Forward genetics to identify Listeria monocytogenes components that activate mammalian cytosolic surveillance pathways

Listeria monocytogenes is Gram-positive facultative intracellular pathogen that is ubiquitously present in the environment. During infection, L. monocytogenes is phagocytosed by host cells and then rapidly escapes from the phagosome into the host cytosol. In the cytosol the bacterium is able to replicate and spread to neighboring cells without leaving the intracellular compartment. Maintenance of the intracellular niche is critical for L. monocytogenes pathogenesis, but there have been several reports indicating cytosolic bacteria can induce inflammasome-mediated host cell death. Additionally, our laboratory previously identified a host transcriptional profile induced by L. monocytogenes upon entry into the cytosol termed the cytosolic surveillance pathway, characterized by the robust upregulation of IFN-beta; and co-regulated genes. Although the host components of the signaling pathways leading to cell death and IFN-beta; production are becoming better defined, the bacterial ligands were largely unknown. To gain insight into the bacterial components and ligands required for stimulation of these two pathways, we performed a forward genetic screen and isolated mutants that affected induction of host cell death and/or IFN-beta. We identified a transposon insertion in lmo2473 that caused hyperstimulation of inflammasome-mediated cell death, pyroptosis. Further characterization revealed these mutants, and to a lesser extent wild-type L. monocytogenes, lysed in the macrophage cytosol. Intracellular bacteriolysis released bacterial DNA that was detected by the cytosolic DNA sensor AIM2 to trigger pyroptosis. Tight regulation of other bacterial factors capable of stimulating inflammasomes, such as flagellin, likely contribute to the low levels of pyroptosis induced by L. monocytogenes. We also isolated a mutant that harbored a transposon insertion in the gene lmo0052, renamed pdeA. Biochemical characterization of the gene product determined PdeA was a cyclic di-AMP phosphodiesterase. Inactivation of this protein resulted in accumulation of the nucleotide and increased stimulation of IFN-beta;. As cyclic di-AMP was only recently identified as a bacterial second messenger, we used mutants that modulate enzymatic processing of c-di-AMP to interrogate the role of its signaling in L. monocytogenes physiology and pathogenesis. This work implicates c-di-AMP, not only as the IFN-beta;-stimulatory ligand, but also as a critical molecule involved in bacterial growth, response to stress, and cell wall stability. In summary, using a forward genetic approach, we have identified mechanisms by which host cells recognize and respond to L. monocytogenes invasion of the cytosol. Other mutants isolated in this screen merit further characterization to potentially unveil additional bacterial components involved in these responses