Sarah E. F. D'ORazio

Certain strains of the facultative intracellular bacterial pathogen Listeria monocytogenes are thought to invade cranial nerves in the gut and disseminate directly to the brainstem to cause rhombencephalitis in both humans and ruminants. Bacteria with actin tails were previously observed within neurons of naturally infected sheep, but the mechanism for how these neurotropic strains access the nervous system has not been well characterized. Using a foodborne mouse model of listeriosis, we show here that bypassing the gut phase of infection prevents colonization of the brain, confirming that invasion of the nervous system happens in the intestinal tract. L. monocytogenes did not efficiently invade neuronal cell lines, although they could replicate exponentially in the cytosol and form actin tails. Instead, the neurotropic strains displayed a preferential ability to invade enteric glial cells, a specialized subset of glia that support neurons and are critical for intestinal homeostasis. Using an in vitro co-culture system, we demonstrated that neurotropic L. monocytogenes could readily invade enteric glial cells and use ActA-mediated actin-based motility to spread to adjacent neurons. These results suggest that invasion of enteric glial cells is a novel virulence strategy that can promote brainstem infection following foodborne transmission of L. monocytogenes . IMPORTANCE: This study provides further evidence for dissemination of neurotropic strains of L. monocytogenes from the gut directly to the brain via axonal migration using foodborne mouse model of listeriosis. It is the first report showing that enteric glial cells, a specialized subset of cells in the gut that support intestinal neurons, are susceptible to pathogenic bacterial infection.

Listeria monocytogenes are facultative intracellular bacterial pathogens that cause foodborne disease in humans. The bacteria can use the surface protein InlA to invade intestinal epithelial cells or transcytose across M cells in the gut, but it is not well understood how the bacteria traffic from the underlying lamina propria to the draining mesenteric lymph nodes (MLN). Previous studies indicated that L. monocytogenes associated with both monocytes and dendritic cells in the intestinal lamina propria. We show here that CCR2 ^−/− mice had a significant reduction in Ly6C ^hi monocytes in the MLN but no change in bacterial burden following foodborne infection; thus, dissemination of L. monocytogenes associated with monocytes is not required for colonization of the MLN. To block CCR7-mediated trafficking of dendritic cells from the lamina propria, we treated mice with anti-VEGFR3 antibody (clone AFL4) prior to and during infection but did not see a change in dendritic numbers in the MLN as had been previously reported with other anti-VEGFR3-specific antibodies. However, increasing the number of circulating dendritic cells by treating mice with rFlt3L resulted in a significant increase in L. monocytogenes in the lymph nodes that drain the small intestine and the spleen. Whole-mount fluorescent microscopy of lymphatic vessels following ligated loop infection revealed both free-floating L. monocytogenes and cell-associated bacteria within lymphatic vessels. Together, these results suggest that L. monocytogenes can use multiple, redundant mechanisms to disseminate from the gut tissue to the MLN. IMPORTANCE: Consumption of the foodborne bacterial pathogen Listeria monocytogenes results in a wide spectrum of human disease from mild self-limiting gastroenteritis to life-threatening infections of the bloodstream, brain, and placenta. It is not well understood how the bacteria migrate from the intestines to the draining mesenteric lymph nodes, which are thought to serve as the last barrier to prevent systemic infections. Results presented here reveal multiple redundant mechanisms L. monocytogenes can use to disseminate from the ileum or colon to the mesenteric lymph nodes.

Lymph node stromal cells (LNSC) are an often overlooked component of the immune system, but play a crucial role in maintaining tissue homeostasis and orchestrating immune responses. Our understanding of the functions these cells serve in the context of bacterial infections remains limited. We previously showed that Listeria monocytogenes, a facultative intracellular foodborne bacterial pathogen, must replicate within an as-yet-unidentified cell type in the mesenteric lymph node (MLN) to spread systemically. Here, we show that L. monocytogenes could invade, escape from the vacuole, replicate exponentially, and induce a type I IFN response in the cytosol of two LNSC populations infected in vitro, fibroblastic reticular cells (FRC) and blood endothelial cells (BEC). Infected FRC and BEC also produced a significant chemokine and pro-inflammatory cytokine response after in vitro infection. Flow cytometric analysis confirmed that GFP⁺ L. monocytogenes were associated with a small percentage of MLN stromal cells in vivo following foodborne infection of mice. Using fluorescent microscopy, we showed that these cell-associated bacteria were intracellular L. monocytogenes and the number of infected FRC and BEC changed over the course of a three-day infection in mice. Ex vivo culturing of these infected LNSC populations revealed viable, replicating bacteria that grew on agar plates. These results highlight the unexplored potential of FRC and BEC to serve as suitable growth niches for L. monocytogenes during foodborne infection and to contribute to the pro-inflammatory environment within the MLN that promotes clearance of listeriosis.

Apolipoprotein E (ApoE) is a lipid transport protein that is hypothesized to suppress proinflammatory cytokine production, particularly after stimulation with Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). Studies using transgenic ApoE human replacement mice (APOE) expressing one of three different allelic variants suggest that there is a hierarchy in terms of responsiveness to proinflammatory stimuli such as APOE4/E4 > APOE3/E3 > APOE2/E2. In this study, we test the hypothesis that APOE genotype can also predict susceptibility to infection with the facultative intracellular gram-positive bacterium Listeria monocytogenes . We found that bone-marrow-derived macrophages isolated from aged APOE4/E4 mice expressed elevated levels of nitric oxide synthase 2 and were highly resistant to in vitro infection with L. monocytogenes compared to APOE3/E3 and APOE2/E2 mice. However, we did not find statistically significant differences in cytokine or chemokine output from either macrophages or whole splenocytes isolated from APOE2/E2, APOE3/E3, or APOE4/E4 mice following L. monocytogenes infection. In vivo , overall susceptibility to foodborne listeriosis also did not differ by APOE genotype in either young (2 mo old) or aged (15 mo old) C57BL/6 mice. However, we observed a sex-dependent susceptibility to infection in aged APOE2/E2 male mice and a sex-dependent resistance to infection in aged APOE4/E4 male mice that was not present in female mice. Thus, these results suggest that APOE genotype does not play an important role in innate resistance to infection with L. monocytogenes but may be linked to sex-dependent changes that occur during immune senescence.

The mesenteric lymph nodes (MLN) function as a barrier to systemic spread for both commensal and pathogenic bacteria in the gut. Listeria monocytogenes , a facultative intracellular foodborne pathogen, readily overcomes this barrier and spreads into the bloodstream, causing life-threatening systemic infections. We show here that intracellular replication protected L. monocytogenes from clearance by monocytes and neutrophils and promoted colonization of the small intestine-draining MLN (sMLN) but was not required for dissemination to the colon-draining MLN (cMLN).

We report the whole-genome sequence of Listeria monocytogenes UKVDL9 and an edited draft genome sequence of L. monocytogenes 2010L-2198. Both are neurotropic lineage III strains; UKVDL9 was isolated from a sheep brain, and 2010L-2198 was isolated from a human subject with rhombencephalitis.

Progress in understanding the two naturally occurring central nervous system (CNS) manifestations of listeriosis (meningitis/meningoencephalitis and rhombencephalitis) has been limited by the lack of small animal models that can readily distinguish between these distinct infections. We report here that certain neurotropic strains of Listeria monocytogenes can spread to the brains of young otherwise healthy mice and cause neurological deficits without causing a fatal bacteremia. The novel strains described here fall within phylogenetic lineage III, a small collection of L. monocytogenes isolates that have not been well characterized to date. The animal model reported here mimics many features of human rhombencephalitis and will be useful for studying the mechanisms that allow L. monocytogenes to disseminate to the brain stem following natural foodborne transmission.

Listeria monocytogenes is a foodborne pathogen that causes serious, often deadly, systemic disease in susceptible individuals such as neonates and the elderly. These facultative intracellular bacteria have been an invaluable tool in immunology research for more than three decades. Intravenous (i.v.) injection is the most commonly used transmission route in mice, but oral models of infection have also been developed in recent years, and these may be more appropriate for many studies. This article includes detailed instructions for use of either foodborne or i.v. inoculation of mice and discusses the rationale for choosing either model. Additionally, a protocol is provided for enrichment of neutrophils and monocytes from the infected liver in a manner that allows for determination of bacterial burden while still providing sufficient cells for use in flow cytometric analysis or in vitro assays. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Foodborne L. monocytogenes infectionSupport Protocol 1: Preparing L. monocytogenes for foodborne infectionBasic Protocol 2: Intravenous L. monocytogenes infectionSupport Protocol 2: Preparing L. monocytogenes for intravenous infectionBasic Protocol 3: Enrichment of non‐parenchymal cells from the infected liver

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.

Inbred mouse strains differ in their susceptibility to infection with the facultative intracellular bacteriumListeria monocytogenes, largely due to delayed or deficient innate immune responses. Previous antibody depletion studies suggested that neutrophils (polymorphonuclear leukocytes [PMN]) were particularly important for clearance in the liver, but the ability of PMN from susceptible and resistant mice to directly killL. monocytogeneshas not been examined. In this study, we showed that PMN infiltrated the livers of BALB/c/By/J (BALB/c) and C57BL/6 (B6) mice in similar numbers and that both cell types readily migrated toward leukotriene B4 in anin vitrochemotaxis assay. However, CFU burdens in the liver were significantly higher in BALB/c mice than in other strains, suggesting that PMN in the BALB/c liver might not be able to clearL. monocytogenesas efficiently as B6 PMN. Unprimed PMN harvested from either BALB/c or B6 bone marrow killedL. monocytogenesdirectlyex vivo, and pretreatment with autologous serum significantly enhanced killing efficiency for both.L. monocytogeneswere internalized within 10 min and rapidly triggered intracellular production of reactive oxygen species in a dose-dependent manner. However, PMN from gp91^phox-deficient mice also readily killedL. monocytogenes, which suggested that nonoxidative killing mechanisms may be sufficient for bacterial clearance. Together, these results indicate that there is not an intrinsic defect in the ability of PMN from susceptible BALB/c mice to killL. monocytogenesand further suggest that if PMN function is impaired in BALB/c mice, it is likely due to locally produced modulating factors present in the liver during infection.