B. parapertussis hide and persist alive inside immune cells

GORGOJO, JUAN PABLO; OVIEDO, JUAN MARCOS; RODRIGUEZ, MARÍA EUGENIA

Mar del plata, Buenos Aires

Congreso; LIX Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica y LXII Reunión Científica Anual de la Sociedad Argentina de Inmunología; 2014

Sociedad Argentina de Investigación Clínica y Sociedad Argentina de Inmunología. SAI/SAIC

Polymixin B protection assays have previously shown that in the absence of opsonic antibodies B. parapertussis survives the innate interaction with macrophages and persist intracellularly up to 48 h post infection. However, the actual location of live bacteria has never been determined. In order to investigate this issue we performed a fluorescent in situ hybridization (FISH) assay to image live B. parapertussis inside macrophages derived from human blood. Confocal analysis showed that 14 % of the macrophages contained a high number of intracellular live B. parapertussis (~10 bacteria per cell). As assessed by immunostaining combined with FISH live bacteria in these macrophages represented 78% of the intracellular bacteria. Additional experiments demonstrated that solely those bacteria that were not colocalizing with the acid tropic dye Lysotracker were alive, confirming that the evasion of phagolysosomal fusion is required for B. parapertussis intracellular survival. In agreement with the hypothesis that lipid raft enrichment of the phagosome is an effective immune evasion mechanism to avoid the degradative pathway we found that live intracellular B. parapertussis were found in flotillin enriched phagosomes (a lipid raft marker) and retained early endosome characteristics. Since the bacterial survival within compartments that remain integral to the endosomal network carries the risk of continued sampling of bacterium-derived peptides and their presentation to T cells, we next analyzed the cell surface HLA-DR expression in macrophages infected with B. parapertussis. Using flow cytometry we observed that B. parapertussis macrophage infection inhibits HLA-DR increase in response to INF. Taken together these results suggest that B. parapertussis has evolved mechanisms that allows it to hide inside host macrophages even under inflammatory conditions.