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

MÉNDEZ AAE; BERTONATI B; ZHAO Z; ARGÜELLO JM; SONCINI FC; CHECA SK

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General; 2023

Sociedad Argentina de Microbiología General

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) excess. Cu-sensitive Salmonella mutants show a reduced survival in macrophages comparing with the wild-type strain. Most of the Cu-resistant determinants and cuproproteins reside in the pathogen´s cell envelope, which is also the main target for Cu toxicity. 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 in a CpxRA-dependent manner. 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 that 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 CpxRA two component system that controls scsABCD transcription. Focused on ScsD, we performed a series of in silico, in vitro and in vivo studies on the protein to test its localization, regulation, stability and copper binding capability. Alpha fold-simulation revealed that it is a periplasmic protein anchored to the Salmonella inner membrane. This protein accumulated in the membrane fraction under conditions of Cu treatment, both in the presence and in the absence of the CpxRA. This suggests the presence of an alternative mechanism of Scs expression regulation which responds to Cu but is independent of CpxRA. We cloned and expressed ScsD periplasmic soluble domain in E. coli to analyze its secondary structure by circular dichroism, confirming our in silico analysis. Moreover, its stability in solution was determined by thermal shift assay and its copper binding ability was confirmed by a competition assay between a copper chelating reagent (BCS) and both the wild type protein and a mutant in a specific cysteine from its putative copper binding motif (MBP-ScsDp-CxxS74). In sum, these results contribute to shed light on the role of the ScsABCD system and particularly the ScsD protein in the homeostasis of the S. enterica envelope.