NF-κB/ROS and ERK pathways regulate NLRP3 inflammasome activation in Listeria monocytogenes infected BV2 microglia cells.

Listeria monocytogenes is a food-borne pathogen responsible for neurolisteriosis, which is potentially lethal in immunocompromised individuals. Microglia are the main target cells for L. monocytogenes in central nervous system (CNS). However, the precise mechanisms by which they trigger neuroinflammatory processes remain unknown. The BV2 microglial cell line and a murine model of L. monocytogenes infection were used for experiments in this study. Listeria monocytogenes induced pyroptosis and nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3) inflammasome activation in BV2. Pharmacological inhibition of the NLRP3 inflammasome attenuated L. monocytogenes-induced pyroptosis. Moreover, inhibition of nuclear factor kappa-B (NF-κB) and extracellular regulated protein kinases (ERK) pathways induced a decrease in caspase1 activation and mature IL-1ß-17 secretion. Our collective findings support critical involvement of the NLRP3 inflammasome in L. monocytogenes-induced neuroinflammation and, to an extent, ROS production. In addition, ERK and NF-κB signaling play an important role in activation of the NLRP3 inflammasome, both in vitro and in vivo.

Laboratory-based surveillance of invasive listeriosis in Northern Italy over a fourteen-year period: epidemiological and clinical results.

INTRODUCTION: Invasive listeriosis is a rare foodborne disease with a significant impact on public health worldwide, because of the severity of its clinical manifestations and high fatality rate. In this study, we provide a snapshot of epidemiology of listeriosis in Lombardy Region, Northern Italy, reviewing enhanced surveillance data collected over fourteen years, after the implementation of a voluntary laboratory-based surveillance system for the referral of clinical isolates of Listeria monocytogenes to a regional reference laboratory, since 2005. METHODS: Invasive listeriosis cases data from 2005 to 2018 were extracted from the regional laboratory-based surveillance system database and compared with the regional mandatory notification disease system data. RESULTS: Over the fourteen-year period under study, 533 Listeria monocytogenes isolates were detected by the laboratory surveillance system, 55 of which from pregnancy-related cases. The median age of non-pregnancy-associated patients was 71 years, with 64.6% of cases observed in the elderly. Cases with underlying medical risk conditions accounted for 92.1%, and the fatality rate was 26.2%. By integrating data from the mandatory notification system and the laboratory-based surveillance system, a total of 935 cases were recorded. The collection of data through the laboratory surveillance system allowed to increase the surveillance sensitivity by 18%. CONCLUSIONS: Our results documented the growing epidemiological relevance of listeriosis through the analysis of two information sources, the regional mandatory notification system and the regional laboratory-based surveillance system. The data we obtained were consistent with the literature, except for pregnancy-related cases, which are often underdiagnosed. This study highlighted the importance of laboratory-based surveillance system, which led to a significant increase in the sensitivity of the mandatory notification system.

Rapidly dynamic host cell heterogeneity in bacterial adhesion governs susceptibility to infection by Listeria monocytogenes.

Interactions between host cells and individual pathogenic bacteria determine the clinical severity of disease during systemic infection in humans. Vascular endothelial cells, which line the lumen of blood vessels, represent a critical barrier for a bacterium in the bloodstream. These cells adopt a myriad of phenotypes that may modulate their susceptibility to infection; however, the precise determinants of their heterogeneity in susceptibility are not known. Here, we show that heterogeneity in susceptibility to Listeria monocytogenes infection among primary human vascular endothelial cells can be attributed entirely to robust, preexisting host cell heterogeneity in bacterial adhesion, and we find no evidence for significant heterogeneity in later steps of infection. High susceptibility to adhesion decays rapidly, within 30-60 min. Thus, rapidly fluctuating, nongenetic variability in bacterial adhesion diversifies susceptibility to infection, both among host cells and within individual cells over time.

Maternal-neonatal listeriosis.

Listeriosis is a rare and severe foodborne infection caused by Listeria monocytogenes. It manifests as septicemia, neurolisteriosis, and maternal-fetal infection. In pregnancy, it may cause maternal fever, premature delivery, fetal loss, neonatal systemic and central nervous system infections. Maternal listeriosis is mostly reported during the 2nd and 3rd trimester of pregnancy, as sporadic cases or in the context of outbreaks. Strains belonging to clonal complexes 1, 4 and 6, referred to as hypervirulent, are the most associated to maternal-neonatal infections. Here we review the clinical, pathophysiological, and microbiological features of maternal-neonatal listeriosis.

Listeria monocytogenes, a model in infection biology.

Listeria monocytogenes causes listeriosis, a systemic infection which manifests as bacteremia, often complicated by meningoencephalitis in immunocompromised individuals and the elderly, and fetal-placental infection in pregnant women. It has emerged over the past decades as a major foodborne pathogen, responsible for numerous outbreaks in Western countries, and more recently in Africa. L. monocytogenes' pathogenic properties have been studied in detail, thanks to concomitant advances in biological sciences, in particular molecular biology, cell biology and immunology. L. monocytogenes has also been instrumental to basic advances in life sciences. L. monocytogenes therefore stands both a tool to understand biology and a model in infection biology. This review briefly summarises the clinical and some of the pathophysiological features of listeriosis. In the context of this special issue, it highlights some of the major discoveries made by Pascale Cossart in the fields of molecular and cellular microbiology since the mid-eighties regarding the identification and characterisation of multiple bacterial and host factors critical to L. monocytogenes pathogenicity. It also briefly summarises some of the key findings from our laboratory on this topic over the past years.

Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

Epidemiology and Clinical Manifestations of Listeria monocytogenes Infection.

Listeria monocytogenes is a Gram-positive pathogenic bacterium which can be found in soil or water. Infection with the organism can develop after ingestion of contaminated food products. Small and large outbreaks of listeriosis have been described. Listeria monocytogenes can cause a number of clinical syndromes, most frequently sepsis, meningitis, and rhombencephalitis, particularly in immunocompromised hosts. The latter syndrome mimics the veterinary infection in ruminants called "circling disease". Neonatal infection can occur as a result of maternal chorioamnionitis ("early onset" sepsis) or through passage through a birth canal colonized with Listeria from the gastrointestinal tract. ("late onset" meningitis). Treatment of listeriosis is usually with a combination of ampicillin and an aminoglycoside but other regimens have been used. The mortality rate is high, reflecting the combination of an immunocompromised host and an often delayed diagnosis.

Co-infection with Streptococcus pneumoniae and Listeria monocytogenes in an immunocompromised patient.

A 34-year-old HIV-positive man with a history of chronic substance abuse was admitted with dual infection of Streptococcus pneumoniae and Listeria monocytogenes. Combined bacteraemia with S. pneumoniae and L. monocytogenes is very rare. To the best of our knowledge, this is the first such case documented at our institution and in South Africa. Ampicillin should be added to antibiotic regimens to improve patient outcome if L. monocytogenes infection is suspected. Co-infections that occur with L. monocytogenes may have conflicting antibiotic treatment options. This case report emphasises the need for a good relationship between the local microbiology pathologist and physician to select appropriate antibiotic treatment before definitive results are available.

Host cell perforation by listeriolysin O (LLO) activates a Ca2+-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing.

Host cell invasion is an indispensable step for a successful infection by intracellular pathogens. Recent studies identified pathogen-induced host cell plasma membrane perforation as a novel mechanism used by diverse pathogens (Trypanosoma cruzi, Listeria monocytogenes, and adenovirus) to promote their internalization into target cells. It was concluded that T. cruzi and adenovirus damage the host cell plasma membrane to hijack the endocytic-dependent membrane resealing machinery, thereby invading the host cell. We studied L. monocytogenes and its secreted pore-forming toxin listeriolysin O (LLO) to identify key signaling events activated upon plasma membrane perforation that lead to bacterial internalization. Using various approaches, including fluorescence resonance energy transfer imaging, we found that the influx of extracellular Ca2+ subsequent to LLO-mediated plasma membrane perforation is required for the activation of a conventional protein kinase C (cPKC). cPKC is positioned upstream of Rac1 and the Arp2/3 complex, which activation leads to F-actin--dependent bacterial internalization. Inhibition of this pathway did not prevent membrane resealing, revealing that perforation-dependent L. monocytogenes endocytosis is distinct from the resealing machinery. These studies identified the LLO-dependent endocytic pathway of L. monocytogenes and support a novel model for pathogen uptake promoted by plasma membrane injury that is independent of membrane resealing.

Pathway Interaction Network Analysis Identifies Dysregulated Pathways in Human Monocytes Infected by Listeria monocytogenes.

In our study, we aimed to extract dysregulated pathways in human monocytes infected by Listeria monocytogenes (LM) based on pathway interaction network (PIN) which presented the functional dependency between pathways. After genes were aligned to the pathways, principal component analysis (PCA) was used to calculate the pathway activity for each pathway, followed by detecting seed pathway. A PIN was constructed based on gene expression profile, protein-protein interactions (PPIs), and cellular pathways. Identifying dysregulated pathways from the PIN was performed relying on seed pathway and classification accuracy. To evaluate whether the PIN method was feasible or not, we compared the introduced method with standard network centrality measures. The pathway of RNA polymerase II pretranscription events was selected as the seed pathway. Taking this seed pathway as start, one pathway set (9 dysregulated pathways) with AUC score of 1.00 was identified. Among the 5 hub pathways obtained using standard network centrality measures, 4 pathways were the common ones between the two methods. RNA polymerase II transcription and DNA replication owned a higher number of pathway genes and DEGs. These dysregulated pathways work together to influence the progression of LM infection, and they will be available as biomarkers to diagnose LM infection.

How the study of Listeria monocytogenes has led to new concepts in biology.

The opportunistic intracellular bacterial pathogen Listeria monocytogenes has in 30 years emerged as an exceptional bacterial model system in infection biology. Research on this bacterium has provided considerable insight into how pathogenic bacteria adapt to mammalian hosts, invade eukaryotic cells, move intracellularly, interfere with host cell functions and disseminate within tissues. It also contributed to unveil features of normal host cell pathways and unsuspected functions of previously known cellular proteins. This review provides an updated overview of our knowledge on this pathogen. In many examples, findings on L. monocytogenes provided the basis for new concepts in bacterial regulation, cell biology and infection processes.

Listeria Placental Infection.

The Gram-positive facultative intracellular bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection. Pregnant women are at risk of contracting listeriosis, which can potentially lead to miscarriage, stillbirth, preterm birth, and congenital neonatal infections. While other systemic bacterial infections may result in adverse pregnancy outcomes at comparable frequencies, L. monocytogenes has particular notoriety because fetal complications largely occur in the absence of overt illness in the mother, delaying medical intervention. Here, we briefly review the pathophysiology and mechanisms of maternofetal listeriosis, discussed in light of a recent mBio report on Listeria transplacental infection in a nonhuman primate model.

Listeria monocytogenes induces an interferon-enhanced activation of the integrated stress response that is detrimental for resolution of infection in mice.

Type I interferons (IFNs) induce a detrimental response during Listeria monocytogenes (L. monocytogenes) infection. We were interested in identifying mechanisms linking IFN signaling to negative host responses against L. monocytogenes infection. Herein, we found that infection of myeloid cells with L. monocytogenes led to a coordinated induction of type I IFNs and activation of the integrated stress response (ISR). Infected cells did not induce Xbp1 splicing or BiP upregulation, indicating that the unfolded protein response was not triggered. CHOP (Ddit3) gene expression was upregulated during the ISR activation induced by L. monocytogenes. Myeloid cells deficient in either type I IFN signaling or PKR activation had less upregulation of CHOP following infection. CHOP-deficient mice showed lower expression of innate immune cytokines and were more resistant than wild-type counterparts following L. monocytogenes infection. These findings indicate that L. monocytogenes infection induces type I IFNs, which activate the ISR through PKR, which contributes to a detrimental outcome in the infected host.

Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation.

Acute infections are associated with a set of stereotypic behavioral responses, including anorexia, lethargy, and social withdrawal. Although these so-called sickness behaviors are the most common and familiar symptoms of infections, their roles in host defense are largely unknown. Here, we investigated the role of anorexia in models of bacterial and viral infections. We found that anorexia was protective while nutritional supplementation was detrimental in bacterial sepsis. Furthermore, glucose was necessary and sufficient for these effects. In contrast, nutritional supplementation protected against mortality from influenza infection and viral sepsis, whereas blocking glucose utilization was lethal. In both bacterial and viral models, these effects were largely independent of pathogen load and magnitude of inflammation. Instead, we identify opposing metabolic requirements tied to cellular stress adaptations critical for tolerance of differential inflammatory states. VIDEO ABSTRACT.

Listeriolysin O mediates cytotoxicity against human brain microvascular endothelial cells.

Penetration of the brain microvascular endothelial layer is one of the routes Listeria monocytogenes use to breach the blood-brain barrier. Because host factors in the blood severely limit direct invasion of human brain microvascular endothelial cells (HBMECs) by L. monocytogenes, alternative mechanisms might be used by this bacterium to penetrate the endothelial cell layer. In this study, we evaluated the cytotoxicity of proteins secreted by L. monocytogenes against HBEMCs using a live/dead staining method. Interestingly, the integrity of the plasma membrane of HBMECs was impaired by proteins secreted by the EGD wild-type strain but not proteins secreted by the isogenic ΔprfA strain. Therefore, we investigated the cytotoxicity of proteins secreted by several isogenic mutant strains (ΔplcA, Δmpl and Δhly) incapable of producing the prfA-regulated bacterial products PlcA, Mpl and LLO, respectively. Results from both fluorescent microscopy and flow cytometry analyses showed that proteins secreted by the Δhly strain were not cytotoxic to HBMECs, whereas those secreted by the ΔplcA and Δmpl strains were cytotoxic. These results suggest that LLO-mediated cytotoxicity against brain microvascular endothelial cells enables L. monocytogenes to effectively penetrate the brain microvascular endothelial layer.

Papel inmunomodulador de las dietas lipídicas en un modelo murino inmunosuprimido e infectado con Listeria monocytogenes / Immunomodulatory role of dietary lipids in an immunosuppressed mouse model and infected with listeria monocytogenes

Introducción: La capacidad inmunomoduladora de los ácidos grasos de la dieta en situaciones de inmunosupresión puede diferir de acuerdo con el tipo de ácido graso presente. Objetivo: Analizar el efecto de diferentes tipos de dietas lipídicas, en la resistencia de animales inmunosuprimidoso no, frente a una infección experimental con Listeria monocytogenes. Métodos: Ratones Balb/c fueron divididos en cuatro grupos experimentales, según su tratamiento inmunosupresor: control (PBS), Ciclofos famida (CPA), GK 1.5 yRB6-8C5. Cada grupo fue subdividido en cuatro subgrupos según la dieta lipídica utilizada: control con aceite de maíz 5% (BG); aceite de oliva 20% (AO); aceite de pescado20% (AP) y aceite de girasol 20% (AG). Los animales se alimentaron durante un mes antes del tratamiento y posteriormente infectados con L. monocytogenes. Resultados: Mostramos incrementos en el número de bacterias viables en bazo e hígado, y bajos porcentajes de supervivencia en todos los grupos de ratones inmunosuprimidos y también en el grupo PBS alimentado con AP. Además, se observaron incrementos en la linfoproliferación, de bazos de ratones alimentados con AO y tratados con CPA. Discusión: La dieta AP, produce una disminución en la resistencia del hospedador en situaciones de inmunosupresión. Por el contrario, las dietas AO y AG muestran mayor eficacia en la eliminación de L. monocytogenes y mayores ventajas en animales inmunosuprimidos. El tratamiento con RB6-8C5, produce una reducción en la supervivencia de los ratones de los grupos estudiados, lo que induce a establecer que los granulocitos juegan un papel fundamental en el control de la infección (AU)

Introduction: Dietary fatty acids immunomodulatory capacity in immunosuppression conditions may differ according to the type of fatty acid present in the diet. Objective: To analyze the effect of different types of dietary lipids on the immune resistance of immunosuppressed and immunocompetent animals, against experimental infection with a virulent strain of Listeria monocytogenes .Methods: Balb/c mice were divided into four experimental groups, according to their immunosuppressive treatment: control (PBS), cyclophosphamide (CPA), GK1.5 and RB6-8C5. Each group was subdivided into four groups according to the lipid diet used which: control, with corn oil 5% (BG); olive oil 20% (AO); fish oil 20%(AP) and sunflower oil 20% (AG). The animals were fed for a month before treatment and subsequently infected with L. monocytogenes. Results: We show increases in the number of viable bacteria in spleen and liver, and low survival rates in all groups of immunosuppressed mice and also in the PBS group and fed with AP. Furthermore, increases in the lymphocyte proliferation were observed, in the spleen of mice fed with AO and treated with CPA. Discussion: The AP diet produces a significant decrease the host resistance in situations of immunosuppression .On the contrary, the AO and AG diets show major efficiency in the elimination of L. monocytogenes and major immunological advantages in immunosuppressed mice. Treatment with RB6-8C5, produces a reduction in the survival of the mice in all groups studied, which leads us to establish that granulocytes play a key role in the control of infection (AU)

Listeria monocytogenes ActA: a new function for a 'classic' virulence factor.

Listeria monocytogenes (Lm) is ubiquitous and widespread in the environment. It is responsible for one of the most severe human foodborne infection. Lm is a facultative intracellular bacterium that can cross the intestinal barrier, disseminate via the bloodstream and reach the liver, spleen, central nervous system and fetus. The bacterial surface protein ActA is one of the most critical and best characterized virulence factors of Lm. It fulfills many essential functions within host cells, allowing Lm escape from autophagy and recruiting an actin polymerization complex that promotes Lm actin-based motility, cell-to-cell spread and dissemination within host tissues. We have recently shown that ActA also acts extracellularly. It mediates Lm aggregation and biofilm formation in vitro and in vivo, and long-term colonization of the gut lumen. This new property of ActA favors Lm transmission and may participate in the selective pressure on Lm to maintain ActA.