Visualizing zinc-starved slow-replicating Mycobacterium tuberculosis in neutrophil-rich lung lesions

The months-long treatment for tuberculosis is partially attributed to a minor Mycobacterium tuberculosis (Mtb) subpopulation exhibiting phenotypic drug resistance, although origins of these bacilli in hosts remain unknown. Previously, we observed that mycobacteria respond to growth-restrictive limitations of zinc by accumulating Mrf, and inducing Mpy recruitment to the decoding center of the ribosome, thereby hibernating the ribosome in an inactive state. Mrf accumulation - regulated at both transcriptional and post-translational levels by Zur and Clp, respectively, in a zinc-dependent manner - facilitates the formation of stable Mpy-70S ribosome complex in zinc-starved cells. Here, using a translational fusion of Mrf with a fluorescent-reporter protein we visualized zinc-starved mycobacteria with a physiology conducive for Mpy-dependent ribosome hibernation. About half of zinc-starved mycobacterial population expressed microscopically detectable levels of the reporter and exhibited Mpy-dependent slowdown of growth and isoniazid tolerance in vitro. In chronic, high-burden mouse lung infections, ~3% of Mtb bacilli expressed the reporter, and the frequency increased to ~35% upon isoniazid treatment. Transcriptomics of mouse lungs suggests an important role of S100A8/A9 and ZIP2 in neutrophils in zinc mobilization during Mtb infections. Moreover, depletion of neutrophils, or loss of S100A9 caused significant attenuation of zinc starvation response in Mtb. Altogether, the study offers a new approach for visualizing and manipulating slow-replicating Mtb in host tissues.