Project Summary Tick-borne diseases are on the increase, and are responsible for nearly all of the vector-transmitted disease in the US. Vector-borne pathogens face the dual challenge of adaptation to two very different host environments: the arthropod vector and the mammalian host. Tick-borne pathogens in the order Rickettsiales accomplish this adaptation with the limited toolset encoded by a minimal genome. However, this transition is little studied for any tick-borne pathogen, due to the need for specialized training when working with ticks; organisms that, compared to insect vectors, have long and fastidious rearing requirements. Upon entry into the tick, the pathogen must infect the midgut, then transit into the hemolymph before entering the salivary glands, from where it can be transmitted to a new mammalian host. The challenge faced by these pathogens is to survive intracellularly, avoid the innate immune defenses of the tick, and disseminate and replicate in the midgut and salivary glands. Rickettsial pathogens encode a Type IV Secretion System (T4SS), which has undergone duplication of some of the genes that encode this nanomachine, namely virB2, 4, 6, 8 and 9. While most organisms contain but a single copy of each gene, the reason for multiple copies of these genes has puzzled researchers. VirB2 makes up the pilus of the T4SS, and it has recently been shown that in two Anaplasma species, the virB2 paralogs demonstrated tick or mammalian specific expression patterns leading us to hypothesize that changing the VirB2 component of the T4SS is necessary to achieve transmission. To examine this hypothesis, we will study Anaplasma phagocytophilum, a zoonotic pathogen. A. phagocytophilum has eight paralogous genes encoding VirB2. In studies conducted with cell cultured organisms four paralogs shown to be transcribed in a tick specific manner, while two are transcribed in a mammalian context, with two paralogs remaining silent. We have access to a transposon (Tn) knockout library, in which we have identified 10 Tn mutants in the tick-specific virB2 genes. The availability of these Tn mutants allows us to examine the contribution of the virB2 paralogs to tick transmission in this otherwise genetically intractable organism. In this study, we will first test whether host specific transcription is seen in vivo, and if this carries through to different protein composition for the T4SS in the different hosts. Next, we will use the Tn mutants to determine whether these knockout virB2 mutants have an altered growth phenotype in tick cell culture and finally we will evaluate the mutants for their ability for be transmitted by ticks. Understanding how these pathogens achieve vector-borne transmission will open the door to strategies to interfere with the spread of disease.