Abstract
While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models. However, the role of macrophages in P. aeruginosa clearance in vivo remains poorly studied. The major outer membrane porin OprF has been recently shown to be involved in P. aeruginosa fate within cultured macrophages and analysis of an oprF mutant may thus provide insights to better understand the relevance of this intramacrophage stage during infection. In the present study, we investigated for the first time the virulence of a P. aeruginosa oprF mutant in a vertebrate model that harbors functional macrophages, the zebrafish (Danio rerio) embryo, which offers powerful tools to address macrophage–pathogen interactions. We established that P. aeruginosa oprF mutant is attenuated in zebrafish embryos in a macrophage-dependent manner. Visualization and quantification of P. aeruginosa bacteria phagocytosed by macrophages after injection into closed cavities suggested that the attenuated phenotype of oprF mutant is not linked to higher macrophage recruitment nor better phagocytosis than wild-type strain. Using cultured macrophages, we showed an intramacrophage survival defect of P. aeruginosa oprF mutant, which is correlated with elevated association of bacteria with acidic compartments. Notably, treatment of embryos with bafilomycin, an inhibitor of acidification, increased the sensibility of embryos towards both wild-type and oprF mutant, and partially suppressed the attenuation of oprF mutant. Taken together, this work supports zebrafish embryo as state-of-the-art model to address in vivo the relevance of P. aeruginosa intramacrophage stage. Our results highlight the contribution of macrophages in the clearance of P. aeruginosa during acute infection and suggest that OprF protects P. aeruginosa against macrophage clearance by avoiding bacterial elimination in acidified phagosomes.
Highlights
While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models
Recent studies support the ability of P. aeruginosa to reside, at least transiently, in macrophages and bacterial factors that play a role in the intramacrophage stage, such as OprF, are starting to be identified
We have evaluated for the first time the role of OprF in P. aeruginosa virulence in a vertebrate model harbouring functional macrophages, the zebrafish embryo model
Summary
While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models. The major outer membrane porin OprF has been recently shown to be involved in P. aeruginosa fate within cultured macrophages and analysis of an oprF mutant may provide insights to better understand the relevance of this intramacrophage stage during infection. We explored the behavior of an oprF mutant in connexion with intramacrophage survival during P. aeruginosa acute infection in a non-mammalian vertebrate animal model. P. aeruginosa has been shown to be phagocytosed by macrophages upon acute infection in zebrafish e mbryos[21,22], allowing to study in vivo the bacterial factors involved in P. aeruginosa survival into macrophages and evaluate their importance in the outcome of infection. We first investigated the behavior of oprF mutant strain in vivo upon infection of zebrafish embryo, before moving to further analysis with cultured macrophages. Our results highlight the contribution of macrophages in the clearance of P. aeruginosa during acute infection and support a role of OprF in the ability of P. aeruginosa to avoid localization in acidified phagosomes and resist elimination by macrophages
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