Progress in the functional analysis of rickettsial genes through directed mutagenesis of Rickettsia prowazekii phospholipase D

Rickettsioses have afflicted humans worldwide throughout the course of history. Rickettsia prowazekii is the etiological agent of epidemic typhus, a disease transmitted by body lice and capable of massive outbreaks under conditions of compromised hygiene, such as famine, mass migration and war. Fastidious growth requirements and an obligately intracellular lifestyle, preferably within the cytoplasm of the host cell, pose a number of challenges in genetic manipulation of rickettsiae. Driskell et al. describe the successful application of a combination of molecular approaches to generate, isolate and characterize a R. prowazekii mutant lacking a 93-bp sequence of the pld gene responsible for phospholipase D activity. In initial studies, this Δpld mutant of R. prowazekii is shown to be capable of infecting macrophage-like RAW 264.7 cells in culture and quickly escaping from the phagosome into the cytosol. However, in guinea pigs infected by intraperitoneal inoculation, the pld deletion mutant exhibits attenuation of virulence and the ability to induce protective immune responses against virulent R. prowazekii. The fundamental importance of this study lies in the generation of a site-directed gene mutant for subsequent evaluation of the target gene's function(s) in rickettsial pathogenesis and immune defense mechanisms. The results also lend support to the possibility that rickettsiae may either possess multiple phosholipases with different substrate specificities or as yet unknown alternative mechanisms for quick phagosomal escape into the host cytoplasm. Studies aimed at detailed characterization of the combinatorial mutant generated in this study and possibly other complete knockouts for genes with putative functions using relevant in vitro and in vivo models of infection are necessary to further elucidate their roles in the biology of rickettsiae.