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1.  A Small-Molecule Screen Identifies the Antipsychotic Drug Pimozide as an Inhibitor of Listeria monocytogenes Infection▿  
We developed a screening procedure to identify small-molecule compounds that altered infection by Listeria monocytogenes to gain insights into bacterial/host cellular processes required for intracellular pathogenesis. A small-molecule library of 480 compounds with known biological functions was screened, and 21 compounds that altered the L. monocytogenes infection of murine bone marrow-derived macrophages (BMM) were identified. The identified compounds affected various cellular functions, such as actin polymerization, kinase/phosphatase activity, calcium signaling, and apoptosis. Pimozide, an FDA-approved drug used to treat severe Tourette's syndrome and schizophrenia, was further examined and shown to decrease the bacterial uptake and vacuole escape of L. monocytogenes in BMM. The inhibitory effect of pimozide on internalization was not specific for L. monocytogenes, as the phagocytosis of other bacterial species (Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Escherichia coli K12) was significantly inhibited in the presence of pimozide. The invasion and cell-to-cell spread of L. monocytogenes during the infection of nonprofessional phagocytic cells also was decreased by pimozide treatment. Although pimozide has been reported to be an antagonist of mammalian cell calcium channels, the infection of BMM in a calcium-free medium did not relieve the inhibitory effects of pimozide on L. monocytogenes infection. Our results provide a generalizable screening approach for identifying small-molecule compounds that affect cellular pathways that are required for intracellular bacterial pathogenesis. We also have identified pimozide, a clinically approved antipsychotic drug, as a compound that may be suitable for further development as a therapeutic for intracellular bacterial infections.
PMCID: PMC2630664  PMID: 19015342
2.  Invasive Extravillous Trophoblasts Restrict Intracellular Growth and Spread of Listeria monocytogenes 
PLoS Pathogens  2011;7(3):e1002005.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that can infect the placenta, a chimeric organ made of maternal and fetal cells. Extravillous trophoblasts (EVT) are specialized fetal cells that invade the uterine implantation site, where they come into direct contact with maternal cells. We have shown previously that EVT are the preferred site of initial placental infection. In this report, we infected primary human EVT with L. monocytogenes. EVT eliminated ∼80% of intracellular bacteria over 24-hours. Bacteria were unable to escape into the cytoplasm and remained confined to vacuolar compartments that became acidified and co-localized with LAMP1, consistent with bacterial degradation in lysosomes. In human placental organ cultures bacterial vacuolar escape rates differed between specific trophoblast subpopulations. The most invasive EVT—those that would be in direct contact with maternal cells in vivo—had lower escape rates than trophoblasts that were surrounded by fetal cells and tissues. Our results suggest that EVT present a bottleneck in the spread of L. monocytogenes from mother to fetus by inhibiting vacuolar escape, and thus intracellular bacterial growth. However, if L. monocytogenes is able to spread beyond EVT it can find a more hospitable environment. Our results elucidate a novel aspect of the maternal-fetal barrier.
Author Summary
Infection of the placenta and fetus is an important cause of pregnancy complications and fetal and neonatal morbidity and mortality. Listeria monocytogenes is an intracellular bacterial pathogen that causes pregnancy-related infections in humans. The pathogenesis of listeriosis during pregnancy is poorly understood. We have previously shown that transmission of L. monocytogenes from maternal cells and tissues to fetal cells occurs in the uterine implantation site, and that a small subpopulation of specialized fetal cells called extravillous trophoblasts are the preferred initial site of infection. Here we use primary human placental organ and cell culture systems to characterize the intracellular fate of L. monocytogenes in extravillous trophoblasts. We found that these cells entrap bacteria in vacuolar compartments where they are degraded and therefore reduce bacterial dissemination into deeper structures of the placenta. Our study provides new insights into the nature of the maternal-fetal barrier. Extravillous trophoblasts that are accessible to infection with intracellular pathogens from infected maternal cells have host defense mechanisms that constitute a bottleneck in maternal-fetal transmission.
PMCID: PMC3048367  PMID: 21408203
3.  The Pore-Forming Toxin Listeriolysin O Is Degraded by Neutrophil Metalloproteinase-8 and Fails To Mediate Listeria monocytogenes Intracellular Survival in Neutrophils 
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor secreted by the facultative intracellular pathogen Listeria monocytogenes. This toxin facilitates L. monocytogenes intracellular survival in macrophages and diverse nonphagocytic cells by disrupting the internalization vesicle, releasing the bacterium into its replicative niche, the cytosol. Neutrophils are innate immune cells that play an important role in the control of infections, yet it was unknown if LLO could confer a survival advantage to L. monocytogenes in neutrophils. We report that LLO can enhance the phagocytic efficiency of human neutrophils and is unable to protect L. monocytogenes from intracellular killing. To explain the absence of L. monocytogenes survival in neutrophils, we hypothesized that neutrophil degranulation leads to the release of LLO-neutralizing molecules in the forming phagosome. In support of this, L. monocytogenes is a potent inducer of neutrophil degranulation, since its virulence factors, such as LLO, facilitate granule exocytosis. Within the first few minutes of interaction with L. monocytogenes, granules can fuse with the plasma membrane at the bacterial interaction site before closure of the phagosome. Furthermore, granule products directly degrade LLO, irreversibly inhibiting its activity. The matrix metalloproteinase-8, stored in secondary granules, was identified as an endoprotease that degrades LLO, and blocking neutrophil proteases increased L. monocytogenes intracellular survival. In conclusion, we propose that LLO degradation by matrix metalloproteinase-8 during phagocytosis protects neutrophil membranes from perforation and contributes to maintaining L. monocytogenes in a bactericidal phagosome from which it cannot escape.
PMCID: PMC3876913  PMID: 24319266
4.  Lack of PPARγ in Myeloid Cells Confers Resistance to Listeria monocytogenes Infection 
PLoS ONE  2012;7(5):e37349.
The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγflox/flox). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6Chi monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.
PMCID: PMC3357414  PMID: 22629382
5.  Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection 
mBio  2013;4(3):e00282-13.
Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.
Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.
PMCID: PMC3663569  PMID: 23716572
6.  A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication 
Journal of Bacteriology  2013;195(15):3331-3340.
Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.
PMCID: PMC3719552  PMID: 23687268
7.  Effect of recombinant human gamma interferon on intracellular activities of antibiotics against Listeria monocytogenes in the human macrophage cell line THP-1. 
Listeria monocytogenes is a facultative intracellular pathogen which enters cells by endocytosis and reaches phagolysosomes from where it escapes and multiplies in the cytosol of untreated cells. Exposure of macrophages to gamma interferon (IFN-gamma) restricts L. monocytogenes to phagosomes and prevents its intracellular multiplication. We have tested whether IFN-gamma also modulates the susceptibility of L. monocytogenes to antibiotics. We selected drugs from three different classes displaying marked properties concerning their cellular accumulation and subcellular distribution, namely, ampicillin (not accumulated by cells but present in cytosol), azithromycin (largely accumulated by cells but mostly restricted to lysosomes), and sparfloxacin (accumulated to a fair extent but detected only in cytosol). We used a continuous line of myelomonocytic cells (THP-1 macrophages), which display specific surface receptors for IFN-gamma, and examined the activity of these antibiotics against L. monocytogenes Hly+ (virulent variant) and L. monocytogenes Hly- (a nonvirulent variant defective in hemolysin production). Untreated THP-1 and phorbol myristate acetate-differentiated THP-1 were permissive for infection and multiplication of intracellular L. monocytogenes Hly+ (virulent variant). All three antibiotics tested were bactericidal against this Listeria strain when added to an extracellular concentration of 10x their MIC. After preexposure of THP-1 to IFN-gamma, L. monocytogenes Hly+ was still phagocytosed but no longer grew intracellularly. The activity of ampicillin became almost undetectable (antagonistic effect), and that of azithromycin was unchanged (additive effect with that of IFN-gamma), whereas that of sparfloxacin was markedly enhanced (synergy). A similar behavior (lack of bacterial growth, associated with a loss of activity of ampicillin, an enhanced activity of sparfloxacin, and unchanged activity of azithromycin) was observed in cells infected with L. monocytogenes Hly-. This modulation of antibiotic activity, which we ascribe to the change of subcellular localization of L. monocytogenes caused by IFN-gamma or by the lack of virulence factor, could result from a change in bacterial responsiveness to antibiotics, a modification of the drug activity, or differences in drug bioavailabilities between cytosol and phagosomes.
PMCID: PMC163296  PMID: 8723471
8.  The Listeria monocytogenes σB Regulon and Its Virulence-Associated Functions Are Inhibited by a Small Molecule 
mBio  2011;2(6):e00241-11.
The stress-responsive alternative sigma factor σB is conserved across diverse Gram-positive bacterial genera. In Listeria monocytogenes, σB regulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor of L. monocytogenes σB activity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC50 = 3.5 µM) downregulated the majority of genes previously identified as members of the σB regulon in L. monocytogenes 10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigB strain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σB. Downregulated genes included key virulence and stress response genes, such as inlA, inlB, bsh, hfq, opuC, and bilE. From a functional perspective, FPSS also inhibited L. monocytogenes invasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σB activity in both L. monocytogenes and Bacillus subtilis indicates its utility as a specific inhibitor of σB across multiple Gram-positive genera.
The σB transcription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission of L. monocytogenes and other Gram-positive bacteria. Regulation of σB activity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σB across Gram-positive bacterial genera, i.e., in both Listeria monocytogenes and Bacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks in L. monocytogenes and other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.
PMCID: PMC3225968  PMID: 22128349
9.  Targeting Listeria Monocytogenes rpoA and rpoD Genes Using Peptide Nucleic Acids 
Nucleic Acid Therapeutics  2013;23(5):363-367.
Treating intracellular pathogens remains a considerable medical challenge because of the inefficient intracellular delivery of antimicrobials and the frequent emergence of bacterial resistance to therapeutic agents deemed the drugs of last resort. We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K cell penetrating peptide to target RNA polymerase α subunit (rpoA) and RNA polymerase sigma 70 (rpoD) in the intracellular pathogen Listeria monocytogenes. The PNAs tested displayed a concentration dependent inhibition of L. monocytogenes growth in pure culture at the micromolar level and significantly reduced intracellular L. monocytogenes in infected cell culture and Caenorhabditis elegans whole animal model. In vitro, the combined PNAs treatment was synergistic resulting in a clearance of L. monocytogenes at 0.5× the individual PNA concentration. This study demonstrates the potential of anti-rpoA PNA as an antibacterial agent and will provide the basis for improving and developing these PNAs to better target intracellular pathogens like Listeria. This study also establishes C. elegans as a potential model for the screening of PNAs.
PMCID: PMC3760085  PMID: 23859300
10.  Small Molecule Deubiquitinase Inhibitors Promote Macrophage Anti-Infective Capacity 
PLoS ONE  2014;9(8):e104096.
The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity.
PMCID: PMC4122495  PMID: 25093325
11.  Listeria Pathogenesis and Molecular Virulence Determinants 
Clinical Microbiology Reviews  2001;14(3):584-640.
The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.
PMCID: PMC88991  PMID: 11432815
12.  Cytolysin-Dependent Escape of the Bacterium from the Phagosome Is Required but Not Sufficient for Induction of the Th1 Immune Response against Listeria monocytogenes Infection: Distinct Role of Listeriolysin O Determined by Cytolysin Gene Replacement▿  
Infection and Immunity  2007;75(8):3791-3801.
Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-γ), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-γ production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-γ production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-γ induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-γ-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-γ that is essentially required to generate a Th1-dependent immune response.
PMCID: PMC1951982  PMID: 17517863
13.  Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells 
Cellular microbiology  2009;11(9):1382-1398.
Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of infected host cells. The ability to rapidly escape the phagocytic vacuole is essential for efficient intracellular replication. In the murine model of infection, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for vacuolar dissolution, as LLO-deficient (ΔLLO) mutants remain trapped within vacuoles. In contrast, in many human cell types ΔLLO L. monocytogenes are capable of vacuolar escape at moderate to high frequencies. To better characterize the mechanism of LLO-independent vacuolar escape in human cells, we conducted an RNA interference (RNAi) screen to identify vesicular trafficking factors that play a role in altering vacuolar escape efficiency of ΔLLO L. monocytogenes. RNAi knockdown of 18 vesicular trafficking factors resulted in increased LLO-independent vacuolar escape. Our results suggest that knockdown of one factor, RABEP1 (rabaptin-5), decreased the maturation of vacuoles containing ΔLLO L. monocytogenes. Thus, we provide evidence that increased vacuolar escape of ΔLLO L. monocytogenes in human cells correlates with slower vacuolar maturation. We also determined that increased LLO-independent dissolution of vacuoles during RABEP1 knockdown required the bacterial broad-range phospholipase PC-PLC. We hypothesize that slowing the kinetics of vacuolar maturation generates an environment conducive for vacuolar escape mediated by the bacterial phospholipases.
PMCID: PMC2759110  PMID: 19500109
Listeria monocytogenes; listeriolysin O; vacuolar maturation; rabaptin-5; PC-PLC
14.  In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection 
PLoS Pathogens  2009;5(5):e1000449.
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.
Author Summary
The facultative intracellular bacterial pathogen Listeria monocytogenes is the etiological agent of a severe foodborne disease. In humans it causes a variety of manifestations ranging from asymptomatic intestinal carriage and gastroenteritis to invasive and disseminated severe diseases. Septicemia, meningoencephalitis, and infection of the foetus in pregnant women are the most serious clinical features of listeriosis. Virulence is a trait that only manifests in a susceptible host, involving a highly coordinated interaction between bacterial factors and host components. This article reports the use of in vivo genome expression profiling as a powerful approach to gain a detailed understanding of the Listeria responses and the molecular cross-talk taking place in infected mice. We showed that, during infection, L. monocytogenes shifts the expression of its entire genome to promote virulence, subverting host defenses and adapting to host conditions. This first analysis of L. monocytogenes gene expression in vivo significantly enhances our understanding of the means by which intracellular pathogens promote infection.
PMCID: PMC2679221  PMID: 19478867
15.  Inhibition of Calpain Blocks the Phagosomal Escape of Listeria monocytogenes 
PLoS ONE  2012;7(4):e35936.
Listeria monocytogenes is a Gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1β, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.
PMCID: PMC3338540  PMID: 22563421
16.  Nonhematopoietic Cells Control the Outcome of Infection with Listeria monocytogenes in a Nucleotide Oligomerization Domain 1-Dependent Manner▿  
Infection and Immunity  2009;77(7):2908-2918.
We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1−/− mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480+ Gr1+ inflammatory monocytes and lower numbers of F480− Gr1+ neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1−/− mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1−/− BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1−/− BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.
PMCID: PMC2708577  PMID: 19398545
17.  Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles 
PLoS Pathogens  2015;11(3):e1004707.
Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol.
Author Summary
Experimental evidence has established that bacteria do not always exist as isolated single celled organisms, but instead communicate with each other through the secretion of small molecules that enable individual cells to coordinate complex traits and behaviors. Gram-positive bacteria rely on the secretion of small peptide pheromones to coordinate activities that include biofilm formation, exogenous DNA uptake via competence mechanisms, conjugal transfer of plasmid DNA, and expression of gene products that promote bacterial virulence. Here we provide evidence of a novel use of bacterial peptide pheromone signaling, that being the use of the pPplA peptide by L. monocytogenes to detect the confines of host cell vacuoles. Secretion and import of pPplA is required for efficient escape of L. monocytogenes from its initial membrane-bound host cell compartment, and bacteria lacking the peptide pheromone are severely attenuated for virulence in mice. The pPplA peptide pheromone is thus used by individual bacteria within a confined membrane bound space to coordinate the expression of gene products required by L. monocytogenes for intracellular growth and survival.
PMCID: PMC4379056  PMID: 25822753
18.  Differential Modulation of Intracellular Survival of Cytosolic and Vacuolar Pathogens by Curcumin 
Antimicrobial Agents and Chemotherapy  2012;56(11):5555-5567.
Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.
PMCID: PMC3486527  PMID: 22890770
19.  Simvastatin Enhances Protection against Listeria monocytogenes Infection in Mice by Counteracting Listeria-Induced Phagosomal Escape 
PLoS ONE  2013;8(9):e75490.
Statins are well-known cholesterol lowering drugs targeting HMG-CoA-reductase, reducing the risk of coronary disorders and hypercholesterolemia. Statins are also involved in immunomodulation, which might influence the outcome of bacterial infection. Hence, a possible effect of statin treatment on Listeriosis was explored in mice. Statin treatment prior to subsequent L. monocytogenes infection strikingly reduced bacterial burden in liver and spleen (up to 100-fold) and reduced histopathological lesions. Statin-treatment in infected macrophages resulted in increased IL-12p40 and TNF-α and up to 4-fold reduced bacterial burden within 6 hours post infection, demonstrating a direct effect of statins on limiting bacterial growth in macrophages. Bacterial uptake was normal investigated in microbeads and GFP-expressing Listeria experiments by confocal microscopy. However, intracellular membrane-bound cholesterol level was decreased, as analyzed by cholesterol-dependent filipin staining and cellular lipid extraction. Mevalonate supplementation restored statin-inhibited cholesterol biosynthesis and reverted bacterial growth in Listeria monocytogenes but not in listeriolysin O (LLO)-deficient Listeria. Together, these results suggest that statin pretreatment increases protection against L. monocytogenes infection by reducing membrane cholesterol in macrophages and thereby preventing effectivity of the cholesterol-dependent LLO-mediated phagosomal escape of bacteria.
PMCID: PMC3782446  PMID: 24086542
20.  Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection 
PLoS ONE  2010;5(1):e8610.
Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.
Methodology/Principal Findings
However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5−/−). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5−/− mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5−/− BMDMs.
We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.
PMCID: PMC2797616  PMID: 20062534
21.  Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth 
PLoS Pathogens  2014;10(2):e1003946.
Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.
Author Summary
Infection with the bacterial pathogen Mycobacterium tuberculosis causes the disease tuberculosis (TB) that imposes significant worldwide morbidity and mortality. Approximately 2 billion people are infected with M. tuberculosis, and almost 1.5 million people die annually from TB. With increasing drug resistance and few novel drug candidates, our inability to effectively treat all infected individuals necessitates a deeper understanding of the host-pathogen interface to facilitate new approaches to treatment. In addition, the current anti-tuberculosis regimen requires months of strict compliance to clear infection; targeting host immune function could play a strategic role in reducing the duration and complexity of treatment while effectively treating drug-resistant strains. Here we use a microscopy-based screen to identify molecules that target host pathways and inhibit the growth of M. tuberculosis in macrophages. We identified several host pathways not previously implicated in tuberculosis. The identified inhibitors prevent growth either by blocking host pathways exploited by M. tuberculosis for virulence, or by activating immune responses that target intracellular bacteria. Fluoxetine, used clinically for treating depression, induces autophagy and enhances production of TNF-α. Similarly, gefitinib, used clinically for treating cancer, inhibits M. tuberculosis growth in macrophages. Importantly, gefitinib treatment reduces bacterial replication in the lungs of M. tuberculosis-infected mice.
PMCID: PMC3930586  PMID: 24586159
Macrophage activation often contributes to the strong immune response elicited upon infection. The ability of macrophages to become activated was discovered when sub-lethal primary infections of mice with the bacterium Listeria monocytogenes provided protection against secondary infections through nonhumoral immunity. L. monocytogenes infect and propagate in macrophages by escaping the phagosome into the cytosol, where they avoid humoral immune mediators. Activated macrophages kill L. monocytogenes by blocking phagosomal escape. The timing of the antimicrobial activities within the phagosome is crucial to the outcome. In non-activated macrophages, bacterial factors generally prevail, and L. monocytogenes can escape from the vacuoles and grow within cytoplasm. Activated macrophages generate reactive oxygen or nitrogen intermediates early after bacterial uptake, which prevent the bacteria from escaping vacuoles into cytoplasm. The heterogeneity in the interactions between L. monocytogenes and the macrophage indicate a complex relationship between the host and the pathogen governed by chemistries that promote and inhibit escape from vacuoles. This review examines the mechanisms used by activated and non-activated macrophages to kill microbes, and how those mechanisms are employed against L. monocytogenes.
PMCID: PMC2851543  PMID: 17127272
listeriolysin O; ROI; RNI; NOS2; phagosome maturation; cholesterol-dependent cytolysin; interferon-gamma; Rab5; lysosome
23.  Distinct TLR- and NLR-Mediated Transcriptional Responses to an Intracellular Pathogen 
PLoS Pathogens  2008;4(1):e6.
How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2−/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens.
Author Summary
Macrophages are critical cells of the innate immune system, contributing to immediate and robust defense against microbial infections. Macrophages detect pathogens using host receptors located on the cell surface, in phagosomal vacuoles, and in the cytosol. While fundamental to innate immunity, it is not clear if these different receptors merely provide redundant mechanisms for sensing microbial infection, or if instead they induce distinct gene expression programs that may allow for threat-specific host responses. We addressed this question by dissecting the macrophage transcriptional responses to the model intracellular bacterial pathogen Listeria monocytogenes. Using genetic and genomic approaches, we found that the macrophage response to L. monocytogenes trapped in phagosomal compartments was distinct and separable from the response to live bacteria replicating in the host cytosol. The macrophage response to cytosolic bacteria was recapitulated by bacterial nucleic acid and cell wall fragments, and induced surprisingly few primary response genes. These findings highlight a mechanism by which the innate immune system may specifically sense intracellular bacteria, as the macrophage response to Mycobacterium tuberculosis was similarly regulated.
PMCID: PMC2186359  PMID: 18193943
24.  L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane 
PLoS Pathogens  2015;11(5):e1004919.
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.
Author Summary
Listeria monocytogenes is a foodborne bacterial pathogen that preferentially infects immunocompromised hosts, eliciting a severe and often lethal disease. In humans, clinical manifestations range from asymptomatic intestinal carriage and gastroenteritis to harsher systemic states of the disease such as sepsis, meningitis or encephalitis, and fetal infections. The surface of L. monocytogenes is decorated with wall teichoic acids (WTAs), a class of carbohydrate-based polymers that contributes to cell surface-related events with implications in physiological processes, such as bacterial division or resistance to antimicrobial peptides (AMPs). The addition of other molecules to the backbone of WTAs modulates their chemical properties and consequently their functionality. In this context, we studied the role of WTA tailoring mechanisms in L. monocytogenes, whose WTAs are strictly decorated with monosaccharides. For the first time, we link WTA glycosylation with AMP resistance by showing that the decoration of L. monocytogenes WTAs with l-rhamnose confers resistance to host defense peptides. We suggest that this resistance is based on changes in the permeability of the cell wall that delay its crossing by AMPs and therefore promote the protection of the bacterial membrane integrity. Importantly, we also demonstrate the significance of this WTA modification in L. monocytogenes virulence.
PMCID: PMC4441387  PMID: 26001194
25.  Intracellular Gene Expression Profile of Listeria monocytogenes †  
Infection and Immunity  2006;74(2):1323-1338.
Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.
PMCID: PMC1360297  PMID: 16428782

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