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 as a probe of immune function.

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.     

组蛋白翻译后修饰技术研究进展

翻译后修饰调控着真核生物大部分蛋白质的活性,这些修饰的解读对研究生物功能是必不可少的。组蛋白翻译后修饰是蛋白质翻译后修饰中研究的较好一类小分子碱性蛋白,易被各种生物大分子修饰,尤其易发生在N-末端的尾部。不同组合式修饰构成了"组蛋白密码",在细胞的发育、生长、分化和动态平衡中,组蛋白密码影响着染色体的结构状态,进而调控基因的表达状态。组蛋白翻译后修饰的研究可作为一种模式来解析蛋白质复杂的修饰状态及研究其分子功能。翻译后修饰分析技术的发展对组蛋白密码的解析是至关重要的。重点讨论组蛋白修饰分析技术的发展和应用。     

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen–host interactions

Enteric pathogen–host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane‐bound toll‐like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro‐inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase‐1 and are regulated by related caspases, such as caspase‐11, ‐4, ‐5 and ‐8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome‐mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.     

Recent findings and technological advances in phosphoproteomics for cells and tissues

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.     

The actin comet guides the way: How Listeria actin subversion has impacted cell biology,infection biology and structural biology

The discovery of the role of ActA to polymerise actin at one pole of Listeria monocytogenes represents a key event in the field of cellular microbiology. It uncovered much more than the molecular principle behind actin‐based motility of Listeria within the cytosol of infected cells, and it changed the way how actin dynamics could be studied and eventually understood. The ActA discovery took place at a time when cell biology, biochemistry and microbiology came together in a very fruitful fashion. Here, we provide an overview of the science that took place around this event. Then, we outline the wide array of research fields that have been impacted by this finding. This ranges from structural and biophysical investigations on actin and its dynamics, the role of actin polymerisation during infection with different pathogens, to actin‐dynamics during various pathologies. Like a comet in the sky, Pascale Cossart's work on ActA has inspired and will inspire generations of (life) scientists.     

Influence of staphylococcal enterotoxin B (SEB) on the course of murine listeriosis

Abstract Confrontation of the immune system with bacterial superantigens leads to an initial activation of the immune system followed by a state of profound immunosuppression. To investigate the role of a superantigen in an acute infection with a facultatively intracellular bacterium, we have studied the effect of staphylococcal enterotoxin B on the course of murine listeriosis. Intraperitoneal injection of SEB led to a statistically significant growth restriction of Listeria monocytogenes in the organs of mice infected intravenously or intraperitoneally when treatment with SEB and infection with L. monocytogenes were given simultaneously or when the mice were treated two days before infection. No effect of SEB on murine listeriosis was found when SEB was given more than two days before infection or one or more days after infection. We conclude that initial immunostimulation by SEB which is indicated by a massive liberation of all interleukins measured (IL1α, IL6, TNFα, IL2, IFNγ, IL4) is responsible for the growth restriction of L. monocytogenes in the organs of treated mice. Apoptosis of Vβ8 positive T cells which was accompanied by a 30% reduction of these cells at day 7 after treatment seems to be totally compensated.     

Controlling Listeria monocytogenes in Cottage cheese through heterologous production of enterocin A by Lactococcus lactis

Aims: Enterocin A is an example of a class IIa bacteriocin with potent anti‐listerial activity. This study was initiated with a view to harnessing this activity, through heterologous production by a lactococcal starter strain, to limit levels of Listeria monocytogenes in a food (Cottage cheese). Methods and Results: Plasmid pEnt02 (containing entA, I, T and D genes under the control of a constitutive promoter) was introduced into a Lactococcus lactis strain capable of fermenting lactose. When this bacteriocin‐producing starter was used in combination with a non‐enterocin A producer, thereby compensating for an associated reduction in acid production, during a Cottage cheese fermentation, a decrease in L. monocytogenes (tagged with lux genes for convenience) levels was evident. Conclusions: Enterocin A, heterologously produced by a food grade lactic acid bacteria (LAB), was therefore shown to have potential for use as a biocontrol agent in food. Significance and Impact of the Study: Many of the most active anti‐listerial compounds identified to date are enterocins. However, because of Enterococcus‐associated concerns, the use of these antimicrobials in a food setting has been curtailed. Although enterocins have been heterologously produced in LAB to overcome this problem, this study represents the first occasion upon which the benefits of such heterologous production have been demonstrated in a food context.     

FERONIA mutation induces high levels of chloroplast‐localized Arabidopsides which are involved in root growth

The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT‐ICR)‐MS approach was used for initial screening of wild‐type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non‐wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole‐time‐of‐flight (Q‐TOF) MS by direct injection and LC‐MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild‐type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix‐assisted laser desorption/ionization MS imaging (MALDI‐MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild‐type and fer mutant leaves. More significantly, five micron high‐spatial resolution MALDI‐MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.     

Investigation of relationships between marine diatoms and the bacterium Listeria monocytogenes

Relationships between marine diatoms and the bacterium Listeria monocytogenes have been studied by routine algological methods and high-resolution video-enhanced differential interference contrast light microscopy. The study showed that the relationship between the listeria and the benthic diatom Navicula sp. has a parasitic character, whereas the relationship between the listeria and the planktonic diatom Phaeodactylum tricornutum is protocooperative.     

Listeria monocytogenes induces IFNβ expression through an IFI16‐, cGAS‐ and STING‐dependent pathway

Listeria monocytogenes is a gram‐positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria‐derived cyclic‐di‐AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic‐di‐AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.     

A novel real-time PCR-based method for the detection of Listeria monocytogenes in food

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.     

Enhanced in vitro engulfment of Listeria monocytogenes by rabbit polymorphonuclear leukocytes in the presence of sera from immune rabbits

Abstract The role of immune serum in the engulfment of Listeria monocytogenes by polymorphonuclear leukocytes (PMNLs) of rabbits was documented employing an in vitro assay. Three serovarieties of L. monocytogenes , viz. serovars 1/2a, 4a and 4b, were separately incubated with PMNLs of nonimmune rabbit and high titre homologous or heterologous antisera. Normal rabbit serum was used as control. The number of L. monocytogenes per neutrophil was counted in stained smears in each assay and opsonic indices were calculated. Higher opsonic indices, i.e. 2.50, 2.09 and 2.56, for the three strains respectively, were obtained when bacteria were engulfed in the presence of homologous antisera as compared to when the bacterial cells were incubated with heterologous antisera, opsonic indices being in the range of 0.87 to 1.63. These results are indicative of a possible role of specific serum factors in at least the internalization of L. monocytogenes by PMNLs of rabbits and therefore in the host defenses against Listerial infections.     

Metaproteomic and Metabolomic Approaches for Characterizing the Gut Microbiome

The gut microbiome has been shown to play a significant role in human healthy and diseased states. The dynamic signaling that occurs between the host and microbiome is critical for the maintenance of host homeostasis. Analyzing the human microbiome with metaproteomics, metabolomics, and integrative multi‐omics analyses can provide significant information on markers for healthy and diseased states, allowing for the eventual creation of microbiome‐targeted treatments for diseases associated with dysbiosis. Metaproteomics enables functional activity information to be gained from the microbiome samples, while metabolomics provides insight into the overall metabolic states affecting/representing the host–microbiome interactions. Combining these functional ‐omic platforms together with microbiome composition profiling allows for a holistic overview on the functional and metabolic state of the microbiome and its influence on human health. Here the benefits of metaproteomics, metabolomics, and the integrative multi‐omic approaches to investigating the gut microbiome in the context of human health and diseases are reviewed.     

From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome‐like compartments called Coxiella‐containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once‐neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.     

Listeria monocytogenes infection in pregnant mice: Abnormalities in the function of non-adherent accessory light density dendritic cells

Abstract Pregnant A/J mice were found to be more susceptible to the lethal effect of Listeria monocytogenes bacteria than virgin females. However, during the first four days of post-infection there was no difference in the elimination of Listeria from the spleens of pregnant and virgin mice. This suggests that the increase in the susceptibility of pregnant mice to pathogenic activity of L. monocytogenes was related to the diminution in Listeria -specific cellular reactions. Indeed, we found that non-adherent light density dendritic cells (DCs) from pregnant mice showed a marked reduction in the ability to form clusters with L. monocytogenes immune T lymphocytes and it is known that cell cluster formation between antigen presenting cells (APC) and responding T cells is required for antigen recognition as well as for cell proliferation. DCs from pregnant mice also demonstrated the decrease and an instability in the expression of H-2 class II molecules which play a crucial role in the recognition of exogenous antigens. The abnormalities demonstrated in the function of the light density dendritic cells from the spleens of pregnant mice could compromise cellular reactions to L. monocytogenes bacteria possibly resulting in increased susceptibility of pregnant mice to experimental listeriosis.