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Abstract Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions.

Abstract L. monocytogenes causes listeriosis, a foodborne disease that is particularly dangerous for immunocompromised individuals and fetuses. Several virulence factors of this bacterial pathogen belong to a family of leucine-rich repeat (LRR)-containing proteins called internalins. Among these, InlP is known for its role in placental infection. We report here a function of InlP in mammalian cell nucleus organization. We demonstrate that bacteria do not produce InlP under in vitro culture conditions. When ectopically expressed in human cells, InlP translocates into the nucleus and changes the morphology of nuclear speckles, which are membrane-less organelles storing splicing factors. Using yeast two-hybrid screen, immunoprecipitation and pull-down experiments, we identify the tumor suppressor and splicing factor RBM5 as a major nuclear target of InlP. InlP inhibits RBM5-induced cell death and stimulate the formation of RBM5-induced nuclear granules, where the SC35 speckle protein redistributes. Taken together, these results suggest that InlP acts as a nucleomodulin controlling compartmentalization and function of RBM5 in the nucleus and that L. monocytogenes has developed a mechanism to target the host cell splicing machinery.

Enterococcus faecalis is a bacterial species present at a subdominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is one of the rare pathobionts identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unraveling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.

Staphylococcus aureus is an opportunistic pathogen that causes a range of devastating diseases including chronic osteomyelitis, which partially relies on the internalization and persistence of S. aureus in osteoblasts. The identification of the mechanisms of the osteoblast response to intracellular S. aureus is thus crucial to improve the knowledge of this infectious pathology. Since the signal from specifically infected bacteria-bearing cells is diluted and the results are confounded by bystander effects of uninfected cells, we developed a novel model of long-term infection. Using a flow cytometric approach we isolated only S. aureus-bearing cells from mixed populations that allows to identify signals specific to intracellular infection. Here we present an in-depth analysis of the effect of long-term S. aureus infection on the transcriptional program of human osteoblast-like cells. After RNA-seq and KEGG and Reactome pathway enrichment analysis, the remodeled transcriptomic profile of infected cells revealed exacerbated immune and inflammatory responses, as well as metabolic dysregulations that likely influence the intracellular life of bacteria. Numerous genes encoding epigenetic regulators were downregulated. The later included genes coding for components of chromatin-repressive complexes (e.g., NuRD, BAHD1 and PRC1) and epifactors involved in DNA methylation. Sets of genes encoding proteins of cell adhesion or neurotransmission were also deregulated. Our results suggest that intracellular S. aureus infection has a long-term impact on the genome and epigenome of host cells, which may exert patho-physiological dysfunctions additionally to the defense response during the infection process. Overall, these results not only improve our conceptual understanding of biological processes involved in the long-term S. aureus infections of osteoblast-like cells, but also provide an atlas of deregulated host genes and biological pathways and identify novel markers and potential candidates for prophylactic and therapeutic approaches.

Many bacterial species, including several pathogens, can enter a so-called “viable but non-culturable” (VBNC) state when subjected to stress. Bacteria in the VBNC state are metabolically active but have lost their ability to grow on standard culture media, which compromises their detection by conventional techniques based on bacterial division. Under certain conditions, VBNC bacteria can regain their growth capacity and, for pathogens, their virulence potential, through a process called resuscitation. Here, we review the current state of knowledge of the VBNC state of Listeria monocytogenes (Lm), a Gram-positive pathogenic bacterium responsible for listeriosis, one of the most dangerous foodborne zoonosis. After a brief summary of characteristics of VBNC bacteria, we highlight work on VBNC Lm in the environment and in agricultural and food industry settings, with particular emphasis on the impact of antimicrobial treatments. We subsequently discuss recent data suggesting that Lm can enter the VBNC state in the host, raising the possibility that VBNC forms contribute to the asymptomatic carriage of this pathogen in wildlife, livestock and even humans. We also consider the resuscitation and virulence potential of VBNC Lm and the danger posed by these bacteria to at-risk individuals, particularly pregnant women. Overall, we put forth the hypothesis that VBNC forms contribute to adaptation, persistence, and transmission of Lm between different ecological niches in the One-Health continuum, and suggest that screening for healthy carriers, using alternative techniques to culture-based enrichment methods, should better prevent listeriosis risks.

Listeria monocytogenes causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through its important functions in immunity. However, recent evidence suggests that during long-term infection of hepatocytes, a subpopulation of Listeria may escape eradication by entering a persistence phase in intracellular vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first optimized cell models of persistent infection in human hepatocyte cell lines HepG2 and Huh7 and primary mouse hepatocytes (PMH). In these cells, Listeria efficiently entered the persistence phase after three days of infection, while inducing a potent interferon response, of type I in PMH and type III in HepG2, while Huh7 remained unresponsive. RNA-sequencing analysis identified a common signature of long-term Listeria infection characterized by the overexpression of a set of genes involved in antiviral immunity and the under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. Infection also altered the expression of cholesterol metabolism-associated genes in HepG2 and Huh7 cells. The decrease in APP transcripts was correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1β). Collectively, these results reveal that long-term infection with Listeria profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.

Pathogenic bacteria secrete a variety of proteins that manipulate host cell function by targeting components of the plasma membrane, cytosol, or organelles. In the last decade, several studies identified bacterial factors acting within the nucleus on gene expression or other nuclear processes, which has led to the emergence of a new family of effectors called “nucleomodulins”. In human and animal pathogens, Listeria monocytogenes for Gram-positive bacteria and Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, Legionella pneumophila, Shigella flexneri, and Escherichia coli for Gram-negative bacteria, have led to pioneering discoveries. In this review, we present these paradigms and detail various mechanisms and core elements (e.g., DNA, histones, epigenetic regulators, transcription or splicing factors, signaling proteins) targeted by nucleomodulins. We particularly focus on nucleomodulins interacting with epifactors, such as LntA of Listeria and ankyrin repeat- or tandem repeat-containing effectors of Rickettsiales, and nucleomodulins from various bacterial species acting as post-translational modification enzymes. The study of bacterial nucleomodulins not only generates important knowledge about the control of host responses by microbes but also creates new tools to decipher the dynamic regulations that occur in the nucleus. This research also has potential applications in the field of biotechnology. Finally, this raises questions about the epigenetic effects of infectious diseases.

Intracellular bacterial pathogens are generally classified into two types: those that exploit host membrane trafficking to construct specific niches in vacuoles (i.e., “vacuolar pathogens”), and those that escape from vacuoles into the cytosol, where they proliferate and often spread to neighboring cells (i.e., “cytosolic pathogens”). However, the boundary between these distinct intracellular phenotypes is tenuous and may depend on the timing of infection and on the host cell type. Here, we discuss recent progress highlighting this phenotypic duality in Listeria monocytogenes, which has long been a model for cytosolic pathogens, but now emerges as a bacterium also capable of residing in vacuoles, in a slow/non-growing state. The ability of L. monocytogenes to enter a persistence stage in vacuoles might play a role during the asymptomatic incubation period of listeriosis and/or the carriage of this pathogen in asymptomatic hosts. Moreover, persistent vacuolar Listeria could be less susceptible to antibiotics and more difficult to detect by routine techniques of clinical biology. These hypotheses deserve to be explored in order to better manage the risks related to this food-borne pathogen.

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

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.

ABSTRACT For nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogen Listeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strain in vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infection in vivo, highlighting the fact that in vitro invasion assays are not sufficient for evaluating the pathogenic potential of L. monocytogenes strains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using different L. monocytogenes strains. IMPORTANCE Over the past 3 decades, Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studying Listeria use primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator of Listeria virulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may use Listeria as a tool to study the pathophysiology of listeriosis and immune responses.

ABSTRACT The nucleus has emerged as a key target for nucleomodulins, a family of effectors produced by bacterial pathogens to control host transcription or other nuclear processes. The virulence factor LntA from Listeria monocytogenes stimulates interferon responses during infection by inhibiting BAHD1, a nuclear protein involved in gene silencing by promoting heterochromatin formation. So far, whether the interaction between LntA and BAHD1 is direct and sufficient for inhibiting BAHD1 activity is unknown. Here, we functionally characterized the molecular interface between the two proteins in vitro and in transfected or infected human cells. Based on the known tridimensional structure of LntA, we identified a dilysine motif (K180/K181) in the elbow region of LntA and a central proline-rich region in BAHD1 as crucial for the direct LntA-BAHD1 interaction. To better understand the role played by the dilysine motif in the functionality of LntA, we solved the crystal structure of a K180D/K181D mutant to a 2.2-Å resolution. This mutant highlights a drastic redistribution of surface charges in the vicinity of a groove, which likely plays a role in nucleomodulin target recognition. Mutation of the strategic dilysine motif also abolished the recruitment of LntA to BAHD1-associated nuclear foci and impaired the LntA-mediated stimulation of interferon responses upon infection. Last, the strict conservation of residues K180 and K181 in LntA sequences from 188 L. monocytogenes strains of different serotypes and origins further supports their functional importance. Together, these results provide structural and functional details about the mechanism of inhibition of an epigenetic factor by a bacterial nucleomodulin. IMPORTANCE Pathogens have evolved various strategies to deregulate the expression of host defense genes during infection, such as targeting nuclear proteins. LntA, a secreted virulence factor from the bacterium Listeria monocytogenes, stimulates innate immune responses by inhibiting a chromatin-associated repressor, BAHD1. This study reveals the structural features of LntA required for BAHD1 inhibition. LntA interacts directly with a central domain of BAHD1 via a surface patch of conserved positive charges, located nearby a groove on the elbow region of LntA. By demonstrating that this patch is required for LntA function, we provide a better understanding of the molecular mechanism allowing a bacterial pathogen to control host chromatin compaction and gene expression.

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been "murinized" to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlA(m)) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlA(m)-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlA(m)-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts.