Against the Odds: Genetic Manipulation of Obligate Intracellular Rickettsia

Members of the genus Rickettsia comprise a diverse group of obligate intracellular bacteria that grow directly within the cytosol of eukaryotic host cells, unbounded by a vacuolar membrane.  R. prowazekii and R. rickettsii, which are vectored by human body lice and ticks, respectively, cause the severe human bacterial diseases, epidemic typhus and Rocky Mountain spotted fever.  While the obligate nature of rickettsial growth and the requirement for Biosafety Level 3 laboratory practices place significant barriers on the genetic manipulation of these unusual pathogens, significant progress has been made.  Transformation systems have been developed and the ability to generate both transposon insertional mutants and specific gene knockouts via homologous recombination has been accomplished. In addition, fluorescent markers have been introduced into rickettsiae on transposons as well as on newly characterized plasmid vectors, allowing fluorescence activated cell sorting to be used for the identification and purification of selected rickettsial populations.  Although the study of bacteria that replicate only in an environment where cytoplasm is on both sides of their membrane remains challenging, the tools are now available to dissect rickettsial obligate intracellular growth and virulence.