Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA^m) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA^m), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA^m significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa. Ingestion of Listeria monocytogenes-contaminated food can be life-threatening for immune compromised individuals, can cause severe gastroenteritis in otherwise healthy people, and is also thought to occur frequently with little consequence. The factors that determine susceptibility to this infection are unknown, due to the lack of an appropriate animal model that closely mimics this wide range of human disease. Mice are highly resistant to oral L. monocytogenes infection, and the prevailing view has been that a low affinity between the bacterial surface protein InlA and E-cadherin expressed on the gut mucosa was largely responsible for limited invasion of the murine intestines. We used a novel food borne model of listeriosis to show that a mouse-adapted InlA offers little advantage over wild type InlA for initial colonization of the gut, and indeed, even bacteria lacking InlA can establish intestinal infection in mice. Thus, other aspects of the murine gastrointestinal environment appear to be the key to innate resistance against oral transmission. Surprisingly, our study uncovered a novel function for InlA later in the infection, when the bacteria begin to spread systemically. The natural feeding model presented here using susceptible and resistant strains of mice should be very useful for future studies investigating both mechanisms of microbial pathogenesis and host responses to oral infection.

It could be argued that we understand the immune response to infection withListeria monocytogenesbetter than the immunity elicited by any other bacteria.L. monocytogenesare Gram-positive bacteria that are genetically tractable and easy to cultivatein vitro, and the mouse model of intravenous (i.v.) inoculation is highly reproducible. For these reasons, immunologists frequently use the mouse model of systemic listeriosis to dissect the mechanisms used by mammalian hosts to recognize and respond to infection. This article provides an overview of what we have learned over the past few decades and is divided into three sections: “Innate Immunity” describes how the host initially detects the presence ofL. monocytogenesand characterizes the soluble and cellular responses that occur during the first few days postinfection; “Adaptive Immunity” discusses the exquisitely specific T cell response that mediates complete clearance of infection and immunological memory; “Use of AttenuatedListeriaas a Vaccine Vector” highlights the ways that investigators have exploited our extensive knowledge of anti-Listeriaimmunity to develop cancer therapeutics.

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlA ^m -expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens of L. monocytogenes were recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication of L. monocytogenes was essential during the intestinal phase of infection. We found that lipoate protein ligase-deficient L. monocytogenes (Δ lplA1 ) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of the L. monocytogenes burden in the gastrointestinal tract is extracellular, but the small proportion of intracellular L. monocytogenes is essential for dissemination to the MLN and systemic organs.

This unit describes general procedures for the lab cultivation and storage of the Gram‐positive facultative intracellular bacterium Listeria monocytogenes. The basic protocols are relevant for a wide scope of applications including microbial genetics and both in vitro and in vivo infection studies. Commonly used L. monocytogenes strains, serotypes, and growth parameters are also discussed. Curr. Protoc. Microbiol. 31:9B.2.1‐9B.2.7. ©2013 by John Wiley & Sons, Inc.

IFN-γ is critical for innate immunity against Listeria monocytogenes (L. monocytogenes), and it has long been thought that NK cells are the major source of IFN-γ during the first few days of infection. However, it was recently shown that a significant number of CD44highCD8+ T cells also secrete IFN-γ in an Ag-independent fashion within 16 h of infection with L. monocytogenes. In this report, we showed that infection with other intracellular pathogens did not trigger this early IFN-γ response and that cytosolic localization of Listeria was required to induce rapid IFN-γ production by CD44highCD8+ T cells. Infection of C57BL/6 mice with an Escherichia coli strain expressing listeriolysin O (LLO), a pore-forming toxin from L. monocytogenes, also resulted in rapid IFN-γ expression by CD8+ T cells. These results suggest that LLO expression is essential for induction of the early IFN-γ response, although it is not yet clear whether LLO plays a direct role in triggering a signal cascade that leads to cytokine production or whether it is required simply to release other bacterial product(s) into the host cell cytosol. Interestingly, mouse strains that displayed a rapid CD8+ T cell IFN-γ response (C57BL/6, 129, and NZB) all had lower bacterial burdens in the liver 3 days postinfection compared with mouse strains that did not have an early CD8+ T cell IFN-γ response (BALB/c, A/J, and SJL). These data suggest that participation of memory CD8+ T cells in the early immune response against L. monocytogenes correlates with innate host resistance to infection.

Type I IFN (IFN-α/β) is thought to enhance growth of the foodborne intracellular pathogen Listeria monocytogenes by promoting mechanisms that dampen innate immunity to infection. However, the type I IFN response has been studied primarily using methods that bypass the stomach and, therefore, fail to replicate the natural course of L. monocytogenes infection. In this study, we compared i.v. and foodborne transmission of L. monocytogenes in mice lacking the common type I IFN receptor (IFNAR1^−/−). Contrary to what was observed using i.v. infection, IFNAR1^−/− and wild-type mice had similar bacterial burdens in the liver and spleen following foodborne infection. Splenocytes from wild-type mice infected i.v. produced significantly more IFN-β than did those infected by the foodborne route. Consequently, the immunosuppressive effects of type I IFN signaling, which included T cell death, increased IL-10 secretion, and repression of neutrophil recruitment to the spleen, were all observed following i.v. but not foodborne transmission of L. monocytogenes. Type I IFN was also previously shown to cause a loss of responsiveness to IFN-γ through downregulation of the IFN-γ receptor α-chain on macrophages and dendritic cells. However, we detected a decrease in surface expression of IFN-γ receptor α-chain even in the absence of IFN-α/β signaling, suggesting that in vivo, this infection-induced phenotype is not type I IFN–dependent. These results highlight the importance of using the natural route of infection for studies of host–pathogen interactions and suggest that the detrimental effects of IFN-α/β signaling on the innate immune response to L. monocytogenes may be an artifact of the i.v. infection model.