A bacterial virulence factor interacts with the splicing factor RBM5 and stimulates formation of nuclear RBM5 granules.

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.

Corrigendum: An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

[This corrects the article DOI: 10.3389/fcimb.2021.761945.].

Transcriptome Architecture of Osteoblastic Cells Infected With Staphylococcus aureus Reveals Strong Inflammatory Responses and Signatures of Metabolic and Epigenetic Dysregulation.

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.

The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes.

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.

The Viable But Non-Culturable State of Listeria monocytogenes in the One-Health Continuum.

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.

An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

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-1ß). 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.

AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion.

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.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner.

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Microscopy of Intracellular Listeria monocytogenes in Epithelial Cells.

The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.

BAHD1 haploinsufficiency results in anxiety-like phenotypes in male mice.

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.

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins.

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.

Targeting host epigenetic machinery: The Listeria paradigm.

By modifying the host cell transcription programme, pathogenic bacteria disrupt a wide range of cellular processes and take control of the host's immune system. Conversely, by mobilising a network of defence genes, the host cells trigger various responses that allow them to tolerate or eliminate invaders. The study of the molecular basis of this crosstalk is crucial to the understanding of infectious diseases. Although research has long focused on the targeting of eukaryotic DNA-binding transcription factors, more recently, another powerful way by which bacteria modify the expression of host genes has emerged: chromatin modifications in the cell nucleus. One of the most prolific bacterial models in this area has been Listeria monocytogenes, a facultative intracellular bacterium responsible for serious food-borne infections. Here, we aim to highlight the contribution of this model to the field of bacteria-mediated chromatin modifications. We will first recall the general principles of epigenetic regulation and then illustrate five mechanisms that mobilise the epigenetic machinery in response to Listeria factors, either through bacterial molecular patterns, a toxin, an invasion protein, or nucleomodulins. Strategies used by Listeria to control the expression of host genes at the chromatin level, by activation of cytosolic signalling pathways or direct targeting of epifactors in the nucleus, have contributed to the emergence of a new discipline combining cellular microbiology and epigenetics: "patho-epigenetics."

To Be Cytosolic or Vacuolar: The Double Life of Listeria monocytogenes.

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 switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

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.

Spatial Organization of Cell Wall-Anchored Proteins at the Surface of Gram-Positive Bacteria.

Bacterial surface proteins constitute an amazing repertoire of molecules with important functions such as adherence, invasion, signalling and interaction with the host immune system or environment. In Gram-positive bacteria, many surface proteins of the "LPxTG" family are anchored to the peptidoglycan (PG) by an enzyme named sortase. While this anchoring mechanism has been clearly deciphered, less is known about the spatial organization of cell wall-anchored proteins in the bacterial envelope. Here, we review the question of the precise spatial and temporal positioning of LPxTG proteins in subcellular domains in spherical and ellipsoid bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis) and in the rod-shaped bacterium Listeria monocytogenes. Deposition at specific sites of the cell wall is a dynamic process tightly connected to cell division, secretion, cell morphogenesis and levels of gene expression. Studying spatial occupancy of these cell wall-anchored proteins not only provides information on PG dynamics in responses to environmental changes, but also suggests that pathogenic bacteria control the distribution of virulence factors at specific sites of the surface, including pole, septa or lateral sites, during the infectious process.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

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.

Overexpression of the Heterochromatinization Factor BAHD1 in HEK293 Cells Differentially Reshapes the DNA Methylome on Autosomes and X Chromosome.

BAH domain-containing protein 1 (BAHD1) is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi), but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq) to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT) to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1). We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX). We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX). Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats, and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these "BAHD1-Associated differentially methylated Domains" (BADs) overlapped with lamina-associated domains (LADs). Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

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.

Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.