Bacterial reporter–paired scRNA sequencing reveals cross talk between zinc starvation and zinc toxicity in macrophage antibacterial defense

Mechanisms by which macrophages deploy antibacterial zinc toxicity are poorly understood. To gain insight into this antimicrobial pathway, we developed bacterial reporter-paired single-cell RNA sequencing of human monocyte-derived macrophages (HMDM) infected with an <i>Escherichia coli</i> zinc-stress reporter strain. We identified HMDM subpopulations harboring zinc-stressed <i>E. coli</i> and corresponding mammalian genes predicted to be associated with either zinc toxicity or survival of zinc-stressed bacteria. Consistent with the latter, <i>SLC30A4</i> that encodes zinc exporter ZNT4 was enriched in one subpopulation of HMDM containing zinc-stressed <i>E. coli</i> and its overexpression in human macrophages increased intracellular <i>E. coli</i> survival. At a population level, <i>SLC30A4</i> expression was rapidly downregulated in human macrophages responding to <i>E. coli</i> and its ectopic expression in macrophages attenuated zinc starvation of intracellular <i>E. coli</i>. This is consistent with a model in which macrophages switch off SLC30A4 to engage zinc starvation, while also deploying zinc toxicity against bacteria adapting to a low-zinc environment. Consistent with this, intramacrophage <i>E. coli</i> rapidly upregulated <i>znuA</i> messenger RNA (mRNA) that is induced during zinc limitation, with <i>zntA</i> mRNA that is induced during zinc stress peaking later. Moreover, <i>E. coli</i> cultured under conditions of zinc limitation displayed greatly enhanced zinc sensitivity. Susceptibility of zinc-sensitive <i>E. coli</i> to killing by macrophages was also attenuated when zinc uptake by <i>E. coli</i> was inactivated, confirming the coordinated actions of zinc starvation and zinc toxicity in macrophage antibacterial responses. Strategies that enhance zinc starvation of intracellular bacteria could be exploited in the design of host-directed therapeutics that amplify macrophage-mediated antibacterial zinc toxicity.