Mycoplasma genitalium (MG) is an emerging sexually transmitted pathogen that causes non-gonococcal urethritis in men and has been significantly associated with a range of reproductive tract diseases in women. Clinically, MG eradication remains a difficult task, and high rates of treatment failure indicate MG can cause persistent infection. It is unclear how MG persists in the host genital tract. Although antigenic variation has long been believed to contribute to MG persistence, this mechanism fails to explain MG persistent infection following standard antibiotic treatment. Our studies indicate that MG is capable of host cell invasion in vitro and in vivo and that intracellular MG can survive long-term in the presence of certain antibiotics and intact immune system. These findings have led us to hypothesize that MG invasion of host cells is required for persistent infection. On the basis of our success of obtaining MG mutants that are deficient in cell invasion but retain intact cytadherence capabilities, this proposal is aimed to gain insight into the mechanisms of MG persistent infection. We propose to (1) identify MG genes required for host cell invasion; and (2) determine whether MG invasion is required for MG persistence and pathogenesis. The proposed studies will substantiate the role of MG invasion and intracellular localization in persistent infection and pathogenesis. Knowledge obtained will set the stage for further dissecting MG-host interactions and improving clinical management of MG infection.