. We showed that two systems were involved in response to infection-related stresses, allowing survival of vegetative cells to exposure to oxygen, inflammation-related molecules and bile salts. A supplementary TrxB copy present in some C. difficile strains also contributes to this stress-response arsenal. One of the conserved stress-response Trx system was also found to be present in the spore via a dual transcriptional control by different sigma factors. This system contributes to spore survival to hypochlorite and ensure proper germination in the presence of oxygen. Finally, we found that the third Trx system was contributing to sporulation. This involvement was likely linked to the recycling of the glycine-reductase, a Stickland pathway enzyme that allows consumption of glycine, a spore co-germinant. Altogether, our results showed that the multiplicity of Trx systems produced under the control of different regulatory signals and networks and the diversity of TrxBs meet specific needs of Clostridia, i.e., adaptation to strong stress exposure, sporulation and Stickland pathways. More broadly, this multiplicity responds to cell compartmentation and differentiation, which can be transposed to other multiple-TrxBs organisms such as Cyanobacteria or eukaryotes.