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1.
Proc Natl Acad Sci U S A ; 121(17): e2320311121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38635627

RESUMO

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.


Assuntos
Infecções do Sistema Nervoso Central , Listeria monocytogenes , Listeriose , Camundongos , Animais , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Encéfalo/microbiologia
2.
PLoS Pathog ; 19(6): e1011088, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37352334

RESUMO

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Camundongos , Animais , Galectina 3/genética , Tuberculose/metabolismo , Galectinas/genética , Galectinas/metabolismo , Macrófagos , Salmonella typhimurium , Camundongos Knockout
3.
Proc Natl Acad Sci U S A ; 119(13): e2122173119, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35316134

RESUMO

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.


Assuntos
Proteínas de Bactérias , Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo , Listeria monocytogenes , Proteínas de Membrana Transportadoras , Riboflavina , Proteínas de Bactérias/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas de Membrana Transportadoras/metabolismo , Riboflavina/metabolismo
4.
Infect Immun ; 92(3): e0042223, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38289071

RESUMO

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.


Assuntos
Listeria monocytogenes , Animais , Cisteína/metabolismo , Dissulfeto de Glutationa/genética , Dissulfeto de Glutationa/metabolismo , Compostos de Enxofre/metabolismo , Glutationa , Enxofre/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mamíferos
5.
Nature ; 562(7725): 140-144, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30209391

RESUMO

Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell1. Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms2-4 but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.


Assuntos
Transporte de Elétrons , Flavinas/metabolismo , Bactérias Gram-Positivas/metabolismo , Aerobiose , Animais , Benzoquinonas/metabolismo , Respiração Celular , Eletrodos , Transporte de Elétrons/genética , Elétrons , Feminino , Firmicutes/enzimologia , Firmicutes/genética , Firmicutes/metabolismo , Trato Gastrointestinal/microbiologia , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Ferro/química , Listeria monocytogenes/enzimologia , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Camundongos , NADH Desidrogenase/metabolismo
6.
PLoS Pathog ; 17(8): e1009819, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34407151

RESUMO

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.


Assuntos
Proteínas de Bactérias/metabolismo , Glutationa/metabolismo , Lactoilglutationa Liase/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Aldeído Pirúvico/metabolismo , Virulência , Animais , Proteínas de Bactérias/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Inativação Metabólica , Lactoilglutationa Liase/genética , Listeriose/metabolismo , Camundongos , Mutação , Aldeído Pirúvico/química , Substâncias Redutoras/química , Ativação Transcricional
7.
Proc Natl Acad Sci U S A ; 117(38): 23774-23781, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32878997

RESUMO

Intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality, and each has evolved specialized strategies to establish and maintain their replicative niche. Listeria monocytogenes is a facultative intracellular pathogen that secretes a pore-forming cytolysin called listeriolysin O (LLO), which disrupts the phagosomal membrane and, thereby, allows the bacteria access to their replicative niche in the cytosol. Nonsynonymous and synonymous mutations in a PEST-like domain near the LLO N terminus cause enhanced LLO translation during intracellular growth, leading to host cell death and loss of virulence. Here, we explore the mechanism of translational control and show that there is extensive codon restriction within the PEST-encoding region of the LLO messenger RNA (mRNA) (hly). This region has considerable complementarity with the 5' UTR and is predicted to form an extensive secondary structure that overlaps the ribosome binding site. Analysis of both 5' UTR and synonymous mutations in the PEST-like domain that are predicted to disrupt the secondary structure resulted in up to a 10,000-fold drop in virulence during mouse infection, while compensatory double mutants restored virulence to WT levels. We showed by dynamic protein radiolabeling that LLO synthesis was growth phase-dependent. These data provide a mechanism to explain how the bacteria regulate translation of LLO to promote translation during starvation in a phagosome while repressing it during growth in the cytosol. These studies also provide a molecular explanation for codon bias at the 5' end of this essential determinant of pathogenesis.


Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , RNA Bacteriano/química , RNA Mensageiro/química , Regiões 5' não Traduzidas/genética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Replicação do DNA/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose , Camundongos , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética
8.
Infect Immun ; 90(11): e0020722, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36317877

RESUMO

Biosyntheses of para-aminobenzoic acid (PABA) and its downstream folic acid metabolites are essential for one-carbon metabolism in all life forms and the targets of sulfonamide and trimethoprim antibiotics. In this study, we identified and characterized two genes (pabA and pabBC) required for PABA biosynthesis in Listeria monocytogenes. Mutants in PABA biosynthesis were able to grow normally in rich media but not in defined media lacking PABA, but growth was restored by the addition of PABA or its downstream metabolites. PABA biosynthesis mutants were attenuated for intracellular growth in bone marrow-derived macrophages, produced extremely small plaques in fibroblast monolayers, and were highly attenuated for virulence in mice. PABA biosynthesis genes were upregulated upon infection and induced during growth in broth in a strain in which the master virulence regulator, PrfA, was genetically locked in its active state (PrfA*). To gain further insight into why PABA mutants were so attenuated, we screened for transposon-induced suppressor mutations that formed larger plaques. Suppressor mutants in relA, which are predicted to have higher levels of (p)ppGpp, and mutants in codY, which is a GTP-binding repressor of many biosynthetic genes, partially rescued the plaque defect but, notably, restored the capacity of the mutants to escape from phagosomes and induce the polymerization of host cell actin. However, these suppressor mutant strains remained attenuated for virulence in mice. These data suggest that even though folic acid metabolites exist in host cells and might be available during infection, de novo synthesis of PABA is required for L. monocytogenes pathogenesis.


Assuntos
Listeria monocytogenes , Camundongos , Animais , Ácido 4-Aminobenzoico/metabolismo , Virulência/genética , Supressão Genética , Ácido Fólico/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica
9.
PLoS Pathog ; 16(7): e1008622, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634175

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Listeriose/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Endossomos/metabolismo , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(52): 26892-26899, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31818955

RESUMO

Mineral-respiring bacteria use a process called extracellular electron transfer to route their respiratory electron transport chain to insoluble electron acceptors on the exterior of the cell. We recently characterized a flavin-based extracellular electron transfer system that is present in the foodborne pathogen Listeria monocytogenes, as well as many other Gram-positive bacteria, and which highlights a more generalized role for extracellular electron transfer in microbial metabolism. Here we identify a family of putative extracellular reductases that possess a conserved posttranslational flavinylation modification. Phylogenetic analyses suggest that divergent flavinylated extracellular reductase subfamilies possess distinct and often unidentified substrate specificities. We show that flavinylation of a member of the fumarate reductase subfamily allows this enzyme to receive electrons from the extracellular electron transfer system and support L. monocytogenes growth. We demonstrate that this represents a generalizable mechanism by finding that a L. monocytogenes strain engineered to express a flavinylated extracellular urocanate reductase uses urocanate by a related mechanism and to a similar effect. These studies thus identify an enzyme family that exploits a modular flavin-based electron transfer strategy to reduce distinct extracellular substrates and support a multifunctional view of the role of extracellular electron transfer activities in microbial physiology.

11.
Cell Microbiol ; 22(4): e13175, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32185899

RESUMO

Listeria monocytogenes is a rapidly growing, Gram-positive, facultative intracellular pathogen that has been used for over 5 decades as a model to study basic aspects of infection and immunity. In a murine intravenous infection model, immunisation with a sublethal infection of L. monocytogenes initially leads to rapid intracellular multiplication followed by clearance of the bacteria and ultimately culminates in the development of long-lived cell-mediated immunity (CMI) mediated by antigen-specific CD8+ cytotoxic T-cells. Importantly, effective immunisation requires live, replicating bacteria. In this review, we summarise the cell and immunobiology of L. monocytogenes infection and discuss aspects of its pathogenesis that we suspect lead to robust CMI. We suggest five specific features of L. monocytogenes infection that positively impact the development of CMI: (a) the bacteria have a predilection for professional antigen-presenting cells; (b) the bacteria escape from phagosomes, grow, and secrete antigens into the host cell cytosol; (c) bacterial-secreted proteins enter the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation; (d) the bacteria do not induce rapid host cell death; and (e) cytosolic bacteria induce a cytokine response that favours CMI. Collectively, these features make L. monocytogenes an attractive vaccine vector for both infectious disease applications and cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Citocinas/imunologia , Citosol/imunologia , Citosol/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/patogenicidade , Camundongos , Fagossomos/microbiologia , Linfócitos T Citotóxicos/imunologia
12.
Nature ; 517(7533): 170-3, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25567281

RESUMO

Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Glutationa/metabolismo , Espaço Intracelular/metabolismo , Espaço Intracelular/microbiologia , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Regulação Alostérica/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glutationa/farmacologia , Espaço Intracelular/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Macrófagos/metabolismo , Mutação/genética , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Seleção Genética/genética , Supressão Genética/genética , Virulência/genética
13.
Proc Natl Acad Sci U S A ; 115(2): E210-E217, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29279409

RESUMO

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenesL. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.


Assuntos
Autofagia , Listeria monocytogenes/fisiologia , Macrófagos/microbiologia , Fagocitose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citosol/metabolismo , Citosol/microbiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/genética , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Mutação , Fagossomos/metabolismo , Fagossomos/microbiologia , Imagem com Lapso de Tempo/métodos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
14.
Proc Natl Acad Sci U S A ; 115(32): 8179-8184, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038013

RESUMO

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Listeria monocytogenes/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Listeria monocytogenes/genética , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Resultado do Tratamento , Vacinação/métodos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Infect Immun ; 88(12)2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33020211

RESUMO

Stimulator of interferon genes (STING) acts as a cytoplasmic signaling hub of innate immunity that is activated by host-derived or bacterially derived cyclic dinucleotides. Listeria monocytogenes is a foodborne, facultative intracellular pathogen that secretes c-di-AMP and activates STING, yet the in vivo role of the STING pathway during bacterial pathogenesis remains unclear. In this study, we found that STING-deficient mice had increased weight loss and roughly 10-fold-increased systemic bacterial burden during L. monocytogenes-induced enterocolitis. Infection with a L. monocytogenes mutant impaired in c-di-AMP secretion failed to elicit a protective response, whereas a mutant with increased c-di-AMP secretion triggered enhanced protection. Type I interferon (IFN) is a major output of STING signaling; however, disrupting IFN signaling during L. monocytogenes-induced enterocolitis did not recapitulate STING deficiency. In the absence of STING, the intestinal immune response was associated with a reduced influx of inflammatory monocytes. These studies suggest that in barrier sites such as the intestinal tract, where pathogen-associated molecular patterns are abundant, cytosolic surveillance systems such as STING are well positioned to detect pathogenic bacteria.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Enterocolite/imunologia , Enterocolite/microbiologia , Listeria monocytogenes/metabolismo , Listeriose/imunologia , Proteínas de Membrana/metabolismo , Animais , Enterocolite/metabolismo , Citometria de Fluxo , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
16.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31792073

RESUMO

Isoprenoids are an essential and diverse class of molecules, present in all forms of life, that are synthesized from an essential common precursor derived from either the mevalonate pathway or the nonmevalonate pathway. Most bacteria have one pathway or the other, but the Gram-positive, facultative intracellular pathogen Listeria monocytogenes is unusual because it carries all the genes for both pathways. While the mevalonate pathway has previously been reported to be essential, here we demonstrate that the nonmevalonate pathway can support growth of strains 10403S and EGD-e, but only anaerobically. L. monocytogenes lacking the gene hmgR, encoding the rate-limiting enzyme of the mevalonate pathway, had a doubling time of 4 h under anaerobic conditions, in contrast to the 45 min doubling time of the wild type. In contrast, deleting hmgR in two clinical isolates resulted in mutants that grew significantly faster, doubling in approximately 2 h anaerobically, although they still failed to grow under aerobic conditions without mevalonate. The difference in anaerobic growth rate was traced to three amino acid changes in the nonmevalonate pathway enzyme GcpE, and these changes were sufficient to increase the growth rate of 10403S to the rate observed in the clinical isolates. Despite an increased growth rate, virulence was still dependent on the mevalonate pathway in 10403S strains expressing the more active GcpE allele.


Assuntos
Anaerobiose/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Ácido Mevalônico/metabolismo , Transdução de Sinais/genética , Terpenos/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos/genética , Listeria monocytogenes/genética , Virulência/genética
17.
Cell Microbiol ; 21(3): e12988, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30511471

RESUMO

Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000-fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including mitogen-activated protein kinase activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.


Assuntos
Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Fagossomos/microbiologia , Fatores de Virulência/metabolismo , Animais , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Virulência
18.
Infect Immun ; 87(8)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31235641

RESUMO

Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection.


Assuntos
Vacinas Bacterianas/imunologia , Listeria monocytogenes/imunologia , Animais , Feminino , Imunoterapia , Listeria monocytogenes/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Vacinas Atenuadas/imunologia , Virulência
19.
Cell Microbiol ; 20(9): e12854, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29726107

RESUMO

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin-based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin-based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time-lapse microscopy using green fluorescent protein-LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin-based motility moved away from LC3-positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin-based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.


Assuntos
Actinas/metabolismo , Autofagia , Citosol/microbiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Movimento (Física) , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Análise Mutacional de DNA , Evasão da Resposta Imune , Macrófagos/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Multimerização Proteica , Imagem com Lapso de Tempo
20.
Mol Microbiol ; 104(2): 212-233, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28097715

RESUMO

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Listeria monocytogenes/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Fosfatos de Dinucleosídeos/genética , Fosfatos de Dinucleosídeos/fisiologia , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica/genética , Listeria monocytogenes/crescimento & desenvolvimento , Osmorregulação/fisiologia , Pressão Osmótica , Fósforo-Oxigênio Liases/metabolismo , Piruvato Carboxilase/metabolismo , Sistemas do Segundo Mensageiro , Supressão Genética
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