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

Preeti Garai,Malika Moussouni,Sophie Bleves,Anne-Béatrice Blanc-Potard,Laurence Berry,Joan Mecsas

doi:10.1371/journal.ppat.1007812

Abstract

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.

Highlights

Pathogenic bacteria are commonly classified as intracellular or extracellular pathogens [1]
We visualized the fate of P. aeruginosa within cultured macrophages, revealing macrophage lysis driven by intracellular P. aeruginosa
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

Summary

Introduction

Pathogenic bacteria are commonly classified as intracellular or extracellular pathogens [1]. Intracellular bacterial pathogens, such as Mycobacterium tuberculosis or Salmonella enterica, can replicate within host cells, including macrophages. Yersinia spp. subvert the functions of phagocytes from the outside, these bacteria subvert macrophage functions within the cell [2]. 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 [7]

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Results

Discussion

Conclusion