Aggregate formation by Pseudomonas aeruginosa upon host cell contact

PAOLA LEPANTO; JÉSSICA ROSSELLO; DAVID BRYANT; KEITH MOSTOV; ARLINET KIERBEL

Simposio; 14th Bay Area Microbial Pathogenesis Symposium, University of California San Francisco, San Francisco, U.S; 2011

Background: Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is capable of causing acute infections in individuals with wounds or those with immune defects, as well as chronic infections with high mortality in cystic fibrosis (CF) patients. Biofilm formation is a key factor in P. aeruginosa CF-associated airway infections. Transition from a planktonic state to surface-associated multicellular structures or aggregates is an important early step in biofilm development. But the process by which P. aeruginosa makes such transition during infections has not been described to date. After associating with the host cell surface, P. aeruginosa can become intracellular. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) is necessary for P. aeruginosa entry from the apical surface of polarized epithelial cells and that large membrane protrusions enriched for phosphatidylinositol 3,4,5-trisphosphate (PIP3) formed at the apical site of bacterial attachment. In those studies we observed P. aeruginosa often attached to the apical surface as bacterial aggregates. Material and Method: Polarized MDCK epithelial cells grown on filters were infected with an invasive strain of P. aeruginosa. Fixed samples were analyzed by Scanning electron and confocal microscopy. Dynamics of bacterial attachment was followed by live-cell time-lapse video microscopy. Silencing of host cell genes was generated using short hairpin RNAs delivered by lentiviral vectors. Results: In the present work we investigated the origin of P. aeruginosa aggregates and their implication in the host cell response elicited by infection. We found that P. aeruginosa makes the transition from planktonic to host cell-attached multicellular structures by recruitment of bacteria to localized spots on the cell surface. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell?cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with the previously described host membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates PI3K activation, PIP3-enriched protrusion formation and aggregate internalization Conclusion: Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Processes such as adhesion and internalization, as well as other infection mechanisms, take a new dimension when studied in this context. We are currently extending our studies to understand activation of Type Three Secretion System of bacteria contacting host cells as multicellular structures.