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Summary Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize gene expression variation that underlies distinct infection outcomes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers, monitoring infection phenotypes. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host Type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.

Background & Aims Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting anti-microbial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. Methods We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1f/f x Villin-cre) or CD11c+ cells (Atg16l1f/f x CD11c-cre); these mice were used to assess cell type-specific, anti-bacterial autophagy. All responses were compared to Atg16l1f/f mice (controls). Mice were infected with Salmonella enterica serovar Typhimurium; cecum and small intestine tissues were collected for immunofluorescence, histology, and quantitative reverse transcription PCR analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on anti-bacterial responses in human epithelial cells. Results Autophagy was induced in small intestine and cecum following infection with S Typhimurium, and required Atg16l1. S Typhimurium colocalized with microtubule-associated protein 1 light chain 3 beta (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1f/f x Villin-cre mice. Atg16l1f/f x Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMP. Consistent with these defective immune responses, Atg16l1f/f x Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1f/f x CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. Conclusions Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It is also required to prevent systemic infection of mice with enteric bacteria.