ScsD, a periplasmic protein implicated in copper/redox homeostasis in the Salmonella enterica envelope

MÉNDEZ, A. A. E.; BERTONATI, B.; SONCINI, F.C.; CHECA, S. K.

Rosario

Congreso; LIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2023

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Salmonella enterica is a species that includes a large group of food-borne and facultative intracellular pathogens causinginfections that ranges from self-limited gastroenteritis to severe invasive illness in susceptible hosts. During its infective cycle,Salmonella adapts to and survive in different conditions, including the menacing host environment. Once inside theSalmonella-containing vacuole (SCV) within infected phagocytic cells, copper, a highly reactive and toxic metal, activelyaccumulates to contain the multiplication and dissemination of the pathogen. These ions exacerbate redox stress, primarily in thebacterial cell envelope, where many of the Cu distribution proteins and cuproproteins reside. ScsABCD, a putative thioredoxinsystem in S. enterica, absent in Escherichia coli but present in other enteropathogens, contributes to both Cu and redox stresstolerance. Our previous work showed that this operon is induced by Cu. ScsB, ScsC and ScsD carry putative Cu-binding motifsin their periplasmic thioredoxin-like domains. ScsB and ScsC form a redox pair resembling described IM-bound reductases andperiplasmic oxidase/isomerase partners, such as DsbD/DsbC, present in Salmonella but not involved in Cu tolerance. RegardingSalmonella ScsD, its AlphaFold 2 structure resembles that of the membrane-linked thiol:disulfide interchange proteins such asDsbE/CcmG and TlpA, involved in cytochrome c and aa3 biogenesis. Considering that we have determined the function ofSalmonella ScsC and ScsD under Cu+ stress depend on ScsB and that DsbE is a DsbD partner in E. coli, we propose ScsD asanother ScsB redox partner. In this work, we focused our studies on Salmonella ScsD. We tagged the scsD chromosomal genewith a 3xFLAG epitope to determine its intracellular localization and expression. We confirmed that it accumulates in themembrane fraction under conditions of copper treatment, both in the presence or absence of the CpxR/A two component systemthat controls scsABCD transcription. This suggests the presence of an alternative mechanism of Scs function regulation whichresponds to Cu but is independent of CpxR/A. ScsD periplasmic soluble domain containing the putative copper binding motifwas cloned and expressed as an MBP fusion protein in E. coli to perform in vitro studies. Its secondary structure was analyzed bycircular dichroism, supporting the in silico structural analysis. To evaluate ScsD Cu(I) binding and its ability to form a redox pairwith ScsB, a mutant of the ScsD Cu-binding motif, C71xxS74, was generated using Quick-change PCR. As expected, the domainbounds Cu(I) ion and the mutation of this signature Cys in thioredoxins prevented Cu(I) binding. Moreover, the redox trap assayperformed between both ScsB and ScsD Cu-binding motif mutants is an indicative of them forming a redox pair. These resultsshed light on the role of the ScsABCD system and particularly the ScsD protein in the metal/redox homeostasis of the S. entericaenvelope.