A Universal Screen for Genetic and Phenotypic Factors Affecting Phagocytic Susceptibility of P. aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects burn wounds and the lungs of cystic fibrosis (CF) patients.  Chronic P. aeruginosa infection is noted for its correlation with a worsening prognosis for CF patients.  P. aeruginosa isolated from patients exhibit down regulated flagellar genes and motility, enabling the bacterium to evade phagocytosis. The deletion of the FlhF gene from the clinical isolate PAK results in an intriguing phenotype in which biogenesis of the flagellum occurs at atypical positions on the bacterium, which consequently display reduced motility. However, it has not been determined how loss of FlhF alters phagocytosis of the bacteria.  We have begun an investigation into the phagocytic susceptibility of PAK as well as associated isogenic motility mutants, including ΔFlhF.  To screen for P. aeruginosa mutants adapted against phagocytosis, we have generated a modification of the standard gentamicin protection assay using a multi-well plate format. PAK and PA14 control strains are coincubated with dendritic cells (DCs) followed by lysis of the DCs, and finally, growth of the live phagocytosed bacteria in liquid culture within the same plate.  The optical density (OD) is subsequently used to quantitatively measure phagoctyosis since OD is proportional to initial bacterial concentration in the lysate. We recently discovered that ΔFlhF has nearly a 100-fold decrease in phagocytosis compared to the wild type, demonstrating for the first time its phagocytic resistance.  Based on this, current experiments utilizing this method of screening for phagocytic susceptibility are being developed for lysogenized P. aeruginosa.