Sex-dependent circadian alterations of both central and peripheral clock genes expression and gut-microbiota composition during activity-based anorexia in mice.

RATIONALE: Patients with anorexia nervosa (AN) often present sleep disorders and circadian hormonal dysregulation. The role of the microbiota-gut-brain axis in the regulation of feeding behavior has emerged during the last decades but its relationships with the circadian rhythm remains poorly documented. Thus, we aimed to characterize the circadian clock genes expression in peripheral and central tissues in the activity-based anorexia mouse model (ABA), as well as the dynamics of the gut-microbiota composition. METHODS: From day 1 to day 17, male and female C57Bl/6 mice were submitted or not to the ABA protocol (ABA and control (CT) groups), which combines a progressive limited access to food and a free access to a running wheel. At day 17, fasted CT and ABA mice were euthanized after either resting (EoR) or activity (EoA) phase (n = 10-12 per group). Circadian clock genes expression was assessed by RT-qPCR on peripheral (liver, colon and ileum) and central (hypothalamic suprachiasmatic nucleus or SCN) tissues. Cecal bacterial taxa abundances were evaluated by qPCR. Data were compared by two-way ANOVA followed by post-tests. RESULTS: ABA mice exhibited a lower food intake, a body weight loss and an increase of diurnal physical activity that differ according with the sex. Interestingly, in the SCN, only ABA female mice exhibited altered circadian clock genes expression (Bmal1, Per1, Per2, Cry1, Cry2). In the intestinal tract, modification of clock genes expression was also more marked in females compared to males. For instance, in the ileum, female mice showed alteration of Bmal1, Clock, Per1, Per2, Cry1, Cry2 and Rev-erbα mRNA levels, while only Per2 and Cry1 mRNAs were affected by ABA model in males. By contrast, in the liver, clock genes expression was more markedly affected in males compared to females in response to ABA. Finally, circadian variations of gut-bacteria abundances were observed in both male and female mice and sex-dependent alteration were observed in response to the ABA model. CONCLUSIONS: This study shows that alteration of circadian clock genes expression at both peripheral and central levels occurs in response to the ABA model. In addition, our data underline that circadian variations of the gut-microbiota composition are sex-dependent.

Anorexia nervosa is an eating disorder with a female predominance. However, the underlying pathophysiological mechanisms are still incompletely understood. Patients with anorexia nervosa often show alterations in circadian rhythm, including sleep disorders and modifications in hormone circadian rhythm. The circadian rhythm is controlled in the central nervous system, particularly in the suprachiasmatic nucleus, but clocks have also been described in peripheral tissues. To better understand the putative role of circadian rhythm in the pathophysiology of anorexia nervosa, we have conducted an experimental study in a rodent model of anorexia nervosa called "activity-based anorexia" on both males and females. Interestingly, we observed that the expression of genes involved in the circadian rhythm is affected by the activity-based anorexia model in both the suprachiasmatic nucleus and peripheral tissues, such as the small intestine and liver. In addition, gut­microbiota also shows circadian variation. Interestingly, the anorexia-induced alterations of circadian variations (clock genes expression and gut­microbiota composition) are sex- and tissue-dependent. For instance, female mice exhibited more marked alterations in the ileum, whereas, in males, modifications were more pronounced in the liver. This study highlights sex-dependent alterations of circadian clock genes expression and of gut­microbiota in response to the anorexia rodent model. Further experiments should be performed to investigate the contribution of these mechanisms in the etiology of anorexia nervosa and the higher prevalence in females.

Are Escherichia coli causing recurrent cystitis just ordinary Uropathogenic E. coli (UPEC) strains?

Specific determinants associated with Uropathogenic Escherichia coli (UPEC) causing recurrent cystitis are still poorly characterized. The aims of this study were (i) to describe genomic and phenotypic traits associated with recurrence using a large collection of recurrent and paired sporadic UPEC isolates, and (ii) to explore within-host genomic adaptation associated with recurrence using series of 2 to 5 sequential UPEC isolates. Whole genome comparative analyses between 24 recurrent cystitis isolates (RCIs) and 24 phylogenetically paired sporadic cystitis isolates (SCIs) suggested a lower prevalence of putative mobile genetic elements (MGE) in RCIs, such as plasmids and prophages. The intra-patient evolution of the 24 RCI series over time was characterized by SNP occurrence in genes involved in metabolism or membrane transport, and by plasmid loss in 5 out of the 24 RCI series. Genomic evolution occurred early in the course of recurrence, suggesting rapid adaptation to strong selection pressure in the urinary tract. However, RCIs did not exhibit specific virulence factor determinants and could not be distinguished from SCIs by their fitness, biofilm formation, or ability to invade HTB-9 bladder epithelial cells. Taken together, these results suggest a rapid but not convergent adaptation of RCIs that involves both strain- and host-specific characteristics.

Invasion of intestinal cells by Staphylococcus warneri, a member of the human gut microbiota.

Coagulase negative staphylococci (CoNS) are a heterogeneous group of bacteria that colonize different types of human epithelia. These bacteria have a highly variable pathogenic potential ranging from avirulent species to major nosocomial pathogens. Staphylococcus warneri is a CoNS species considered to be nonpathogenic. Here, we identify that S. warneri is a natural member of both human and mouse gut microbiota. In addition, we demonstrate that this bacterium is able to get internalized into human cells. We show that S. warneri efficiently invades several human cell types and, more specifically, intestinal epithelial cells, using actin-dependent mechanisms. In contrast to bona fide pathogens, S. warneri does not actively replicate within intestinal cells or resist killing by macrophages. Together, our results highlight that bacteria from the human gut microbiota that are not associated with a high pathogenic potential, can actively invade intestinal cells and may, in this way, impact intestinal physiology.

A Panax quinquefolius-Based Preparation Prevents the Impact of 5-FU on Activity/Exploration Behaviors and Not on Cognitive Functions Mitigating Gut Microbiota and Inflammation in Mice.

Chemotherapy-related cognitive impairment (CRCI) and fatigue constitute common complaints among cancer patient survivors. Panax quinquefolius has been shown to be effective against fatigue in treated cancer patients. We developed a behavioral C57Bl/6j mouse model to study the role of a Panax quinquefolius-based solution containing vitamin C (Qiseng®) or vitamin C alone in activity/fatigue, emotional reactivity and cognitive functions impacted by 5-Fluorouracil (5-FU) chemotherapy. 5-FU significantly reduces the locomotor/exploration activity potentially associated with fatigue, evokes spatial cognitive impairments and leads to a decreased neurogenesis within the hippocampus (Hp). Qiseng® fully prevents the impact of chemotherapy on activity/fatigue and on neurogenesis, specifically in the ventral Hp. We observed that the chemotherapy treatment induces intestinal damage and inflammation associated with increased levels of Lactobacilli in mouse gut microbiota and increased expression of plasma pro-inflammatory cytokines, notably IL-6 and MCP-1. We demonstrated that Qiseng® prevents the 5-FU-induced increase in Lactobacilli levels and further compensates the 5-FU-induced cytokine release. Concomitantly, in the brains of 5-FU-treated mice, Qiseng® partially attenuates the IL-6 receptor gp130 expression associated with a decreased proliferation of neural stem cells in the Hp. In conclusion, Qiseng® prevents the symptoms of fatigue, reduced chemotherapy-induced neuroinflammation and altered neurogenesis, while regulating the mouse gut microbiota composition, thus protecting against intestinal and systemic inflammation.

Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation.

The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. The effect of gut commensal bacteria on host protein SUMOylation, an essential ubiquitin-like modification involved in various intestinal functions, remains, however, unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in intestinal cells in a pH-dependent manner. We demonstrate that these metabolites inactivate intestinal deSUMOylases and promote the hyperSUMOylation of nuclear matrix-associated proteins. We further show that BCFAs inhibit the NF-κB pathway, decrease pro-inflammatory cytokine expression, and promote intestinal epithelial integrity. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses triggered by the gut microbiota.

Role of microbiota-gut-brain axis dysfunctions induced by infections in the onset of anorexia nervosa.

Anorexia nervosa (AN) is an eating disorder characterized by low food intake, severe body weight loss, intense fear of gaining weight, and dysmorphophobia. This chronic disease is associated with both psychiatric and somatic comorbidities. Over the years, clinical studies have accumulated evidence that viral or bacterial infections may promote the onset of eating disorders such as AN. This review aims to describe how infections and the subsequent immune responses affect food intake regulation in the short term and also how these processes may lead to long-term intestinal disorders, including gut barrier disruption and gut microbiota dysbiosis, even after the clearance of the pathogens. We discuss in particular how infection-mediated intestinal dysbiosis may promote the onset of several AN symptoms and comorbidities, including appetite dysregulation, functional gastrointestinal disorders, and mood disorders.

Identification and Characterization of Human Observational Studies in Nutritional Epidemiology on Gut Microbiomics for Joint Data Analysis.

In any research field, data access and data integration are major challenges that even large, well-established consortia face. Although data sharing initiatives are increasing, joint data analyses on nutrition and microbiomics in health and disease are still scarce. We aimed to identify observational studies with data on nutrition and gut microbiome composition from the Intestinal Microbiomics (INTIMIC) Knowledge Platform following the findable, accessible, interoperable, and reusable (FAIR) principles. An adapted template from the European Nutritional Phenotype Assessment and Data Sharing Initiative (ENPADASI) consortium was used to collect microbiome-specific information and other related factors. In total, 23 studies (17 longitudinal and 6 cross-sectional) were identified from Italy (7), Germany (6), Netherlands (3), Spain (2), Belgium (1), and France (1) or multiple countries (3). Of these, 21 studies collected information on both dietary intake (24 h dietary recall, food frequency questionnaire (FFQ), or Food Records) and gut microbiome. All studies collected stool samples. The most often used sequencing platform was Illumina MiSeq, and the preferred hypervariable regions of the 16S rRNA gene were V3-V4 or V4. The combination of datasets will allow for sufficiently powered investigations to increase the knowledge and understanding of the relationship between food and gut microbiome in health and disease.

Gut microbiota depletion affects nutritional and behavioral responses to activity-based anorexia model in a sex-dependent manner.

BACKGROUND & AIMS: In the last decade, the role of the microbiota-gut-brain axis in eating behavior and anxiety-depressive disorders has gained increasing attention. Although a gut microbiota dysbiosis has been reported in anorectic patients, its pathophysiological role remains poorly understood. Thus, we aimed to characterize the potential role of gut microbiota by evaluating the effects of its depletion in the Activity-Based Anorexia (ABA) mouse model both in male and female mice. METHODS: Male and female C57Bl/6 mice were submitted (ABA group) or not (CT group) to the ABA protocol, which combines access to a running wheel with a progressive limited food access. Gut microbiota was previously depleted or not by a cocktail of antibiotics (ATB) delivered by oral gavages. We monitored body composition, anxiety-like behavior, leptin and adiponectin plasma levels, hypothalamic and hippocampal neuropeptides mRNA levels, as well as dopamine (DRD) and serotonin (5HT1 and 4) receptors mRNA expression. RESULTS: In response to the ABA model, the body weight loss was less pronounced in ATB-treated ABA compared to untreated ABA, while food intake remained unaffected by ATB treatment. ATB-treated ABA exhibited increased fat mass and decreased lean mass compared to untreated ABA both in male and female mice, whereas but plasma adipokine concentrations were affected in a sex-dependent manner. Only male ABA mice showed a reduced anticipatory physical activity in response to ATB treatment. Similarly, anxiety-like behavior was mainly affected in ATB-treated ABA male mice compared to ATB-treated ABA female mice, which was associated with male-specific alterations of hypothalamic CRH mRNA and hippocampal DRD and 5-HT1A mRNA levels. CONCLUSIONS: Our study provides evidence that ATB-induced gut microbiota depletion triggers alterations of nutritional and behavioral responses to the activity-based anorexia model in a sex-dependent manner.

Gut microbiota alteration in a mouse model of Anorexia Nervosa.

BACKGROUND & AIMS: Anorexia Nervosa is a severe disease depending on both biological, psychological and environmental factors. The gut microbiota has recently been proposed as one of the biological factors potentially involved in the onset or maintenance of Anorexia Nervosa. To unravel the potential role of the gut microbiota in this disease, we characterized the dysbiosis occurring in a mouse model of Anorexia and correlated bacteria level changes with different physiological parameters such as body weight, food intake or levels of hypothalamic neuropeptides. METHODS: We used the Activity-Based Anorexia (ABA) mouse model, which combines food restriction and physical activity, and which mimics core features of Anorexia Nervosa. We characterized the gut microbiota alteration in ABA mice by combining 16S rRNA gene sequencing and quantitative PCR analyses of targeted genera or species. RESULTS: We identified 68 amplicon sequence variants (ASVs) with decreased levels and 8 ASVs with increased levels in the cecal content of ABA mice compared to control mice. We observed in particular in ABA mice increases in the abundance of Clostridium cocleatum and several Lactobacillus species and a decrease in the abundance of Burkholderiales compared to control mice. Interestingly, we show that most of the observed gut microbiota alterations are due to food restriction and are not affected by physical activity. In addition, we identified several bacterial groups that correlate with mice body weight, food intake, lean and fat masses as well as with hypothalamic mRNA levels of NPY (Neuropeptide Y) and POMC (Pro-opiomelanocortin). CONCLUSIONS: Our study provides a comprehensive characterization of the gut microbiota dysbiosis occurring in the Activity-Based Anorexia mouse model. These data constitute a valuable resource to further decipher the role of the gut microbiota in the different facets of anorexia pathophysiology, such as functional gastrointestinal disorders, appetite regulation and mood disorders.

Comparison of different modes of antibiotic delivery on gut microbiota depletion efficiency and body composition in mouse.

BACKGROUND: The use of animal models with depleted intestinal microbiota has recently increased thanks to the huge interest in the potential role of these micro-organisms in human health. In particular, depletion of gut bacteria using antibiotics has recently become popular as it represents a low cost and easy alternative to germ-free animals. Various regimens of antibiotics are used in the literature, which differ in composition, dose, length of treatment and mode of administration. In order to help investigators in choosing the most appropriate protocol for their studies, we compared here three modes of antibiotic delivery to deplete gut bacteria in C57Bl/6 mice. We delivered one of the most frequently used combination of antibiotics (a mix of ampicillin, neomycin, metronidazole and vancomycin) either ad libitum in drinking water or by oral gavage once or twice per day. RESULTS: We quantified the global bacterial density, as well as the abundance of specific bacterial and fungal taxa, in mouse feces in response to antibiotics exposure. We observed that oral gavage once a day with antibiotics is not a reliable method as it occasionally triggers hyperproliferation of bacteria belonging to the Escherichia/Shigella taxon and leads, as a consequence, to a moderate decrease in fecal bacterial density. Antibiotics delivery by oral gavage twice a day or in drinking water induces in contrast a robust and consistent depletion of mouse fecal bacteria, as soon as 4 days of treatment, and is associated with an increase in fecal moisture content. Extending exposure to antibiotics beyond 7 days does not improve total bacteria depletion efficiency and promotes fungal overgrowth. We show in addition that all tested protocols impact neither gut microbiota recolonization efficiency, 1 or 2 weeks after the stop of antibiotics, nor mice body composition after 1 week of treatment. CONCLUSIONS: Our study provides key experimental data and highlights important parameters to consider before selecting an appropriate protocol for antibiotic-mediated depletion of gut bacteria, in order to optimize the accuracy and the reproducibility of results and to facilitate comparison between studies.

Stress-induced intestinal barrier dysfunction is exacerbated during diet-induced obesity.

Obesity and irritable bowel syndrome (IBS) are two major public health issues. Interestingly previous data report a marked increase of IBS prevalence in morbid obese subjects compared with non-obese subjects but underlying mechanisms remain unknown. Obesity and IBS share common intestinal pathophysiological mechanisms such as gut dysbiosis, intestinal hyperpermeability and low-grade inflammatory response. We thus aimed to evaluate the link between obesity and IBS using different animal models. Male C57Bl/6 mice received high fat diet (HFD) for 12 weeks and were then submitted to water avoidance stress (WAS). In response to WAS, HFD mice exhibited higher intestinal permeability and plasma corticosterone concentration than non-obese mice. We were not able to reproduce a similar response both in ob/ob mice and in leptin-treated non-obese mice. In addition, metformin, a hypoglycemic agent, limited fasting glycaemia both in unstressed and WAS diet-induced obese mice but only partially restored colonic permeability in unstressed HFD mice. Metformin failed to improve intestinal permeability in WAS HFD mice. Finally, cecal microbiota transplantation from HFD mice in antibiotics-treated recipient mice did not reproduce the effects observed in stressed HFD mice. In conclusion, stress induced a more marked intestinal barrier dysfunction in diet-induced obese mice compared with non-obese mice that seems to be independent of leptin, glycaemia and gut microbiota. These data should be further confirmed and the role of the dietary composition should be studied.

Author Correction: Listeria monocytogenes impairs SUMOylation for efficient infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of the Receptor Used by the Ecotropic Mouse GLN Endogenous Retrovirus.

Approximately 10% of the mouse genome is composed of endogenous retroviruses belonging to different families. In contrast to the situation in the human genome, several of these families correspond to recent, still-infectious elements capable of encoding complete viral particles. The mouse GLN endogenous retrovirus is one of these active families. We previously identified one fully functional provirus from the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identified receptors for mouse ecotropic retroviral envelope proteins. In this study, we have identified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope is due to the presence or absence of an N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains of the receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses a member of the solute carrier superfamily as a receptor.IMPORTANCE Endogenous retroviruses are genomic traces of past infections present in all vertebrates. Most of these elements degenerate over time and become nonfunctional, but the mouse genome still contains several families with full infection abilities. The GLN retrovirus is one of them, and its members encode particles that are able to infect only mouse cells. Here, we identified the cellular protein used as a receptor by GLN for cell entry. It is SLC19A1, the reduced folate carrier. We show that GLN infection is limited to mouse cells due to both a mutation in the mouse gene preventing the glycosylation of SLC19A1 and also other residues conserved within the rat but not in the hamster and human proteins. Like all other gammaretroviruses whose receptors have been identified, GLN uses a member of the solute carrier superfamily for cell entry, highlighting the role of these proteins for retroviral infection in mammals.

Ubiquitin, SUMO, and NEDD8: Key Targets of Bacterial Pathogens.

Manipulation of host protein post-translational modifications (PTMs) is used by various pathogens to interfere with host cell functions. Among these modifications, ubiquitin (UBI) and ubiquitin-like proteins (UBLs) constitute key targets because they are regulators of pathways essential for the host cell. In particular, these PTM modifiers control pathways that have been described as crucial for infection such as pathogen entry, replication, propagation, or detection by the host. Although bacterial pathogens lack eucaryotic-like UBI or UBL systems, many of them produce proteins that specifically interfere with these host PTMs during infection. In this review we discuss the different mechanisms used by bacteria to interfere with host UBI and the two UBLs, SUMO and NEDD8.

Rapid Remodeling of the Host Epithelial Cell Proteome by the Listeriolysin O (LLO) Pore-forming Toxin.

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions.

SUMOylation of human septins is critical for septin filament bundling and cytokinesis.

Septins are cytoskeletal proteins that assemble into nonpolar filaments. They are critical in diverse cellular functions, acting as scaffolds for protein recruitment and as diffusion barriers for subcellular compartmentalization. Human septins are encoded by 13 different genes and are classified into four groups based on sequence homology (SEPT2, SEPT3, SEPT6, and SEPT7 groups). In yeast, septins were among the first proteins reported to be modified by SUMOylation, a ubiquitin-like posttranslational modification. However, whether human septins could be modified by small ubiquitin-like modifiers (SUMOs) and what roles this modification may have in septin function remains unknown. In this study, we first show that septins from all four human septin groups can be covalently modified by SUMOs. We show in particular that endogenous SEPT7 is constitutively SUMOylated during the cell cycle. We then map SUMOylation sites to the C-terminal domain of septins belonging to the SEPT6 and SEPT7 groups and to the N-terminal domain of septins from the SEPT3 group. We finally demonstrate that expression of non-SUMOylatable septin variants from the SEPT6 and SEPT7 groups leads to aberrant septin bundle formation and defects in cytokinesis after furrow ingression. Altogether, our results demonstrate a pivotal role for SUMOylation in septin filament bundling and cell division.

Promyelocytic Leukemia Protein (PML) Controls Listeria monocytogenes Infection.

The promyelocytic leukemia protein (PML) is the main organizer of stress-responsive subnuclear structures called PML nuclear bodies. These structures recruit multiple interactors and modulate their abundance or their posttranslational modifications, notably by the SUMO ubiquitin-like modifiers. The involvement of PML in antiviral responses is well established. In contrast, the role of PML in bacterial infection remains poorly characterized. Here, we show that PML restricts infection by the pathogenic bacterium Listeria monocytogenes but not by Salmonella enterica serovar Typhimurium. During infection, PML undergoes oxidation-mediated multimerization, associates with the nuclear matrix, and becomes de-SUMOylated due to the pore-forming activity of the Listeria toxin listeriolysin O (LLO). These events trigger an antibacterial response that is not observed during in vitro infection by an LLO-defective Listeria mutant, but which can be phenocopied by specific induction of PML de-SUMOylation. Using transcriptomic and proteomic microarrays, we also characterized a network of immunity genes and cytokines, which are regulated by PML in response to Listeria infection but independently from the listeriolysin O toxin. Our study thus highlights two mechanistically distinct complementary roles of PML in host responses against bacterial infection. IMPORTANCE: The promyelocytic leukemia protein (PML) is a eukaryotic protein that can polymerize in discrete nuclear assemblies known as PML nuclear bodies (NBs) and plays essential roles in many different cellular processes. Key to its function, PML can be posttranslationally modified by SUMO, a ubiquitin-like modifier. Identification of the role of PML in antiviral defenses has been deeply documented. In contrast, the role of PML in antibacterial defenses remains elusive. Here, we identify two mechanistically distinct complementary roles of PML in antibacterial responses against pathogens such as Listeria: (i) we show that PML regulates the expression of immunity genes in response to bacterial infection, and (ii) we unveil the fact that modification of PML SUMOylation by bacterial pore-forming toxins is sensed as a danger signal, leading to a restriction of bacterial intracellular multiplication. Taken together, our data reinforce the concept that intranuclear bodies can dynamically regulate important processes, such as defense against invaders.

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol-dependent cytolysins.

Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum-Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore-forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore-forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol-dependent cytolysins.

ISG15 counteracts Listeria monocytogenes infection.

ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity. The role of ISG15 during bacterial infection remains elusive. We show that ISG15 expression in nonphagocytic cells is dramatically induced upon Listeria infection. Surprisingly this induction can be type I interferon independent and depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection in vitro and in vivo. We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect. Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection. Together, our data reveal a previously uncharacterized ISG15-dependent restriction of Listeria infection, reinforcing the view that ISG15 is a key component of the innate immune response.

How bacterial pathogens colonize their hosts and invade deeper tissues.

Bacterial pathogens have evolved a wide range of strategies to colonize and invade human organs, despite the presence of multiple host defense mechanisms. In this review, we will describe how pathogenic bacteria can adhere and multiply at the surface of host cells, how some bacteria can enter and proliferate inside these cells, and finally how pathogens may cross epithelial or endothelial host barriers and get access to internal tissues, leading to severe diseases in humans.