The incidence of tick-borne diseases in the United States (U.S.) has more than doubled since 2004, and a subset of tick-borne diseases are caused by bacteria known as Spotted Fever Group Rickettsia. Rickettsia initially infects the vasculature and surrounding skin, with severe infections spreading to other tissues (e.g., lungs and brain), leading to rashes, fever, or death. Since 2000, the incidence of U.S. Rickettsia infections has increased more than 10-fold, adding to the emerging public health threat of tick-borne diseases. U.S. military personnel are at a particularly high risk to this threat, because they train in areas with higher burdens of infected ticks or are deployed to regions with endemic Rickettsia. Consequently, efforts are underway to develop new animal models, diagnostics, and treatments. However, many of these efforts are hampered by a lack of sophisticated tools and a basic understanding of Rickettsia pathogenesis. To reduce the public health burden of Rickettsia diseases, we need to know which bacterial factors drive infection so that we can develop appropriate therapeutic innervations. In this proposal, we have taken the innovative approach to develop tools for the identification and study of bacterial proteins required during infection. We will use advanced protein identification methods to determine which bacterial proteins promote invasion, growth, and spread in human and tick cells. We also will develop modern genetic tools so we can test the function of specific bacterial proteins during infection. These contributions will fulfill a critical need set forth by the Tick-Borne Disease Research Program to uncover these novel factors of Rickettsia pathogenesis so that we can determine how they impact disease severity and promote immune evasion. Our work will also enable future efforts to design effective diagnostics and vaccine candidates to combat disease by targeting bacterial proteins that are essential for infection.