Reversible transformation of Anaplasma phagocytophilum using Cre-Lox recombination

Project Summary Global change, including climate warming and human population increases and movement, has facilitated range-expansion of arthropod vectors of human pathogens, with mosquitoes, bugs, and ticks increasingly being reported outside their historic ranges. Concomitantly, there has been an explosive expansion of vector-borne disease agents, especially obligate intracellular bacteria transmitted by ticks including the recently described human anaplasmosis agent, Anaplasma phagoctophilum (Ap) that causes a severe and potentially life- threatening illness. Progress in our understanding of this and related organisms has been hindered by the inherent difficulties with manipulation of obligate intracellular bacteria in the order Rickettsiales. Moreover, over 30% of their encoded genes have no matches in the databases, and are thus of unknown function. While bioinformatics algorithms offer an approach to predict form and function, these predictions are unreliable. At the same time, uptake of existing genetic systems has been slow, in part due to their limitations. To alleviate this situation, we propose to employ a modified himar1 transposon-transposase system that enables efficient replacement of the transposon with another cassette containing a wild-type copy of the mutated gene using recombinase mediated cassette exchange (RMCE), a well-established molecular technique, to restore gene function. We will accomplish our planned projects following four specific aims: Aim 1: Generate a collection of Ap mutants recovered in tick vector and human host cell culture using the himar1 transposon system where the transposon is designed with flanking mis-matched lox sequences to allow subsequent replacement of the transposon using RMCE. Aim 2: Determine insertion sites using whole genome sequencing. This will be facilitated through the use of the Ap-HGE1 isolate for which the genome sequence is available. Aims 1 and 2 will be carried out during the Phase I (R61) component of the application, and set the stage/provide the mutants for the subsequent Aims 3 and 4, which will occupy the Phase II (R33) component of the award. Aim 3: Test infectivity of selected mutants and of genetically “restored” mutants in vitro using multiple tick and mammalian cell lines, and verify gene knock-out/knock-in using RT-PCR to demonstrate absence and restored presence of transcripts, and immune-assays to demonstrate absence/restored presence of gene products. Mutants of interest will be selected for RMCE to restore gene function, and successful RMCE will be verified using site-specific PCR and Sanger sequencing coupled with assessment of restored phenotype. Aim 4: Test infectivity of selected mutants and of genetically “restored” mutants in vivo in ticks and mice, including tick transmission studies.