Project Summary/Abstract Chlamydia genital infections are the most commonly reported infection in the U.S. All Chlamydia species cause an unusual intracellular infection in which there is conversion between a dividing form of the bacterium (reticulate body or RB) and the infectious form (elementary body or EB). RB-to-EB conversion is critical for producing infectious progeny that can spread the infection to a new host cell, but the mechanisms that regulate it are unknown. There has been a longstanding assumption in the Chlamydia field that conversion is regulated by an extrinsic factor. However, we have obtained data to support a new regulatory mechanism in which RB size is used as an intrinsic factor to control conversion. Based on temporal measurements of chlamydial size and number obtained with three-dimensional electron microscopy (3D EM), we hypothesize that RBs undergo size reduction through successive rounds of replication and can only convert into an EB below a size threshold. In Aim 1, we will test this size control mechanism by determining whether RB size is altered when the timing of RB-to-EB conversion onset is changed. In Aim 2, we will investigate if the size of the first RB plays a role in starting the timer of RB size reduction that eventually culminates in RB-to-EB conversion. In Aim 3, we will study how RB size could be used to regulate conversion. We propose a titration mechanism in which EUO, a repressor of late chlamydial genes, is titrated away in smaller RBs to promote conversion. In Aim 4, we will study alternative mechanisms that utilize extrinsic signals to control conversion. Using mathematical modeling and 3D EM analysis, we will test a contact-dependent mechanism, which is based on contact of the RB with the inclusion membrane as the external signal, and a chlamydial communication mechanism in which the external signal is produced by other chlamydiae. These studies will provide important information about the mechanism of RB-to-EB conversion that can be applied in new therapeutic strategies to block the developmental cycle and the production of infectious progeny.