Date Published: June 20, 2019
Publisher: Public Library of Science
Author(s): Preeti Garai, Laurence Berry, Malika Moussouni, Sophie Bleves, Anne-Béatrice Blanc-Potard, Joan Mecsas.
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 (ΔpscN) 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.
Pathogenic bacteria are commonly classified as intracellular or extracellular pathogens . Intracellular bacterial pathogens, such as Mycobacterium tuberculosis or Salmonella enterica, can replicate within host cells, including macrophages. In contrast, extracellular pathogens, such as Yersinia spp., Staphylococcus aureus, Pseudomonas aeruginosa or streptococci, avoid phagocytosis or exhibit cytotoxicity towards phagocytic cells, to promote their extracellular multiplication. However, recent data have emphasized that several extracellular pathogens can enter host cells in vivo, resulting in a phase of intracellular residence, which can be of importance in addition to the typical extracellular infection. For example, although Yersinia spp. subvert the functions of phagocytes from the outside, these bacteria also subvert macrophage functions within the cell . Once considered an extracellular pathogen, it is now established that S. aureus can survive within many mammalian cell types including macrophages [3,4] and the intramacrophage fate of S. aureus has been deciphered [5,6]. Moreover, an intracellular phase within splenic macrophages has been recently shown to play a crucial role in initiating dissemination of Streptococcus pneumoniae, providing a divergence from the dogma that considered this bacterial pathogen a classical example of extracellular pathogens .
The ability of professional phagocytes to ingest and kill microorganisms is central to innate immunity and host defense. P. aeruginosa is known to avoid being killed by phagocytes through the destruction of immune cells extracellularly as well as avoidance of phagocytosis . However, P. aeruginosa has been reported to be engulfed by macrophages in animal infection models [21–23]. In addition, P. aeruginosa has been visualized in phagocytes in cell culture models in several studies, where MgtC and OprF have been shown to be involved in the ability of P. aeruginosa to survive in cultured macrophages [25,26]. The virulence of P. aeruginosa mgtC mutant can be restored in zebrafish embryos upon macrophages depletion, suggesting that MgtC acts to evade phagocytes . Interestingly, a similar behavior has been reported for a T3SS mutant in the same infection model . We show here that MgtC and OprF regulate T3SS when P. aeruginosa resides in macrophages and we describe a novel strategy used by P. aeruginosa to escape from macrophages that relies on a T3SS-dependent cell lysis induced by intracellular bacteria (Fig 9).