HflX controls hypoxia-induced non-replicating persistence in slow growing mycobacteria

GTPase HflX is highly conserved in prokaryotes and is a ribosome splitting factor during heat shock in E. coli. Here we report that HflX produced by slow growing M. tuberculosis and M. bovis BCG is a GTPase that plays a critical role in the pathogens transition to a non-replicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model, that precipitated entry into dormancy. The KO displayed the hallmarks of dormant mycobacteria including phenotypic drug resistance, altered morphology, low intracellular ATP and up-regulated dormancy dos regulon. KO-infected mice displayed increased bacterial burden during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic phase. Unlike fast-growing mycobacteria, BCG HlfX was not involved in antibiotic resistance under normoxia. Proteomics, pull-down and ribo-sequencing supported that mycobacterial HflX is a ribosome binding protein that controls the translational activity of the cell. Collectively, our study provides further insights into the mechanisms deployed by mycobacteria to adapt to their hypoxic microenvironment.