Expression of the Rickettsia prowazekii pldor tlyCGene in Salmonella entericaSerovar Typhimurium Mediates Phagosomal Escape

ABSTRACTMembers of the genus Rickettsiapossess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (tlyA, tlyC, pat1, and pld). This study was undertaken to determine which of the selected genes of Rickettsia prowazekiimediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that tlyCand pldwere transcribed during the period of active phagosomal escape but that tlyAand pat1were not. The functionality of both tlyCand pldwas determined by complementation studies of Salmonella, which replicates within endosomes. Complementation of Salmonellaorganisms with either tlyCor pldresulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for tlyCand pldin the process of phagosomal escape by R. prowazekii.