Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Mechanisms of intracellular survival of Pseudomonas aeruginosa in Acantamoeba castellanii and raw 264.7 macrophage cells (#317)

Wai Leong 1 , Carla Lutz 1 , Jia Chi Tan 1 , Diane Mcdougald 1
  1. Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore

Pseudomonas aeruginosa is an opportunistic pathogen associated with cystic fibrosis lung infections and nosocomial infections. Although it is known to induce the formation of membrane blebs in epithelial cells, it is not a recognised intracellular pathogen, and little is known about its survival within host cells. Previously, we have shown that P. aeruginosa is able to survive within Acanthamoeba castellanii, and amoeba co-adapted P. aeruginosa is more fit when grown with amoeba compared to the parental strain. Here, we further investigate mechanisms of intracellular survival and replication of P. aeruginosa in amoeba and macrophage using confocal microscopy.

GFP-tagged strains were used to visualise cells intracellularly. Data show that the co-adapted P. aeruginosa replicate within amoeba. During infection, infected host cells secrete bacteriocidal compounds such as NO, peroxides, and anti-microbial peptides into phagosomes. Phagosome-lysosome fusion occurs and intracellular bacteria are further challenged by a decrease in pH. In our study, infected host cells were stained with Lysotracker (Thermo, USA) and DAF-FM diacetate (Thermo, USA) to visualise lysosomes and NO respectively. Phagosome acidification and NO secretion were lower within amoeba phagosomes containing the wild type P. aeruginosa compared to E. coli OP50. As PA2283 (mgtC) was highly upregulated in the transcriptome of intracellular P. aeruginosa, we tested a mutant of PA2283 and demonstrate that intracellular survival was reduced within both amoeba and raw 264.7 macrophage cells. However, phagosome acidification was restored in amoeba but not in macrophage.