SURFACE-AGGREGATED Pseudomonas aeruginosa IS ELIMINATED AFTER BEING INTERNALIZED INTO EPITHELIAL CELLS

MARIA VICTORIA PEPE; DARÍO CAPASSO; JÉSSICA ROSSELLO; WALTER SPERAT; ARLINET KIERBEL

Congreso; X CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL SAMIGE; 2014

Pseudomonas aeruginosa (PA) is an environmental bacterium of wide distribution. It is also an opportunistic pathogen capable of causing acute as well as chronic infections of high severity. One of the key factors rendering treatment of PA-infections difficult is the formation of biofilms. Biofilms are multicellular communities of bacteria held together by a self-produced extracellular matrix. In our laboratory we study the formation of PA multicellular structures in the context of host cells, as well as the response of host cells to the formation of such structures. In previous studies we demonstrated, by infecting cultures of polarized epithelial cells, that when PA contacts the cell surface, it forms biofilm-like aggregates in a sequential and minute-length process. Also, we showed that those recently formed aggregates could be internalized into epithelial cells. On the basis of these results, we wondered about the fate of PA upon internalization. In order to answer this question we performed standard antibiotic protection assays measuring the number of intracellular bacteria at 3, 6, 9 and 24 hours post-infection (HPI). These assays were carried out using three different antibiotic concentrations, so as to find the suitable antibiotic concentration that eliminates extracellular bacteria but did not cause death of internalized PA by non-specific antibiotic uptake through pinocytosis. An increase in intracellular bacterial number was measured 6 HPI compared to 3 HPI. But at 9 HPI the number of bacteria decreased remarkably, and at 24 HPI no intracellular bacteria were detected. These results suggest that PA is uncapable of surviving within epithelial cells for prolonged periods of time. To assess the mechanisms involved in intracellular PA elimination, subcellular localization studies were performed. 90% of intracellular PA was found in LAMP1 positive-vesicles. It was also found that approximately 50% of PA-containing vesicles were acidified. These findings suggest that PA is transported to lysosomes, where it would be eliminated. Currently, PA metabolic activity within those epithelial subcellular compartments is being studied.