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

Abstract

AbstractTransposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seqin vivois 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 inAkr1c13that renders the resulting RECON-/-mouse resistant to high dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogenListeria monocytogenes in vivo. We identified 139 genes which were required forL. monocytogenesgrowth in mice including novel genes not previously identified for host survival. We identified organ specific requirements forL. monocytogenessurvival and investigated the role of the folate enzyme FolD inL. monocytogenesliver pathogenesis. A mutant lackingfolDwas impaired for growth in murine livers by 2.5-log10compared to wildtype and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant inalsR,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-log10and 3-log10, respectively, but showed modest phenotypes inin vitromodels. We confirmed that dysregulation of the D-allose catabolism operon is responsible for thein vivogrowth defect, as deletion of the operon in the ΔalsRbackground rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants forL. monocytogeneshost infection, which highlights the utility of the RECON-/-mouse model for future screening efforts.ImportanceListeria monocytogenesis the gram-positive bacterium responsible for the food-borne disease Listeriosis. Although infections withL. monocytogenesare limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements forL. monocytogenes in vivosurvival 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 systemicL. monocytogenesinfection. We utilized this model to screen forL. monocytogenesgenes requiredin vivovia Tn-seq. We identified the liver-specific genefolDand a repressor,alsRthat only exhibits anin vivogrowth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.