ScsD, A PERIPLASMIC PROTEIN FROM Salmonella enterica INVOLVED IN COPPER HOMEOSTASIS

MENDEZ ANDREA AE; BERTONATI BIANCA; ZHAO, ZHENZHEN; ARGUELLO, JOSE M; SONCINI, FERNANDO C.; CHECA, SUSANA K

Chapadmalal

Congreso; SAMIGE 2023; 2023

Foodborne diseases are among the most prevalent health problem worldwide. In this regard, Salmonella enterica is one of the most significant pathogens of both humans and animals, that causes infections ranging from self-limited gastroenteritis to severe invasive illness in susceptible hosts. Like other bacterial pathogens, its interactions with hosts are influenced by transition metals, especially by copper (Cu). Cu-sensitive Salmonella mutants show a reduced survival in macrophages comparing with the wild-type strain. Many of the Cu distribution proteins and cuproproteins reside in the pathogen´s cell envelope. The Salmonella scsABCD operon, absent in Escherichia coli but present in other enteropathogens, contributes to both Cu and redox stress tolerance. Previously, we showed that this operon is induced by Cu. Interestingly, ScsB, ScsC and ScsD carry putative Cu-binding motifs in their periplasmic thioredoxin-like domains. ScsB and ScsC form a redox pair, resembling described IM-bound reductases and periplasmic oxidase/isomerase partners, such as DsbD/DsbC. The Dsb system, present in Salmonella, does not contribute to Cu tolerance. Because of its homology to EcCcmG, which is reduced by EcDsbD, ScsD is proposed to receive reduced equivalents from ScsB. In this work, we assessed the intracellular copper levels of the scsABCD deleted mutant in Salmonella as well as the CpxR/A two component system that controls scsABCD transcription. Our data show that, while mutation on the CpxR/A regulatory system leads to reduced intracellular Cu levels both in the presence and absence of external Cu, deletion of the scsABCD locus had little or no effect on intracellular Cu levels. Focused on ScsD, we performed a series of in silico, in vitro and in vivo studies to test the protein localization, regulation, stability and Cu binding capability. Alpha fold modeling revealed that ScsD is a periplasmic protein anchored to the plasma membrane. This protein accumulated in the membrane fraction under conditions of copper treatment, both in the presence or absence of the CpxR/A two component system. This suggests the presence of an alternative mechanism of Scs function regulation which responds to Cu but is independent of CpxR/A. The secondary structure of ScsD periplasmic soluble domain was analyzed by circular dichroism, supporting the in silico structural analysis. The stability of the soluble domain was studied by thermal shift assay. An MBP bound ScsD soluble domain was used to measure Cu(I) binding spectrophotometrically. As expected, the domain bounds Cu(I) ion and the mutation of a signature Cys in thioredoxins (CxxS74) prevented Cu(I) binding. These results shed light on the role of the ScsABCD system and particularly the ScsD protein in the metal/redox homeostasis of the S. enterica envelope.