Role of Salmonella enterica sv Typhimurium copper homeostasis proteins at the host-pathogen interface

Abstract

During infection, pathogenic bacteria must acquire all nutrients from the host. Host availability of essential metals is tightly regulated to limit access of pathogens to these nutrients, termed nutritional immunity. Conversely, immune cells such as macrophages exploit the reactivity of excess copper as a toxic weapon against invading pathogens. Bacterial strains exhibit virulence defects when they carry mutations in their metal homeostasis systems. Pathogenic bacteria can encounter both a bottleneck of high metal availability (metal toxicity), and a bottleneck of low metal availability (metal starvation), at different stages during infection. The spatiotemporal localization of these bottlenecks, what host factors create them, and how bacteria respond to them, remain unclear. In this study we aimed to characterize the role played by copper homeostasis genes at the hostpathogen interface, using the enterobacterial model Salmonella enterica sv Typhimurium. It has been shown that different strains of Salmonella devoid of the Csp3 copper storage protein are killed significantly more efficiently by murine macrophages compared to the parental strain but does not show copper sensitivity in media supplemented with CuSO4. This study also confirmed that the ΔcopA/golTstrains, devoid of the two periplasmic copper efflux pumps presents a reduced intramacrophage viability and impaired growth phenotype. Unexpectedly, combining this mutation with other known copper homeostasis genes increased, rather than decreased, survival of a Δcsp3/copA/golT and especially a Δcsp3/copA/golT/cueO strain in the in vitro phagocytosis assay. This phenotype was particularly evident at longer time points of infection (six and twenty-four hours). Growth profiles of Δcsp3/copA/golT and Δcsp3/copA/golT/cueO describe a copper sensitivity lower compared to the ΔcopA/golT one. Remarkably, data collected in a mouse model of systemic salmonellosis via intraperitoneal route of infection confirmed an enhanced virulence of these strains, most dramatic in Δcsp3/copA/golT/cueO. Further experiments using the intravenous route of infection indicated that the hypervirulent phenotype is associated with increased survival during the intraphagocytic phase of the Salmonella lifecycle and was not a reflection of overgrowth within the peritoneal cavity. Intermediate strains generated to better investigate the role played by cueO in this phenotype, ΔcueO and ΔcopA/golT/cueO, did not show acquisition of increased viability within cultured macrophages and ΔcopA/golT/cueO shows more copper sensitivity than the ΔcopA/golT strain. We therefore assumed that a model comprising a decreased viability within phagocytes as well as in mouse model of systemic infection and increased copper sensitivity growing in media supplemented with Cu, associated to a progressive disruption of copper homeostasis genes, is incomplete. Especially about the role of Csp3 there is some aspects or functions still ununderstood. Based on data obtained in this study we speculated that Csp3 could have a role in supply copper ions to cuproenzymes or that its absence would generate enhanced intracellular Cu avaibility sensed by other system, but not by the cue system. In both cases, the pattern would be relevant during infection. This phenomenon, a sort of operation of rescue, would take place especially whiting macrophages, probably also triggered by other stressors (i.e low pH, antimicrobial peptides, envelope stress), to enhance chances of survival. This hypothesis is supported by the observation of hypervirulent phenotypes within macrophages at longer time points and in mouse model systemic salmonellosis, therefore when the bacterium was able to regulate a secondary strategy to succeed and survive.