Rickettsia felis was originally identified in the United States as a human pathogen in 1991 and is now associated with human infection in North and South America, Europe, Africa, Asia, and Oceania. Our ultimate goal for this research is to elucidate the biological and molecular mechanisms that are critical to rickettsial transmission by fleas in order to better understand the epidemiology of flea-borne rickettsial diseases and identify novel points of intervention. Recent discoveries including transmission of R. felis in the absence of a rickettsemic host and the identification of multiple flea-borne rickettsial agents cocirculating in flea populations have guided the research to determine if sympatric rickettsial agents circulating in flea populations influence the transmission one another. Additionally, the assembly of the cat flea genome now allows for investigation of the flea-derived factors that facilitate or prevent Rickettsia transmission. The experimental focus of this proposal is to delineate horizontal transmission mechanisms through comparative analyses of coinfections using three distinct rickettsial strains in cat flea transmission bioassays. The flea-derived molecules associated with the transmission of Rickettsia by flea hosts are not known. Studies will also employ gene-editing in fleas to identify transmission determinants in a flea transmission system. Two limiting factors for vector/disease management and the barriers to advancing the field are the scant knowledge of 1) basic transmission biology of R. felis and other rickettsial pathogens in the context of coinfections and, 2) the flea-derived determinants of transmission. The need to overcome these barriers is evident as field collected fleas in areas of flea-borne rickettsioses outbreaks have multiple rickettsial agents circulating in the vector population and the fleas are known to be vectors of a number of pathogens, thus providing knowledge with a broad impact. Through completion of the specific aims outlined in this proposal, these studies will overcome the hurdles by delineating the role of coinfections in the transmission of R. felis (Specific Aim 1) and through identification of flea-derived molecules regulated in response to rickettsial infection relates to vector competence (Specific Aim 2). Thus, this is a multifaceted approach to decipher the vector and pathogen-associated factors essential to transmission and will provide a platform to examine other flea-borne bacterial pathogens.