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Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr158) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr158 defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr158 as a novel layer of regulation of actomyosin cytoskeleton organization. [ABSTRACT FROM AUTHOR]

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire. Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage. [ABSTRACT FROM AUTHOR]

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan. Author summary: During their cell cycle, Gram-positive bacteria can be frequently found surrounded by a cellular envelope, which includes glycopolymers such as peptidoglycan, wall teichoic acids or capsular polysaccharides, whose composition is adjusted to the external insults they may find. The clarification of how bacteria tune synthesis of these three macromolecules, which act as defensive layers, ensuring efficient enclosing of bacteria and their protection from the surrounding medium, will permit a better understanding of how bacteria propagate and may pave the way to the intelligent design of anti-infective strategies. In this manuscript, we have determined how Wzg, a candidate for the Streptococcus pneumoniae ligase that attaches capsular polysaccharides to the bacterial cell wall, is directed to the division septum to ensure the full encapsulation of bacteria. Perturbation of this process result in bacteria with reduced protection from external peptidoglycan binding receptors (such as the pneumococcal major hydrolase) and impaired in their ability to survive within the infected host. [ABSTRACT FROM AUTHOR]

Simple Summary: SARS-CoV-2 is the virus that causes COVD-19; there is consensus that this virus infects cells presenting the angiotensin-converting enzyme 2 (ACE2) receptor at the cell surface. In our study, we assessed the expression profiles of cancer cells regarding the expressions of ACE2 and furin (another important player in the infection process). We analyzed a series of formalin-fixed paraffin-embedded samples of tumor tissues from cancer patients who underwent surgery at the beginning of the pandemic, including some cases with known positive results for SARS-CoV-2. Our goal was to explore the possibility of viral infections in cancer tissues by detecting viral RNA and/or proteins using histology-based methods. From this extensive methodologic approach, we show that colon and gastric carcinoma cells present high expression levels of ACE2 and furin, contrasting with a negative expression profile of ACE2 in thyroid carcinoma. Some cases tested positive for SARS-CoV-2 by PCR extracted from tissue sections, but in situ hybridization and immunohistochemistry did not show consistent results of a viral presence in the cancer cells. Our study raises the possibility of ACE2-mediated viral tropism for cancer tissues to be clarified in future studies. SARS-CoV-2 pandemics have been massively characterized on a global scale by the rapid generation of in-depth genomic information. The main entry gate of SARS-CoV-2 in human cells is the angiotensin-converting enzyme 2 (ACE2) receptor. The expression of this protein has been reported in several human tissues, suggesting a correlation between SARS-CoV-2 organotropism and ACE2 distribution. In this study, we selected (a series of) 90 patients who were submitted to surgery for tumor removal between the beginning of the SARS-CoV-2 pandemic and the closure of operating rooms (by the end of March 2020) in two different countries—Portugal and Brazil. We evaluated the expressions of ACE2 and furin (another important factor for virus internalization) in colon (n = 60), gastric (n = 19), and thyroid (n = 11) carcinomas. In a subseries of cases with PCR results for SARS-CoV-2 detection in the peri-operatory window (n = 18), we performed different methodological approaches for viral detections in patient tumor samples. Our results show that colon and gastric carcinomas display favorable microenvironments to SARS-CoV-2 tropism, presenting high expression levels of ACE2 and furin. From the subseries of 18 cases, 11 tested positive via PCR detection performed in tumor blocks; however, a direct association between the ACE2 expression and SARS-CoV-2 infection was not demonstrated in cancer cells using histology-based techniques, such as immunohistochemistry or in situ hybridization. This study raises the possibility of ACE2-mediated viral tropism in cancer tissues to be clarified in future studies. [ABSTRACT FROM AUTHOR]

Research has been conducted on Sinorhizobium meliloti genes. The isolation of these genes via RNA fingerprinting by arbitrarily primed PCR has been carried out.

Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation, and cancer; however, its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane. We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection. [ABSTRACT FROM AUTHOR]

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen. [ABSTRACT FROM AUTHOR]

Innate immunity is the most broadly effective host defense, being essential to clear the majority of microbial infections. Scavenger Receptors comprise a family of sensors expressed in a multitude of host cells, whose dual role during microbial pathogenesis gained importance over recent years. SRs regulate the recruitment of immune cells and control both host inflammatory response and bacterial load. In turn, pathogens have evolved different strategies to overcome immune response, avoid recognition by SRs and exploit them to favor infection. Here, we discuss the most relevant findings regarding the interplay between SRs and pathogens, discussing how these multifunctional proteins recognize a panoply of ligands and act as bacterial phagocytic receptors. [ABSTRACT FROM AUTHOR]

Summary: Wall teichoic acids (WTAs) are important surface glycopolymers involved in various physiological processes occurring in the Gram‐positive cell envelope. We previously showed that the decoration of Listeria monocytogenes (Lm) WTAs with l‐rhamnose conferred resistance against antimicrobial peptides. Here, we show that WTA l‐rhamnosylation also contributes to physiological levels of autolysis in Lm through a mechanism that requires efficient association of Ami, a virulence‐promoting autolysin belonging to the GW protein family, to the bacterial cell surface. Importantly, WTA l‐rhamnosylation also controls the surface association of another GW protein, the invasin internalin B (InlB), that promotes Lm invasion of host cells. Whereas WTA N‐acetylglucosaminylation is not a prerequisite for GW protein surface association, lipoteichoic acids appear to also play a role in the surface anchoring of InlB. Strikingly, while the GW domains of Ami, InlB and Auto (another autolysin contributing to cell invasion and virulence) are sufficient to mediate surface association, this is not the case for the GW domains of the remaining six uncharacterized Lm GW proteins. Overall, we reveal WTA l‐rhamnosylation as a bacterial surface modification mechanism that contributes to Lm physiology and pathogenesis by controlling the surface association of GW proteins involved in autolysis and infection. [ABSTRACT FROM AUTHOR]

The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin β1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression. [ABSTRACT FROM AUTHOR]

Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacteriumListeria monocytogenesis a human foodborne pathogen that remains a serious public health concern. To transport proteins across its cell envelope, this facultative intracellular pathogen engages a set of specialized secretion systems. Here we show thatL. monocytogenesEGDe uses a specialized secretion system, named ESX-1, to secrete EsxA, a homolog of the virulence determinants ESAT-6 and EsxA ofMycobacterium tuberculosisandStaphylococcus aureus, respectively. Our data show that theL. monocytogenesESX-1 secretion system and its substrates are dispensable for bacterial invasion and intracellular multiplication in eukaryotic cell lines. Surprisingly, we found that the EssC-dependent secretion of EsxA has a detrimental effect onL. monocytogenes in vivoinfection. [ABSTRACT FROM AUTHOR]

This study examined the concordance of penile and oral human papillomavirus (HPV) infections in the United States. A total of 1683 men aged 18-59 years who participated in the 2013-2014 National Health and Nutrition Examination Survey and had results of oral and penile HPV DNA testing were examined. The prevalence of any HPV genotype was 45.3% on the penis, 11.2% in the oral cavity, and 8.8% at both sites. The prevalence of HPV in the oral cavity was higher among those with than among those without penile HPV infection (19.3% vs 4.4%; prevalence ratio, 4.37 [95% confidence interval, 2.66-7.16]). The prevalence of ≥1 genotype-concordant HPV infection was 3.2% and was associated with sexual behavior, independent of demographic characteristics and smoking status. Sexual behavior may partly explain the observed association between penile and oral HPV infections. [ABSTRACT FROM AUTHOR]

During infection, plasma membrane ( PM) blebs protect host cells against bacterial pore-forming toxins ( PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA ( NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA-Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA-Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O ( LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing. [ABSTRACT FROM AUTHOR]

Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes, a foodborne pathogenic bacterium, remains a serious public health concern due to its frequent occurrence in food products coupled with a high mortality rate. Bacterial pathogenicity depends greatly on the ability to secrete virulence factors to or beyond the bacterial cell surface. The Tat pathway, one of the secretion systems present in L. monocytogenes, was until now only investigated in silico. In L. monocytogenes strain EGDe two genes constitute this pathway, tatC(lmo0361) and tatA(lmo0362). Here we show that tatC and tatA are cotranscribed in a bicistronic- and growth-phase-dependent manner, being downregulated in the stationary phase. An EGDe tatAC mutant strain (EGDe ΔtatAC) was constructed, confirming that the Tat pathway is not essential for L.monocytogenes survival or biofilm-forming ability. When compared to the wild-type EGDe, deletion of tatAC did not decrease the virulence potential of EGDe ΔtatAC in HT-29 human epithelial cell line and even increased (p < 0.05) the virulence potential for mice. Moreover, we show that tat genes are prevalent in L. monocytogenes strains belonging to genetic lineage II and are generally absent from lineage I, which is more associated with human cases, thus excluding the possibility of using the Tat system as a target for novel antimicrobial compounds targeting L.monocytogenes. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host-pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a food-borne pathogen able to invade non-phagocytic cells. InlA, a L. monocytogenes surface protein, interacts with human E-cadherin to promote bacterial entry. L. monocytogenes internalization is a dynamic process involving co-ordinated actin cytoskeleton rearrangements and host cell membrane remodelling at the site of bacterial attachment. Interaction between E-cadherin and catenins is required to promote Listeria entry, and for the establishment of adherens junctions in epithelial cells. Although several molecular factors promoting E-cadherin-mediated Listeria internalization have been identified, the proteins regulating the transient actin polymerization required at the bacterial entry site are unknown. Here we show that the Arp2/3 complex acts as an actin nucleator during the InlA/E-cadherin-dependent internalization. Using a variety of approaches including siRNA, expression of dominant negative derivatives and pharmacological inhibitors, we demonstrate the crucial role of cortactin in the activation of the Arp2/3 complex during InlA-mediated entry. We also show the requirement of the small GTPase Rac1 and that of Src-tyrosine kinase activity to promote Listeria internalization. Together, these data suggest a model in which Src tyrosine kinase and Rac1 promote recruitment of cortactin and activation of Arp2/3 at Listeria entry site, mimicking events that occur during adherens junction formation. [ABSTRACT FROM AUTHOR]

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds β-catenin, which then recruits α-catenin. α-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of α-catenin. Using a two-hybrid screen, we identified a new partner of α-catenin: ARHGAP10. This protein colocalized with α-catenin at cell–cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and α-catenin recruitment at cell–cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced α-catenin and cortical actin levels at cell–cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell–cell junctions that controls α-catenin recruitment and has a key role during L. monocytogenes uptake. [ABSTRACT FROM AUTHOR]

By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip–Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E-cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; α- and β- catenins play key roles in these processes. We have recently identified an unconventional myosin, myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is among the best-characterized intracellular pathogens. Its virulence factors, and the way they interfere with host cells to hijack host functions and promote the establishment and dissemination of the infection, have been the focus of multiple studies over the last 30 years. During cellular infection, L. monocytogenes was shown to induce host DNA damage and delay the host cell cycle to its own benefit. However, whether the cell cycle stage would interfere with the capacity of Listeria to infect human cultured cell lines was never assessed. We found here that L. monocytogenes preferentially infects cultured cells in G2/M phases. Inside G2/M cells, the bacteria lead to an increase in the overall mitosis duration by delaying the mitotic exit. We showed that L. monocytogenes infection causes a sustained activation of the spindle assembly checkpoint, which we correlated with the increase in the percentage of misaligned chromosomes detected in infected cells. Moreover, we demonstrated that chromosome misalignment in Listeria-infected cells required the function of two Listeria virulence factors, ActA and InlC. Our findings show the pleiotropic role of Listeria virulence factors and their cooperative action in successfully establishing the cellular infection. [ABSTRACT FROM AUTHOR]

The cell wall of Listeria monocytogenes (Lm), a major intracellular foodborne bacterial pathogen, comprises a thick peptidoglycan layer that serves as a scaffold for glycopolymers such as wall teichoic acids (WTAs). WTAs contain non-essential sugar substituents whose absence prevents bacteriophage binding and impacts antigenicity, sensitivity to antimicrobials, and virulence. Here, we demonstrated, for the first time, the triple function of Lm WTA glycosylations in the following: (1) supporting the correct anchoring of major Lm virulence factors at the bacterial surface, namely Ami and InlB; (2) promoting Lm resistance to antimicrobial peptides (AMPs); and (3) decreasing Lm sensitivity to some antibiotics. We showed that while the decoration of WTAs by rhamnose in Lm serovar 1/2a and by galactose in serovar 4b are important for the surface anchoring of Ami and InlB, N-acetylglucosamine in serovar 1/2a and glucose in serovar 4b are dispensable for the surface association of InlB or InlB/Ami. We found that the absence of a single glycosylation only had a slight impact on the sensibility of Lm to AMPs and antibiotics, however the concomitant deficiency of both glycosylations (rhamnose and N-acetylglucosamine in serovar 1/2a, and galactose and glucose in serovar 4b) significantly impaired the Lm capacity to overcome the action of antimicrobials. We propose WTA glycosylation as a broad mechanism used by Lm, not only to properly anchor surface virulence factors, but also to resist AMPs and antibiotics. WTA glycosyltransferases thus emerge as promising drug targets to attenuate the virulence of bacterial pathogens, while increasing their susceptibility to host immune defenses and potentiating the action of antibiotics. [ABSTRACT FROM AUTHOR]

Clostridium perfringens produces an arsenal of toxins that act together to cause severe infections in humans and livestock animals. Perfringolysin O (PFO) is a cholesterol-dependent pore-forming toxin encoded in the chromosome of virtually all C. perfringens strains and acts in synergy with other toxins to determine the outcome of the infection. However, its individual contribution to the disease is poorly understood. Here, we intoxicated human epithelial and endothelial cells with purified PFO to evaluate the host cytoskeletal responses to PFO-induced damage. We found that, at sub-lytic concentrations, PFO induces a profound reorganization of the actomyosin cytoskeleton culminating into the assembly of well-defined cortical actomyosin structures at sites of plasma membrane (PM) remodeling. The assembly of such structures occurs concomitantly with the loss of the PM integrity and requires pore-formation, calcium influx, and myosin II activity. The recovery from the PM damage occurs simultaneously with the disassembly of cortical structures. PFO also targets the endoplasmic reticulum (ER) by inducing its disruption and vacuolation. ER-enriched vacuoles were detected at the cell cortex within the PFO-induced actomyosin structures. These cellular events suggest the targeting of the endothelium integrity at early stages of C. perfringens infection, in which secreted PFO is at sub-lytic concentrations. [ABSTRACT FROM AUTHOR]

Following damage by pore forming toxins (PFTs) host cells engage repair processes and display profound cytoskeletal remodeling and concomitant plasma membrane (PM) blebbing. We have recently demonstrated that host cells utilize similar mechanisms to control cytoskeletal dynamics in response to PFTs and during cell migration. This involves assembly of cortical actomyosin bundles, reorganisation of the endoplasmic reticulum (ER) network, and the interaction between the ER chaperone Gp96 and the molecular motor Non-muscle Myosin Heavy Chain IIA (NMHCIIA). Consequently, Gp96 regulates actomyosin activity, PM blebbing and cell migration, and protects PM integrity against PFTs. In addition, we observed that PFTs increase association of Gp96 and ER vacuoles with the cell surface or within PM blebs loosely attached to the cell body. Similarly, gut epithelial cells damaged by PFTsin vivowere shown to release microvilli structures or directly purge cytoplasmic content. Cytoplasmic purging involves profound cytoskeletal remodeling and ER vacuolation, suggesting that our observations recapitulate recovery processesin vivo. Here, we discuss our findings in light of the current understanding of PM repair mechanisms andin vivorecovery responses to PFTs. [ABSTRACT FROM AUTHOR]

Presents a correction in the article 'Listeria monocytogenes bile salt hydrolase is a virulence factor involved in the intestinal and hepatic phases of listeriosis' by Dussurget et al. published in 'Molecular Biology' vol. 45, number 4.

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp1--Leu170) as sufficient to interact with Vip; however, we also showed that the region Tyr179--Leu390 of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection [ABSTRACT FROM AUTHOR]

Abstract: The use of the food-grade bacterium Lactococcus lactis as antigen delivery vehicle at the mucosal level is an attractive vaccination strategy intensively explored during the last decade. In this study, we developed L. lactis strains which could be used as a DNA delivery vector to combine both advantages of mucosal delivery and of DNA vaccination. To render lactococci capable of invading epithelial cells, the Listeria monocytogenes inlA gene was cloned and expressed in L. lactis under transcriptional control of the native promoter. Western blot and immunofluorescence assays revealed that recombinant lactococci efficiently displayed the cell wall anchored form of InlA. We demonstrated that this expression promotes internalization of L. lactis inlA + into the human epithelial cell line Caco-2. Gentamicin assay showed that invasiveness of L. lactis in these cells is ~100-fold higher for L. lactis inlA + than for wild type (wt) L. lactis strains. Moreover, we showed that L. lactis inlA + is able to enter intestinal cells in vivo, after oral inoculation of guinea pigs. After internalization, L. lactis inlA + was able to deliver a functional eukaryotic gfp gene into epithelial Caco-2 cells; GFP was detected in 1% of internalized cells. The L. lactis inlA + strain will be a useful bacterial vector for the development of new live oral DNA vaccines. It also constitutes an interesting new model to study the role of internalin in bacterial localization in the animal host. [Copyright &y& Elsevier]

The tight and coordinated regulation of virulence gene expression is crucial to ensure the survival and persistence of bacterial pathogens in different contexts within their hosts. Considering this, bacteria do not express virulence factors homogenously in time and space, either due to their associated fitness cost or to their detrimental effect at specific infection stages. To efficiently infect and persist into their hosts, bacteria have thus to monitor environmental cues or chemical cell-to-cell signaling mechanisms that allow their transition from the external environment to the host, and therefore adjust gene expression levels, intrinsic biological activities, and appropriate behaviors. Listeria monocytogenes ( Lm ), a major Gram-positive facultative intracellular pathogen, stands out for its adaptability and capacity to thrive in a wide range of environments. Because of that, Lm presents itself as a significant concern in food safety and public health, that can lead to potentially life-threatening infections in humans. A deeper understanding of the intricate bacterial virulence mechanisms and the signals that control them provide valuable insights into the dynamic interplay between Lm and the host. Therefore, this review addresses the role of some crucial signals behind Lm pathogenic virulence mechanisms and explores how the ability to assimilate and interpret these signals is fundamental for pathogenesis, identifying potential targets for innovative antimicrobial strategies.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Objective: To understand whether thyroid cells can be directly infected by the SARS-CoV-2 virus and to establish a putative correlation with the expression of the host entry machinery: ACE-2, TMPRSS2, and furin., Methods: We assessed the presence of SARS-CoV-2 virus at the gene level by RT-PCR, viral RNA transcripts localization by in situ hybridization, and by detecting viral proteins by immunohistochemistry for the nucleocapsid and the spike proteins. Furthermore, we also described the immunoexpression of key host factors for virus entry in the COVID-19 thyroid samples., Results: We performed RT-PCR for SARS-CoV-2 in all autopsy specimens and detected viral genome positivity in 13 of 15 thyroid tissues and in a lung specimen. In 9 of the 14 positive samples, we were also able to confirm SARS-CoV-2 signal by in situ hybridization. Immunohistochemistry for the viral nucleocapsid and spike protein was also positive for ten and nine of the RT-PCR-positive cases, respectively, but revealed a lower sensitivity. We also described, for the first time in a COVID-19 series, the immunohistochemical expression of ACE-2, TMPRSS2, and furin in the thyroid., Conclusions: Our results obtained in thyroid specimens from deceased COVID-19 patients indicate that thyrocytes can be directly infected by SARS-CoV-2 since we detected the presence of SARS-CoV-2 genome in follicular cells. Nevertheless, we did not find a clear correlation between the presence of viral genome and the expression of the host factors for virus entry, namely ACE-2, TMPRSS2, and furin.

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence factors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively controls the expression of the Agr quorum sensing system (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the role of MouR in Lm virulence.

We report here the draft genome sequence of Listeria monocytogenes 2542, a serotype 4b clinical strain recovered from a placental sample during a cheese-related listeriosis outbreak in Portugal., (Copyright © 2018 Ferreira et al.)

The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes . We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin β1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.