SUMMARY This proposal will test the over-arching hypothesis that extra-intestinal pathogenic Escherichia coli (ExPEC) overcomes inhibition by urogenital Lactobacilli via sequential activation of inter-connected acid resistance (AR) mechanisms. We further postulate that transient internalization of ExPEC into vaginal epithelial cells increases fitness by enhancing induction of acid resistance and other mechanisms of persistence that enable bacteria to gain access and survive in otherwise harsh host environments. The hypothesis to be tested has been formulated based on the following strong preliminary data: (1) We discovered a novel AR mechanism in ExPEC that is controlled via a non-canonical signaling system, BtsS-YpdB and it uses L-serine deamination to neutralize bacterial cytosolic pH. We call this new AR mechanism, AR6. (2) BtsS-YpdB signaling is induced during infection and in response to several Lactobacillus species. (3) Deletion of btsS-ypdB significantly decreases ExPEC acid tolerance and vaginal colonization. (4) Strains lacking L-serine deaminases, or BtsS- YpdB differentially react to the inhibitory actions of representative urogenital L. gasseri and L. delbrueckii isolates. (5) Deletion of btsS-ypdB alters the induction and function of the known acid-sensing system EvgSA that controls the most prominent, known AR mechanism, AR2. We will test these hypotheses using the most prevalent ExPEC pathotype, uropathogenic E. coli. Uropathogenic E. coli is the main cause of urinary tract infections (UTIs), an infection that disproportionately afflicts women. Similarly, ExPEC strains are the leading cause of infection-related stillbirths. Despite the dominant paradigm that the low pH of vagina is protective against pathogens, we and others have shown that colonization of the vagina by ExPEC can serve as a nidus for infection of the urinary tract, the cervix, uterine horns and the gravid uterus. Vaginal colonization is therefore a key step in pathogenesis. While several acid resistance (AR) mechanisms have been identified that are active in the gut, the relative contribution of each AR mechanism during ExPEC infection remains undefined. With our aims, we will: Interrogate the significance of transient bacterial expansion in the host as a priming niche for the amplification of acid resistance and other persistence mechanisms (Aim 1). We will evaluate the individual and combined contributions of AR mechanisms to the colonization potential of ExPEC in the vagina, bladder and gut and will elucidate the connection of the novel AR6 pathway we discovered to the induction and function of AR2 (Aim 2). Finally, building on exciting preliminary data we will investigate the potential of urogenital Lactobacilli strains in their ability to override ExPEC acid resistance, aiming to identify effective probiotic strategies to prevent ExPEC reservoir formation in the vagina (Aim 3). Completion these aims will uncover how ExPEC leverage their multiple AR systems to...