The Cu-induced ScsD reductase is a membrane-bound redox partner of ScsB in the Salmonella envelope

The intracellular environment Salmonella confronts during infection is characterized by multiple redox stressors including reactive oxygen species (ROS) and copper (Cu) ions. Under these conditions, alternative systems of thiol oxidoreductases such as the Cu induced Scs system are required to protect and repair periplasmic proteins. The scsABCD operon encodes three Dsb-like enzymes, ScsB, ScsC, and ScsD, and an accessory protein, ScsA. These proteins are required both for Cu resistance and H2O2 tolerance. ScsB and ScsC function analogously to the canonical DsbD/DsbC redox pair of thiol oxidoreductases. The absence of ScsC was shown to affect the folding/activity of periplasmic proteins involved in amino acid transport and redox homeostasis. Here, we focus in ScsD, the least characterized member of this system. Upon Cu-induced expression, ScsD localizes to the inner membrane, enabling its predicted C-terminal Dsb-like domain to be exposed to the periplasm. Functional analysis indicates that ScsD exists in a reduced state in the Salmonella envelope and serves as a redox partner of ScsB. ScsD exhibits in vivo disulfide reductase activity and restores a deficient disulfide reduction pathway in Salmonella. Similar to ScsC and ScsB, ScsD binds Cu(I) via the Cys residues of its Dsb-like domain; however, this metal interaction appears to lack relevance in Cu detoxification as no impact on intracellular Cu levels was observed. Our results define ScsD as a specialized membrane-bound thiol-disulfide reductase in the Salmonella envelope and highlight the versatility of the Scs system in maintaining periplasmic proteostasis when canonical pathways are compromised by host-imposed Cu stress. ImportanceCopper is a key component of the innate immune system, serving as a primary defense against pathogens like Salmonella. Copper overload targets the bacterial envelope, specifically attacking protein sulfhydryl groups. This causes protein misfolding and inactivation, disrupting essential processes like metabolism, transport and virulence. To survive this stress and restore thiol homeostasis, Salmonella utilizes the scsABCD operon. While the ScsB-ScsC redox pair is well-documented and some protein substrates identified, the role of ScsD remains undefined. This work characterizes ScsD as an inner-membrane-anchored thiol reductase and a new redox partner for ScsB. The ScsD/ScsB pair expands the bacteriums protein quality control capacity, allowing Salmonella to maintain envelope homeostasis within the hostile, copper-rich environment of the host.