The cnf1 gene is associated with an expanding Escherichia coli ST131 H30Rx/C2 subclade and confers a competitive advantage for gut colonization.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.

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.

Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver.

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host.

From Substrate to Fragments to Inhibitor Active In Vivo against Staphylococcus aureus.

Antibiotic resistance is a worldwide threat due to the decreasing supply of new antimicrobials. Novel targets and innovative strategies are urgently needed to generate pathbreaking drug compounds. NAD kinase (NADK) is essential for growth in most bacteria, as it supports critical metabolic pathways. Here, we report the discovery of a new class of antibacterials that targets bacterial NADK. We generated a series of small synthetic adenine derivatives to screen those harboring promising substituents in order to guide efficient fragment linking. This led to NKI1, a new lead compound inhibiting NADK that showed in vitro bactericidal activity against Staphylococcus aureus. In a murine model of infection, NKI1 restricted survival of the bacteria, including methicillin-resistant S. aureus. Collectively, these findings identify bacterial NADK as a potential drug target and NKI1 as a lead compound in the treatment of staphylococcal infections.

Ubiquitination of Listeria Virulence Factor InlC Contributes to the Host Response to Infection.

Listeria monocytogenes is a pathogenic bacterium causing potentially fatal foodborne infections in humans and animals. While the mechanisms used by Listeria to manipulate its host have been thoroughly characterized, how the host controls bacterial virulence factors remains to be extensively deciphered. Here, we found that the secreted Listeria virulence protein InlC is monoubiquitinated by the host cell machinery on K224, restricting infection. We show that the ubiquitinated form of InlC interacts with the intracellular alarmin S100A9, resulting in its stabilization and in increased reactive oxygen species production by neutrophils in infected mice. Collectively, our results suggest that posttranslational modification of InlC exacerbates the host response upon Listeria infection.IMPORTANCE The pathogenic potential of Listeria monocytogenes relies on the production of an arsenal of virulence determinants that have been extensively characterized, including surface and secreted proteins of the internalin family. We have previously shown that the Listeria secreted internalin InlC interacts with IκB kinase α to interfere with the host immune response (E. Gouin, M. Adib-Conquy, D. Balestrino, M.-A. Nahori, et al., Proc Natl Acad Sci USA, 107:17333-17338, 2010, https://doi.org/10.1073/pnas.1007765107). In the present work, we report that InlC is monoubiquitinated on K224 upon infection of cells and provide evidence that ubiquitinated InlC interacts with and stabilizes the alarmin S100A9, which is a critical regulator of the immune response and inflammatory processes. Additionally, we show that ubiquitination of InlC causes an increase in reactive oxygen species production by neutrophils in mice and restricts Listeria infection. These findings are the first to identify a posttranscriptional modification of an internalin contributing to host defense.

A Listeria monocytogenes Bacteriocin Can Target the Commensal Prevotella copri and Modulate Intestinal Infection.

Understanding the role of the microbiota components in either preventing or favoring enteric infections is critical. Here, we report the discovery of a Listeria bacteriocin, Lmo2776, which limits Listeria intestinal colonization. Oral infection of conventional mice with a Δlmo2776 mutant leads to a thinner intestinal mucus layer and higher Listeria loads both in the intestinal content and deeper tissues compared to WT Listeria. This latter difference is microbiota dependent, as it is not observed in germ-free mice. Strikingly, it is phenocopied by pre-colonization of germ-free mice before Listeria infection with Prevotella copri, an abundant gut-commensal bacteria, but not with the other commensals tested. We further show that Lmo2776 targets P. copri and reduces its abundance. Together, these data unveil a role for P.copri in exacerbating intestinal infection, highlighting that pathogens such as Listeria may selectively deplete microbiota bacterial species to avoid excessive inflammation.

An RNA-Binding Protein Secreted by a Bacterial Pathogen Modulates RIG-I Signaling.

RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-ß production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as "social RNAs" to trigger a host response during infection.

Lmo1656 is a secreted virulence factor of Listeria monocytogenes that interacts with the sorting nexin 6-BAR complex.

Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm, it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm, Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm Furthermore, deletion of the lmo1656 gene (Δlmo1656) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host.

OrfX, a Nucleomodulin Required for Listeria monocytogenes Virulence.

Listeria monocytogenes is a bacterial pathogen causing severe foodborne infections in humans and animals. Listeria can enter into host cells and survive and multiply therein, due to an arsenal of virulence determinants encoded in different loci on the chromosome. Several key Listeria virulence genes are clustered in Listeria pathogenicity island 1. This important locus also contains orfX (lmo0206), a gene of unknown function. Here, we found that OrfX is a small, secreted protein whose expression is positively regulated by PrfA, the major transcriptional activator of Listeria virulence genes. We provide evidence that OrfX is a virulence factor that dampens the oxidative response of infected macrophages, which contributes to intracellular survival of bacteria. OrfX is targeted to the nucleus and interacts with the regulatory protein RybP. We show that in macrophages, the expression of OrfX decreases the level of RybP, which controls cellular infection. Collectively, these data reveal that Listeria targets RybP and evades macrophage oxidative stress for efficient infection. Altogether, OrfX is after LntA, the second virulence factor acting directly in the nucleus.IMPORTANCEListeria monocytogenes is a model bacterium that has been successfully used over the last 30 years to refine our understanding of the molecular, cellular, and tissular mechanisms of microbial pathogenesis. The major virulence factors of pathogenic Listeria species are located on a single chromosomal locus. Here, we report that the last gene of this locus encodes a small secreted nucleomodulin, OrfX, that is required for bacterial survival within macrophages and in the infected host. This work demonstrates that the production of OrfX contributes to limiting the host innate immune response by dampening the oxidative response of macrophages. We also identify a target of OrfX, RybP, which is an essential pleiotropic regulatory protein of the cell, and uncover its role in host defense. Our data reinforce the view that the secretion of nucleomodulins is an important strategy used by microbial pathogens to promote infection.

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.

8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode.

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.

Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection.

Listeria monocytogenes is responsible for gastroenteritis in healthy individuals and for a severe invasive disease in immunocompromised patients. Among the three identified L. monocytogenes evolutionary lineages, lineage I strains are overrepresented in epidemic listeriosis outbreaks, but the mechanisms underlying the higher virulence potential of strains of this lineage remain elusive. Here, we demonstrate that Listeriolysin S (LLS), a virulence factor only present in a subset of lineage I strains, is a bacteriocin highly expressed in the intestine of orally infected mice that alters the host intestinal microbiota and promotes intestinal colonization by L. monocytogenes, as well as deeper organ infection. To our knowledge, these results therefore identify LLS as the first bacteriocin described in L. monocytogenes and associate modulation of host microbiota by L. monocytogenes epidemic strains to increased virulence.

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.

Listeria monocytogenes dampens the DNA damage response.

The DNA damage response (DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O (LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.

Riboswitches. Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA.

Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.

A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection.

Pathogens dramatically affect host cell transcription programs for their own profit during infection, but in most cases, the underlying mechanisms remain elusive. We found that during infection with the bacterium Listeria monocytogenes, the host deacetylase sirtuin 2 (SIRT2) translocates to the nucleus, in a manner dependent on the bacterial factor InlB. SIRT2 associates with the transcription start site of a subset of genes repressed during infection and deacetylates histone H3 on lysine 18 (H3K18). Infecting cells in which SIRT2 activity was blocked or using SIRT2(-/-) mice resulted in a significant impairment of bacterial infection. Thus, SIRT2-mediated H3K18 deacetylation plays a critical role during infection, which reveals an epigenetic mechanism imposed by a pathogenic bacterium to reprogram its host.

Impact of lactobacilli on orally acquired listeriosis.

Listeria monocytogenes is a foodborne pathogen that crosses the intestinal barrier and disseminates within the host. Here, we report a unique comprehensive analysis of the impact of two Lactobacillus species, Lactobacillus paracasei CNCM I-3689 and Lactobacillus casei BL23, on L. monocytogenes and orally acquired listeriosis in a gnotobiotic humanized mouse model. We first assessed the effect of treatment with each Lactobacillus on L. monocytogenes counts in host tissues and showed that each decreases L. monocytogenes systemic dissemination in orally inoculated mice. A whole genome intestinal transcriptomic analysis revealed that each Lactobacillus changes expression of a specific subset of genes during infection, with IFN-stimulated genes (ISGs) being the most affected by both lactobacilli. We also examined microRNA (miR) expression and showed that three miRs (miR-192, miR-200b, and miR-215) are repressed during L. monocytogenes infection. Treatment with each Lactobacillus increased miR-192 expression, whereas only L. casei association increased miR-200b and miR-215 expression. Finally, we showed that treatment with each Lactobacillus significantly reshaped the L. monocytogenes transcriptome and up-regulated transcription of L. monocytogenes genes encoding enzymes allowing utilization of intestinal carbon and nitrogen sources in particular genes involved in propanediol and ethanolamine catabolism and cobalamin biosynthesis. Altogether, these data reveal that the modulation of L. monocytogenes infection by treatment with lactobacilli correlates with a decrease in host gene expression, in particular ISGs, miR regulation, and a dramatic reshaping of L. monocytogenes transcriptome.

OatA, a peptidoglycan O-acetyltransferase involved in Listeria monocytogenes immune escape, is critical for virulence.

Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, ß-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.

Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy.

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK(-) bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.

Neonatal lung immune responses show a shift of cytokines and transcription factors toward Th2 and a deficit in conventional and plasmacytoid dendritic cells.

The high incidence of lung-damaging life-threatening respiratory infections in infants may be related to the immaturity of their immune systems. To determine whether lung immune features differ in early life compared with those in adulthood, whole lung as well as lung T lymphocyte and DC responses were investigated in BALB/c neonates versus adults. Higher expression of GATA-3 and rapid and sustained production of type 2 cytokines by lung explants after in vitro exposure to anti-CD3 was the hallmark of the neonatal period, suggestive of a Th2 bias. Neonatal lung GATA-3-producing cells were identified as CD3(+), CD4 and CD8 double-negative T lymphocytes, a subset found at a higher frequency in neonatal than adult lung. The neonatal lungs contained fewer conventional DCs, with a lower ratio of CD103(+) to CD11b(+) DCs, and a much lower number of plasmacytoid DCs in comparison with adult lungs. Yet, when stimulated in vivo by BCG, neonatal lung DCs matured and primed adult naïve CD4(+) T cells toward Th1 as efficiently as adult BCG-primed lung DCs. Conversely, both adult and neonatal BCG-primed lung DCs induced a Th2 cytokine response from neonatal naïve lymph node T cells, suggestive of an intrinsic feature of neonatal T lymphocytes.