Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

AbstractBackgroundArcanobacterium haemolyticumis an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. Thepldgene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition.ResultsRecombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as apldmutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, thepldmutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type orpldmutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated withA. haemolyticumstrains revealed that thepldmutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities.ConclusionsThese data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis ofA. haemolyticuminfections.