RECON gene disruption enhances host resistance to enable genome-wide evaluation of intracellular pathogen fitness during infection.

Unlabelled: Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in that renders the resulting RECON mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen . We identified 135 genes which were required for growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for survival and investigated the role of the folate enzyme FolD in liver pathogenesis. A mutant lacking was impaired for growth in murine livers by 2.5-log compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log and 3-log, respectively, but showed modest phenotypes in models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the growth defect, as deletion of the operon in the ∆ background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for host infection, which highlights the utility of the RECON mouse model for future screening efforts.

Importance: is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON mouse that is restrictive to systemic infection. We utilized this model to screen for genes required via transposon sequencing. We identified the liver-specific gene and a repressor, , that only exhibits an growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic . A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.