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1.
Sci Rep ; 12(1): 21961, 2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36535993

RESUMO

L. monocytogenes causes listeriosis, a foodborne disease that is particularly dangerous for immunocompromised individuals and fetuses. Several virulence factors of this bacterial pathogen belong to a family of leucine-rich repeat (LRR)-containing proteins called internalins. Among these, InlP is known for its role in placental infection. We report here a function of InlP in mammalian cell nucleus organization. We demonstrate that bacteria do not produce InlP under in vitro culture conditions. When ectopically expressed in human cells, InlP translocates into the nucleus and changes the morphology of nuclear speckles, which are membrane-less organelles storing splicing factors. Using yeast two-hybrid screen, immunoprecipitation and pull-down experiments, we identify the tumor suppressor and splicing factor RBM5 as a major nuclear target of InlP. InlP inhibits RBM5-induced cell death and stimulate the formation of RBM5-induced nuclear granules, where the SC35 speckle protein redistributes. Taken together, these results suggest that InlP acts as a nucleomodulin controlling compartmentalization and function of RBM5 in the nucleus and that L. monocytogenes has developed a mechanism to target the host cell splicing machinery.


Assuntos
Proteínas de Ligação a RNA , Proteínas Supressoras de Tumor , Fatores de Virulência , Humanos , Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fatores de Virulência/metabolismo , Listeria monocytogenes
3.
Front Cell Infect Microbiol ; 11: 761945, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34858876

RESUMO

Listeria monocytogenes causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through its important functions in immunity. However, recent evidence suggests that during long-term infection of hepatocytes, a subpopulation of Listeria may escape eradication by entering a persistence phase in intracellular vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first optimized cell models of persistent infection in human hepatocyte cell lines HepG2 and Huh7 and primary mouse hepatocytes (PMH). In these cells, Listeria efficiently entered the persistence phase after three days of infection, while inducing a potent interferon response, of type I in PMH and type III in HepG2, while Huh7 remained unresponsive. RNA-sequencing analysis identified a common signature of long-term Listeria infection characterized by the overexpression of a set of genes involved in antiviral immunity and the under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. Infection also altered the expression of cholesterol metabolism-associated genes in HepG2 and Huh7 cells. The decrease in APP transcripts was correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1ß). Collectively, these results reveal that long-term infection with Listeria profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.


Assuntos
Listeria monocytogenes , Listeria , Listeriose , Animais , Feminino , Hepatócitos , Humanos , Listeria monocytogenes/genética , Camundongos , Infecção Persistente , Gravidez , Secretoma
5.
Nat Commun ; 11(1): 1344, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165618

RESUMO

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


Assuntos
Adenosina/análogos & derivados , Ceco/metabolismo , Microbioma Gastrointestinal , Fígado/metabolismo , Adenosina/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Ceco/microbiologia , Feminino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
6.
Cell Host Microbe ; 26(6): 823-835.e11, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31761719

RESUMO

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


Assuntos
Proteína DEAD-box 58/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proteína DEAD-box 58/imunologia , Células HEK293 , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Inata , Interferon beta/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Camundongos , RNA Bacteriano/metabolismo , Transdução de Sinais/imunologia , Virulência/imunologia
7.
J Biol Chem ; 293(24): 9265-9276, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29666193

RESUMO

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


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Nexinas de Classificação/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Feminino , Listeria monocytogenes/química , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Fatores de Virulência/química
8.
Cell Mol Life Sci ; 75(3): 355-374, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28779209

RESUMO

The mitochondrial network constantly changes and remodels its shape to face the cellular energy demand. In human cells, mitochondrial fusion is regulated by the large, evolutionarily conserved GTPases Mfn1 and Mfn2, which are embedded in the mitochondrial outer membrane, and by OPA1, embedded in the mitochondrial inner membrane. In contrast, the soluble dynamin-related GTPase Drp1 is recruited from the cytosol to mitochondria and is key to mitochondrial fission. A number of new players have been recently involved in Drp1-dependent mitochondrial fission, ranging from large cellular structures such as the ER and the cytoskeleton to the surprising involvement of the endocytic dynamin 2 in the terminal abscission step. Here we review the recent findings that have expanded the mechanistic model for the mitochondrial fission process in human cells and highlight open questions.


Assuntos
Fusão de Membrana/fisiologia , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/fisiologia , Animais , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia
9.
Nat Commun ; 7: 12148, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27401954

RESUMO

Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIß through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself).


Assuntos
Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Citocinese/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas 14-3-3/metabolismo , Células HeLa , Humanos , Lisofosfolipídeos/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Quinase C/metabolismo , Quinases Ativadas por p21/metabolismo
10.
EMBO Rep ; 17(6): 858-73, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27215606

RESUMO

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin-like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1-dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP-induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Septinas/metabolismo , Actomiosina/metabolismo , Evolução Biológica , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas , Técnicas de Silenciamento de Genes , Inativação Gênica , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Septinas/genética
11.
Nat Cell Biol ; 14(4): 343-54, 2012 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-22366688

RESUMO

Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIß, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIß, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.


Assuntos
Proteínas 14-3-3/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dimerização , Humanos , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Quinases Ativadas por p21/metabolismo
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