Development of a Recombinase-Mediated Cassette Exchange System for Gene Knockout and Expression of Non-Native Gene...

Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens.

Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences.

This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice.

The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development.