Acquired cell-mediated immunity to Listeria monocytogenes is induced by infection with live, replicating bacteria that grow in the host cell cytosol, whereas killed bacteria, or those trapped in a phagosome, fail to induce protective immunity. In this chapter, we focus on how L. monocytogenes is sensed by the innate immune system, with the presumption that innate immunity affects the development of acquired immunity. Infection by L. monocytogenes induces three innate immune pathways: an MyD88-dependent pathway emanating from a phagosome leading to expression of inflammatory cytokines; a STING/IRF3-dependent pathway emanating from the cytosol leading to the expression of IFN-β and coregulated genes; and very low levels of a Caspase-1-dependent, AIM2-dependent inflammasome pathway resulting in proteolytic activation and secretion of IL-1β and IL-18 and pyroptotic cell death. Using a combination of genetics and biochemistry, we identified the listerial ligand that activates the STING/IRF3 pathway as secreted cyclic diadenosine monophosphate, a newly discovered conserved bacterial signaling molecule. We also identified L. monocytogenes mutants that caused robust inflammasome activation due to bacteriolysis in the cytosol, release of DNA, and activation of the AIM2 inflammasome. A strain was constructed that ectopically expressed and secreted a fusion protein containing Legionella pneumophila flagellin that robustly activated the Nlrc4-dependent inflammasome and was highly attenuated in mice, also in an Nlrc4-dependent manner. Surprisingly, this strain was a poor inducer of adaptive immunity, suggesting that inflammasome activation is not necessary to induce cell-mediated immunity and may even be detrimental under some conditions. To the best of our knowledge, no single innate immune pathway is necessary to mount a robust acquired immune response to L. monocytogenes infection.

Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD⁺ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD⁺ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD⁺ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.