Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (Delta pscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages. Author summary The ability of professional phagocytes to ingest and kill microorganisms is central to host defense and Pseudomonas aeruginosa has developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen, P. aeruginosa has been reported to be engulfed by macrophages in animal models. Here, we visualized the fate of P. aeruginosa within cultured macrophages, revealing macrophage lysis driven by intracellular P. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival of P. aeruginosa, appeared to play a role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly in mgtC and oprF mutants. We further showed that intramacrophage P. aeruginosa uses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage of P. aeruginosa that could contribute to bacterial dissemination.