Pseudomonas aeruginosa is a Gram-negative bacterium responsible for nosocomial infections and chronic infections in cystic fibrosis patients. It encodes virulence factors secreted through secretion systems (TSS). We and others have shown recently that T3SS is important in inducing alveolar macrophage (AM) phagocytosis [1, 2]. We set up here to study the involvement of LasB, the major T2SS secreted metalloprotease, in modulating host response and particularly AM functions.
In vivo experiments: C57Bl/6 male mice were instilled intranasally with either WT or βlasB PAO1: 1×108cfu were used in a survival study while 2.5×106cfu were used for the study of host responses. For the latter, 2, 4, or 20hours post-P. aeruginosa instillation, lung BALs were performed to measure cell recruitment and neutrophils activation. P. aeruginosa cfu counts were also assessed to estimate in vivo bacterial clearance.
Ex vivo experiments: AMs from C57Bl/6 male mice were infected ex vivo with P. aeruginosa strains (WT & βlasB PAO1, MOI 0.1, 1 or 10) in 10% FCS-antibiotic-free RPMI medium. One and 4hours later, phagocytosis and P. aeruginosa killing, respectively, were assessed by counting cfu from cell supernatants and lysates. In parallel, cytokine levels were assessed in AMs supernatants.
In vivo experiments: mice infected in vivo with βlasB PAO1 died within 42hours post-infection, while all mice infected with WT PAO1 succumbed 18hours before (n=7, P=0.0009). Furthermore, 2, 4 or 20hours post-infection, βlasB PAO1 counts in BALs were halved, compared to that of WT PAO1 (n=8, P<0.05). Interestingly, at all-time points, there was no difference between bacterial strains in terms of BAL inflammatory cells recruitment and neutrophil activation.
Ex vivo experiments: There was no difference in WT and βlasB PAO1 phagocytosis by AMs, and βlasB was killed more efficiently (15%, n=4, P=0.0101). An increase in IL-1β secretion in AMs supernatants from βlasB-infected animals was noted 4hours post-infection (2.8 & 1.9 fold, respectively, n=4, P<0.001) but no difference in TNF-α production was observed (n=4).
We demonstrate here for the first time that LasB, a P. aeruginosa T2SS secreted virulence factor, is lethal to mice and protects significantly this bacterium against AM-mediated P. aeruginosa killing. In vivo, LasB does not influence inflammatory influx but acts through down-regulating IL-1β. We have shown previously  that IL-1 signalling is instrumental in PAO1 killing and we are currently investigating the signalling pathway linking LasB to IL-1β production.Le texte complet de cet article est disponible en PDF.
Keywords : Infection, Inflammation