The second messenger signaling molecule cyclic di-AMP drives developmental cycle progression in Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia alternates between two functional forms during its developmental cycle: elementary body (EB) and reticulate body (RB). However, the molecular mechanisms governing the transitions between these forms are unknown. Here, we present evidence that cyclic di-AMP (c-di-AMP) is a key factor in triggering the transition from RB to EB (i.e., secondary differentiation) in the chlamydial developmental cycle. By overexpressing or knocking down expression of c-di-AMP synthase genes, we made strains producing different levels of c-di-AMP, which we linked to changes in secondary differentiation status. Increases in c-di-AMP resulted in an earlier increase in transcription of EB-associated genes, and this was further manifested in earlier production of EBs. In contrast, when c-di-AMP levels were decreased, developmental cycle progression was delayed. Based on these data, we conclude there is a threshold level of c-di-AMP needed to trigger secondary differentiation in Chlamydia. This study identifies a mechanism by which secondary differentiation is initiated in Chlamydia and reveals a critical role for the second messenger signaling molecule c-di-AMP in this process.