Zinc-limited <i>Mycobacterium tuberculosis</i> stimulate distinct responses in macrophages compared with standard zinc-replete bacteria

Tuberculosis (TB) is notoriously difficult to treat, likely due to the complex host-pathogen interactions driven by pathogen heterogeneity. An understudied area of TB pathogenesis is host responses to <i>Mycobacterium tuberculosis</i> bacteria (Mtb) that are limited in zinc ions. This distinct population resides in necrotic granulomas and sputum and could be the key player in tuberculosis pathogenicity. In this study, we tested the hypothesis that macrophages differentiate between Mtb grown under zinc limitation or in the standard zinc-replete medium. Using several macrophage infection models, such as murine RAW 264.7 and murine bone marrow-derived macrophages (BMDMs), as well as human THP-1-derived macrophages, we show that macrophages infected with zinc-limited Mtb have increased bacterial burden compared with macrophages infected with zinc-replete Mtb. We further demonstrate that macrophage infection with zinc-limited Mtb trigger higher production of reactive oxygen species (ROS) and cause more macrophage death. Furthermore, the increased ROS production is linked to the increased phagocytosis of zinc-limited Mtb, whereas cell death is not. Finally, transcriptional analysis of RAW 264.7 macrophages demonstrates that macrophages have more robust pro-inflammatory responses when infected with zinc-limited Mtb than zinc-replete Mtb. Together, our findings suggest that Mtb's access to zinc affects their interaction with macrophages and that zinc-limited Mtb may be influencing TB progression. Therefore, zinc availability in bacterial growth medium should be considered in TB drug and vaccine developments.