Abstract
ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.
Highlights
The nosocomial, opportunistic human pathogen Pseudomonas aeruginosa uses numerous secretion systems to deliver a variety of virulence factors into host cells contributing to establish infections
We decided to test whether the guanylate cyclase (GC) activity of ExoY could be detected directly in bacterial culture supernatants in the presence of actin as cofactor to activate ExoY
We focused on the GC activity, which is the highest among the tested nucleotidyl cyclase activities with different nucleotide substrates (GTP, ATP, UTP, and CTP) using recombinant purified enzyme activated by actin (Raoux-Barbot et al, 2018)
Summary
The nosocomial, opportunistic human pathogen Pseudomonas aeruginosa uses numerous secretion systems to deliver a variety of virulence factors into host cells contributing to establish infections. This ability together with the capacity to break down a wide range of nutrients from different environments, to out-compete co-existing bacterial species, and to develop. Antibiotic resistance contributes to the mortality rate in hospitalacquired infections and pulmonary infections of cystic fibrosis (CF) patients (Bennett et al, 2019). The expression of the type III secretion system (T3SS), which enables direct delivery of virulence factors into human endothelial or epithelial cells, was shown to change over the course of CF infections (Jain et al, 2004). As strains with a functional T3SS cause higher bacterial burden and mortality in acute respiratory infections (Hauser et al, 2002), the T3SS is considered as a potential therapeutic target (Lee et al, 2007; Aiello et al, 2010; Anantharajah et al, 2016; Sheremet et al, 2018)
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