A heat-induced HagF-HrcA regulatory loop promotes chlamydial fitness and infectious progeny yield under stress

Bacterial survival under environmental stress requires precise regulation of protein homeostasis, yet how this is achieved in organisms with streamlined genomes remains incompletely understood. Here, we identify HagF as a previously unrecognized protein that antagonizes the conserved heat-inducible transcriptional repressor HrcA in Chlamydia . Using genetic and biochemical approaches, including overexpression and CRISPRi-mediated knockdown, we show that under non-stressed conditions, HrcA represses hagF along with its five known targets, including hrcA and four chaperone genes. Upon heat stress, HagF is induced and binds HrcA, attenuating its repressor activity. This negative feedback loop enhances chaperone gene induction, particularly from the hrcA-grpE-dnaK operon, and promotes infectious elementary body yield. Notably, HagF is conserved across members of the order Chlamydiales , highlighting a lineage-specific adaptation. Together, these findings define a regulatory circuit in which a specialized antagonist modulates a widely conserved repressor, enabling Chlamydia to couple heat-responsive gene expression with developmental progression and enhance fitness under restrictive conditions.