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

## TL;DR

Researchers discovered that macrophages use zinc starvation and zinc toxicity together to fight bacteria, offering new insights into immune defense and potential therapies.

## Contribution

The study introduces a novel high-throughput method combining bacterial reporter and scRNA-seq to reveal coordinated zinc-based immune mechanisms in macrophages.

## Key findings

- Macrophages starve intracellular bacteria of zinc to sensitize them to subsequent zinc toxicity.
- SLC30A4 expression in macrophages influences intracellular bacterial survival and zinc starvation.
- Zinc-sensitive bacteria are more vulnerable to macrophage-mediated zinc toxicity.

## Abstract

We developed high-throughput bacterial reporter–paired single-cell RNA sequencing (scRNA-seq) to track both mammalian and bacterial gene expression in single cells and identified macrophage gene signatures associated with zinc toxicity, as well as those associated with survival of intracellular zinc-stressed bacteria. Through this approach, we found that macrophages starve intracellular Escherichia coli of zinc to sensitize them to subsequent zinc toxicity. Zinc starvation and zinc toxicity have not previously been linked within a single antimicrobial response, so these findings represent a major advance in our understanding of innate immune antibacterial defense. The workflow developed here can be adapted to track pathogen responses to the intracellular environment, providing insights into host–pathogen interactions and opportunities for devising host-directed therapies to combat bacterial infections.

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 Escherichia coli zinc-stress reporter strain. We identified HMDM subpopulations harboring zinc-stressed E. coli and corresponding mammalian genes predicted to be associated with either zinc toxicity or survival of zinc-stressed bacteria. Consistent with the latter, SLC30A4 that encodes zinc exporter ZNT4 was enriched in one subpopulation of HMDM containing zinc-stressed E. coli and its overexpression in human macrophages increased intracellular E. coli survival. At a population level, SLC30A4 expression was rapidly downregulated in human macrophages responding to E. coli and its ectopic expression in macrophages attenuated zinc starvation of intracellular E. coli. 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 E. coli rapidly upregulated znuA messenger RNA (mRNA) that is induced during zinc limitation, with zntA mRNA that is induced during zinc stress peaking later. Moreover, E. coli cultured under conditions of zinc limitation displayed greatly enhanced zinc sensitivity. Susceptibility of zinc-sensitive E. coli to killing by macrophages was also attenuated when zinc uptake by E. coli 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.