A Multiorgan Trafficking Circuit Provides Purifying Selection of Listeria monocytogenes Virulence Genes.

Listeria monocytogenes can cause a life-threatening illness when the foodborne pathogen spreads beyond the intestinal tract to distant organs. Many aspects of the intestinal phase of L. monocytogenes pathogenesis remain unknown. Here, we present a foodborne infection model using C57BL/6 mice that have been pretreated with streptomycin. In this model, as few as 100 L. monocytogenes CFU were required to cause self-limiting enterocolitis, and systemic dissemination followed previously reported routes. Using this model, we report that listeriolysin O (LLO) and actin assembly-inducing protein (ActA), two critical virulence determinants, were necessary for intestinal pathology and systemic spread but were dispensable for intestinal growth. Sequence tag-based analysis of microbial populations (STAMP) was used to investigate the within-host population dynamics of wild-type and LLO-deficient strains. The wild-type bacterial population experienced severe bottlenecks over the course of infection, and by 5 days, the intestinal population was highly enriched for bacteria originating from the gallbladder. In contrast, LLO-deficient strains did not efficiently disseminate and gain access to the gallbladder, and the intestinal population remained diverse. These findings suggest that systemic spread and establishment of a bacterial reservoir in the gallbladder imparts an intraspecies advantage in intestinal occupancy. Since intestinal L. monocytogenes is ultimately released into the environment, within-host population bottlenecks may provide purifying selection of virulence genes.IMPORTANCE Listeria monocytogenes maintains capabilities for free-living growth in the environment and for intracellular replication in a wide range of hosts, including livestock and humans. Here, we characterized an enterocolitis model of foodborne L. monocytogenes infection. This work highlights a multiorgan trafficking circuit and reveals a fitness advantage for bacteria that successfully complete this cycle. Because virulence factors play critical roles in systemic dissemination and multiple bottlenecks occur as the bacterial population colonizes different tissue sites, this multiorgan trafficking circuit likely provides purifying selection of virulence genes. This study also serves as a foundation for future work using the L. monocytogenes-induced enterocolitis model to investigate the biology of L. monocytogenes in the intestinal environment

The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity

Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo

The PAMP c-di-AMP Is Essential for Listeria monocytogenes Growth in Rich but Not Minimal Media due to a Toxic Increase in (p)ppGpp

SummaryCyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium

Patterns of Pathogenesis: Discrimination of Pathogenic and Nonpathogenic Microbes by the Innate Immune System

The dominant conceptual framework for understanding innate immunity has been that host cells respond to evolutionarily conserved molecular features of pathogens called pathogen-associated molecular patterns (PAMPs). Here, we propose that PAMPs should be understood in the context of how they are naturally presented by pathogens. This can be experimentally challenging, since pathogens, almost by definition, bypass host defense. Nevertheless, in this review, we explore the idea that the immune system responds to PAMPs in the context of additional signals that derive from common “patterns of pathogenesis” employed by pathogens to infect, multiply within, and spread among their hosts

Activation of the Listeria monocytogenes Virulence Program by a Reducing Environment.

Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivoIMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence

Bacterial Ligands Generated in a Phagosome Are Targets of the Cytosolic Innate Immune System

Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN)–γ leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR) signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-β response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-β production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol

Mice Lacking the Type I Interferon Receptor Are Resistant to Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that induces a cytosolic signaling cascade resulting in expression of interferon (IFN)-β. Although type I IFNs are critical in viral defense, their role in immunity to bacterial pathogens is much less clear. In this study, we addressed the role of type I IFNs by examining the infection of L. monocytogenes in BALB/c mice lacking the type I IFN receptor (IFN-α/βR−/−). During the first 24 h of infection in vivo, IFN-α/βR−/− and wild-type mice were similar in terms of L. monocytogenes survival. In addition, the intracellular fate of L. monocytogenes in macrophages cultured from IFN-α/βR−/− and wild-type mice was indistinguishable. However, by 72 h after inoculation in vivo, IFN-α/βR−/− mice were ∼1,000-fold more resistant to a high dose L. monocytogenes infection. Resistance was correlated with elevated levels of interleukin 12p70 in the blood and increased numbers of CD11b+ macrophages producing tumor necrosis factor α in the spleen of IFN-α/βR−/− mice. The results of this study suggest that L. monocytogenes might be exploiting an innate antiviral response to promote its pathogenesis

Pivotal role of VASP in Arp2/3 complex–mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility

The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is ∼10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases

Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment.

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells