Cyclic di-AMP drives secondary differentiation 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 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. 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, secondary differentiation 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 is the first study to define a mechanism of secondary differentiation in Chlamydia. Overall design: The transformants of dacAop and dacA-KD were infected into HeLa cells. At 10 hpi, the constructs were induced or not with 5 nM aTc. RNA samples were prepared from dacAop-infected cells and dacA-KD-infected cells at 16 hpi and 24 hpi, respectively. 20 µg RNA samples were treated with DNase to remove DNA contamination by using DNA-free Turbo kit (Thermo) according to the manusfacturer's instruction. Ribosomal RNA was depleted from samples using the MICROBEnrich (Thermo) and MICROBExpress kits (Thermo) following the manufacturer's instructions. RNA samples were processed for sequencing by the UNMC Genomics Core Facility.