Characterisation of the Scs system that confers copper tolerance in Salmonella enterica serovar Typhimurium

Gram-negative bacteria have a variety of systems that catalyse the formation of disulphide bonds, which are essential for the folding, activity, and stability of many periplasmic and secreted proteins. The scsABCD (suppressor of copper sensitivity) locus of Salmonella enterica encodes four proteins with thioredoxin-like catalytic motifs. Previous work has shown that Salmonella encounters toxic levels of copper during infection and the scs system provides protection against copper-mediated toxicity. Given that Cu2+ ions are known to promote disulphide oxidation, it was hypothesised that the StScs proteins and copper both influence the thiol redox status of periplasmic proteins in vivo. The current work reports that expression of the soluble periplasmic protein StScsC is copper-specific, and copper was found to oxidise StScsC in vivo. Using a combination of genetic and proteomics approaches, the abundance of various cysteine-containing periplasmic/secreted proteins were found to be elevated by StScsC and copper in the Salmonella periplasm. Copurification and mass spectrometry approaches provide additional evidence that the arginine-sensing periplasmic protein ArtI interacts with StScsC. Intramacrophage survival data demonstrates that loss of StScsC results in a significant decrease in survival. The current work reports a new role for the thioredoxin-like StScsC protein in disulphide folding of ArtI, a periplasmic L-arginine sensing protein. Given the known impact of arginine sensing/uptake upon c-di-GMP signalling and the production of nitric oxide (NO) by host cells, the current work demonstrates a role for the Scs system in facilitating intramacrophage survival through alleviating copper-stress, and implicates StScsC in a broader role in immune evasion.

In addition, the presence of StScs proteins and copper was shown to increase the yield of Herceptin Fab fragments (used to treat breast cancer) in the E. coli periplasm. Hence, the StScs proteins have the potential to facilitate the formation of disulphides in protein therapeutics that can be used in biotechnological platforms.

This work provides novel insights into the in vivo role for the Scs system in Salmonella, and highlights the importance of disulphide stress responses and copper tolerance during infection.