Transposon mutagenesis of Rickettsia for studying Rickettsia-host-vector interactions - Abstract Rickettsia is a genus of arthropod-borne pathogens responsible for typhus and spotted fever diseases. Hallmark of Rickettsia is their reductive genome evolution, obligate intracellular lifecycle within vascular endothelial cells, immune escape in the bloodstream, and transmission via blood-feeding arthropods, with Rickettsia replicating in the gastrointestinal tract or salivary glands of their various vectors (e.g., lice, ticks, mites, or fleas). Recent environmental and sociological changes have supported the invasion and expansion of arthropod vectors into new geographical areas, prompting healthcare alerts for the increasing arthropod-borne rickettsial infections. Owing to the technical difficulties of isolating, propagating, and genetically manipulating Rickettsia, early work studied rickettsial virulence mechanisms by expressing target genes in heterologous organisms (such as Escherichia coli) or characterizing rickettsial variants with different genetic backgrounds. While these studies identified putative virulence genes, they failed to address the biological functions involved in rickettsial pathogenesis, transmission, and host immunity against typhus and spotted fever diseases. To explore the genetic bases of rickettsial pathogenesis, recent studies developed genetic tools for Rickettsia and characterized rickettsial variants. These investigations characterized several Rickettsia mutants with defects in actin-based motility, O-antigen synthesis, and cell-to-cell spread. Nevertheless, genes underpinning rickettsial entry into vascular endothelial cells, intracellular immune evasion and replication, release and survival in the bloodstream, or replication in various cell types of their arthropod vectors remain largely uncharacterized. This application describes an in vitro transposition reaction using purified Tn5-transposase complexed with the kkaebi mini- transposon, which provides for chloramphenicol selection of Rickettsia variants with single chromosomal insertions. DNA sequencing of insertion sites demonstrates the random nature of insertional mutagenesis with the kkaebi-transposon, while chloramphenicol-selection allows for facile isolation of variants that can be analyzed for in vitro replication as well as defects in the pathogenesis and transmission. In the first phase (R61) of this application, kkaebi variants will be generated using two model organisms of pathogenic Rickettsia (R. conorii from the spotted fever group and R. typhi from the typhus group). After the successful completion of the first phase, kkaebi variants will be characterized for phenotypic alterations using biochemical and molecular analyses, tissue culture infections, tick infection models, and mouse infection studies. Overall, this work will generate key experimental resources for the research community to comprehensively identify rickettsial genes essential for pathogenesis and transmission by arthropod vectors.
