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1.
mBio ; 12(6): e0239721, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903051

RESUMO

Chlamydia trachomatis is an obligate intracellular bacterium that has developed sophisticated mechanisms to survive inside its infectious compartment, the inclusion. Notably, Chlamydia weaves an extensive network of microtubules (MTs) and actin filaments to enable interactions with host organelles and enhance its stability. Despite the global health and economic burden caused by this sexually transmitted pathogen, little is known about how actin and MT scaffolds are integrated into an increasingly complex virulence system. Previously, we established that the chlamydial effector InaC interacts with ARF1 to stabilize MTs. We now demonstrate that InaC regulates RhoA to control actin scaffolds. InaC relies on cross talk between ARF1 and RhoA to coordinate MTs and actin, where the presence of RhoA downregulates stable MT scaffolds and ARF1 activation inhibits actin scaffolds. Understanding how Chlamydia hijacks complex networks will help elucidate how this clinically significant pathogen parasitizes its host and reveal novel cellular signaling pathways. IMPORTANCE Chlamydia trachomatis is a major cause of human disease worldwide. The ability of Chlamydia to establish infection and cause disease depends on the maintenance of its parasitic niche, called the inclusion. To accomplish this feat, Chlamydia reorganizes host actin and microtubules around the inclusion membrane. How Chlamydia orchestrates these complex processes, however, is largely unknown. Here, we discovered that the chlamydial effector InaC activates Ras homolog family member A (RhoA) to control the formation of actin scaffolds around the inclusion, an event that is critical for inclusion stability. Furthermore, InaC directs the kinetics of actin and posttranslationally modified microtubule scaffolds by mediating cross talk between the GTPases that control these cytoskeletal elements, RhoA and ADP-ribosylation factor 1 (ARF1). The precise timing of these events is essential for the maintenance of the inclusion. Overall, this study provides the first evidence of ARF1-RhoA-mediated cross talk by a bacterial pathogen to coopt the host cytoskeleton.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/fisiologia , Citoesqueleto/microbiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Fator 1 de Ribosilação do ADP/genética , Actinas/genética , Actinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/genética , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Citoesqueleto/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/microbiologia , Ligação Proteica , Virulência , Proteína rhoA de Ligação ao GTP/genética
2.
Nat Commun ; 10(1): 2747, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227715

RESUMO

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection.


Assuntos
Proteínas de Bactérias/ultraestrutura , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/patogenicidade , Corpos de Inclusão/microbiologia , Fusão de Membrana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HeLa , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas SNARE/química
3.
Microb Cell ; 7(2): 46-58, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-32025513

RESUMO

Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a parasitic vacuole called the inclusion. The nascent inclusion is derived from the host plasma membrane and serves as a platform from which Chlamydia controls interactions with the host microenvironment. To survive inside the host cell, Chlamydia scavenges for nutrients and lipids by recruiting and/or fusing with various cellular compartments. The mechanisms by which these events occur are poorly understood but require host proteins such as the SNARE proteins (SNAP (Soluble N-ethylmaleimide-sensitive factor attachment protein) Receptor). Here, we show that SNAP-23 and Syntaxin 4, two plasma membrane SNAREs, are recruited to the inclusion and play an important role in Chlamydia development. Knocking down SNAP-23 and Syntaxin 4 by CRISPR-Cas9 reduces the amount of infectious progeny. We then demonstrate that the loss of both of these SNARE proteins results in the dysregulation of Chlamydia-induced lipid droplets, indicating that both SNAP-23 and Syntaxin 4 play a critical role in lipid droplet homeostasis during Chlamydia infection. Ultimately, our data highlights the importance of lipid droplets and their regulation in Chlamydia development.

4.
F1000Res ; 6: 2058, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29225789

RESUMO

Both actin and microtubules are major cytoskeletal elements in eukaryotic cells that participate in many cellular processes, including cell division and motility, vesicle and organelle movement, and the maintenance of cell shape. Inside its host cell, the human pathogen Chlamydia trachomatis manipulates the cytoskeleton to promote its survival and enhance its pathogenicity. In particular, Chlamydia induces the drastic rearrangement of both actin and microtubules, which is vital for its entry, inclusion structure and development, and host cell exit. As significant progress in Chlamydia genetics has greatly enhanced our understanding of how this pathogen co-opts the host cytoskeleton, we will discuss the machinery used by Chlamydia to coordinate the reorganization of actin and microtubules.

5.
mBio ; 8(3)2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28465429

RESUMO

The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion.IMPORTANCEChlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified microtubules around the inclusion and that Chlamydia trachomatis hijacks this novel function of ARF to reposition the Golgi complex.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Actinas , Proteínas de Bactérias/genética , Chlamydia trachomatis/genética , Complexo de Golgi/ultraestrutura , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão/microbiologia , Microtúbulos/genética , Processamento de Proteína Pós-Traducional
6.
J Bacteriol ; 198(8): 1347-55, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26883826

RESUMO

UNLABELLED: Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants. IMPORTANCE: Chlamydia trachomatis replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusions are nonfusogenic with vesicles in the endocytic pathway but, in multiply infected cells, fuse with each other to form a single large inclusion. This homotypic fusion is dependent upon the presence of a chlamydial inclusion membrane-localized protein, IncA. Specificity of membrane fusion in eukaryotic cells is regulated by SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins on the cytosolic face of vesicles and target membranes. IncA contains two SNARE-like domains. Newly developed genetic tools for the complementation of targeted mutants in C. trachomatis are used to confirm the minimal requirement of SNARE-like motifs necessary to promote the homotypic fusion of inclusions.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HeLa , Humanos , Proteínas de Membrana/genética , Mutação , Proteínas SNARE/metabolismo
7.
J Biol Chem ; 289(48): 33469-80, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25324548

RESUMO

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.


Assuntos
Proteínas de Bactérias/química , Chlamydia trachomatis/química , Proteínas de Membrana/química , Multimerização Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Dicroísmo Circular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
8.
Microbes Infect ; 16(6): 502-11, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24642003

RESUMO

Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.


Assuntos
Armadilhas Extracelulares/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Strongyloides stercoralis/imunologia , Animais , Células Cultivadas , Eosinófilos/imunologia , Armadilhas Extracelulares/parasitologia , Humanos , Larva/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Cavidade Peritoneal/parasitologia
9.
Traffic ; 15(5): 516-30, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24494924

RESUMO

Mast cells orchestrate the allergic response through the release of proinflammatory mediators, which is driven by the fusion of cytoplasmic secretory granules with the plasma membrane. During this process, SNARE proteins including Syntaxin4, SNAP23 and VAMP8 play a key role. Following stimulation, the kinase IKKß interacts with and phosphorylates the t-SNARE SNAP23. Phosphorylated SNAP23 then associates with Syntaxin4 and the v-SNARE VAMP8 to form a ternary SNARE complex, which drives membrane fusion and mediator release. Interestingly, mast cell degranulation is impaired following exposure to bacteria such as Escherichia coli. However, the molecular mechanism(s) by which this occurs is unknown. Here, we show that E. coli exposure rapidly and additively inhibits degranulation in the RBL-2H3 rat mast cell line. Following co-culture with E. coli, the interaction between IKKß and SNAP23 is disrupted, resulting in the hypophosphorylation of SNAP23. Subsequent formation of the ternary SNARE complex between SNAP23, Syntaxin4 and VAMP8 is strongly reduced. Collectively, these results demonstrate that E. coli exposure inhibits the formation of VAMP8-containing exocytic SNARE complexes and thus the release of VAMP8-dependent granules by interfering with SNAP23 phosphorylation.


Assuntos
Escherichia coli/metabolismo , Mastócitos/metabolismo , Mastócitos/fisiologia , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Escherichia coli/fisiologia , Quinase I-kappa B/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos , Proteínas de Transporte Vesicular/metabolismo
10.
PLoS One ; 8(7): e69769, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23936096

RESUMO

Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Endocitose , Células HeLa , Humanos , Lipossomos/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas SNARE/metabolismo , Relação Estrutura-Atividade
11.
PLoS One ; 7(11): e49886, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185475

RESUMO

Mast cells play a critical role in the innate immune response to bacterial infection. They internalize and kill a variety of bacteria and process antigen for presentation to T cells via MHC molecules. Although mast cell phagocytosis appears to play a significant role during bacterial infection, little is known about the proteins involved in its regulation. In this study, we demonstrate that the SNARE protein SNAP29 is involved in mast cell phagocytosis. SNAP29 is localized in the endocytic pathway and is transiently recruited to Escherichia coli (E. coli)-containing phagosomes. Interestingly, overexpression of SNAP29 significantly increases the internalization and killing of E. coli, while it does not affect mast cell exocytosis of inflammatory mediators. To our knowledge, these data are the first to demonstrate a novel function of SNAP29 in mast cell phagocytosis and have implications in protection against bacterial infection.


Assuntos
Infecções Bacterianas , Imunidade Inata , Mastócitos , Fagocitose , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Adesinas de Escherichia coli/imunologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Escherichia coli , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Fagocitose/imunologia , Fagocitose/fisiologia , Fagossomos/genética , Fagossomos/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/imunologia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/imunologia , Proteínas Qc-SNARE/metabolismo , Ratos , Linfócitos T/imunologia , Linfócitos T/metabolismo
12.
Immunol Res ; 51(2-3): 215-26, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22048902

RESUMO

In developed countries, the prevalence of allergy is on the rise. Although the causes are unknown, it seems that (1) the disappearance of microbiota may play a role in the increase of allergies and (2) exposure to bacterial infections during childhood decreases the incidence of allergies. Although several cell types are involved in the development of allergy, mast cells play a major role in orchestrating inflammation. Upon activation, mast cell secretory granules fuse with the plasma membrane, resulting in the release of a number of inflammatory mediators. In addition to allergy, mast cells contribute to the innate immune response against a variety of bacteria. This is accomplished through the secretion of cytokines and other soluble mediators. Interestingly, there is growing evidence that mast cells exposed to bacteria down-regulate degranulation in response to IgE/Allergen stimulation. This inhibitory effect seems to require direct contact between bacteria and mast cells, but the intracellular mechanism by which bacterial contact suppresses allergic responses is unknown. Here, we review different aspects of mast cell physiology and discuss hypotheses as to how bacteria may influence mast cell degranulation.


Assuntos
Infecções Bacterianas/imunologia , Degranulação Celular , Hipersensibilidade/imunologia , Mastócitos/imunologia , Animais , Infecções Bacterianas/microbiologia , Citotoxicidade Imunológica , Humanos , Hipersensibilidade/microbiologia , Imunoglobulina E/imunologia , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Mastócitos/microbiologia , Modelos Imunológicos
13.
Virulence ; 1(4): 319-24, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21178463

RESUMO

To penetrate host cells through their membranes, pathogens use a variety of molecular components in which the presence of heptad repeat motifs seems to be a prevailing element. Heptad repeats are characterized by a pattern of seven, generally hydrophobic, residues. In order to initiate membrane fusion, viruses use glycoproteins-containing heptad repeats. These proteins are structurally and functionally similar to the SNARE proteins known to be involved in eukaryotic membrane fusion. SNAREs also display a heptad repeat motif called the "SNARE motif". As bacterial genomes are being sequenced, microorganisms also appear to be carrying membrane proteins resembling eukaryotic SNAREs. This category of SNARE-like proteins might share similar functions and could be used by microorganisms to either promote or block membrane fusion. Such a recurrence across pathogenic organisms suggests that this architectural motif was evolutionarily selected because it most effectively ensures the survival of pathogens within the eukaryotic environment.


Assuntos
Motivos de Aminoácidos , Bactérias/patogenicidade , Membrana Celular/metabolismo , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Vírus/patogenicidade , Animais , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/microbiologia , Membrana Celular/virologia , Humanos , Fagocitose , Ligação Proteica , Proteínas SNARE/química , Proteínas Virais de Fusão/metabolismo , Vírus/metabolismo
14.
PLoS One ; 4(10): e7375, 2009 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-19823575

RESUMO

Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.


Assuntos
Bactérias/metabolismo , Proteínas SNARE/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Transporte Biológico , Linhagem Celular , Chlamydia/metabolismo , Endocitose , Glicoproteínas/metabolismo , Legionella/metabolismo , Lipossomos/metabolismo , Microscopia Confocal/métodos , Modelos Biológicos , Fosfoproteínas/metabolismo , Ratos
15.
PLoS Pathog ; 4(3): e1000022, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18369472

RESUMO

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.


Assuntos
Proteínas de Bactérias/genética , Chlamydia trachomatis/fisiologia , Proteínas de Membrana/genética , Mimetismo Molecular , Proteínas SNARE/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chlamydia trachomatis/patogenicidade , Chlamydia trachomatis/ultraestrutura , Interações Hospedeiro-Patógeno/fisiologia , Corpos de Inclusão/microbiologia , Corpos de Inclusão/ultraestrutura , Proteínas de Membrana/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Vacúolos
16.
Cell ; 128(1): 183-95, 2007 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-17218264

RESUMO

Sec1/Munc18 (SM) proteins are required for every step of intracellular membrane fusion, but their molecular mechanism of action has been unclear. In this work, we demonstrate a fundamental role of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery. In a reconstituted system, mammalian SNARE pairs assemble between bilayers to drive a basal fusion reaction. Munc18-1/nSec1, a synaptic SM protein required for neurotransmitter release, strongly accelerates this reaction through direct contact with both t- and v-SNAREs. Munc18-1 accelerates fusion only for the cognate SNAREs for exocytosis, therefore enhancing fusion specificity.


Assuntos
Proteínas Munc18/metabolismo , Proteínas SNARE/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Exocitose , Humanos , Fusão de Membrana , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Mutação/genética , Neurônios/citologia , Ligação Proteica , Subunidades Proteicas/metabolismo , Ratos , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo
17.
J Biol Chem ; 280(22): 21137-43, 2005 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-15799968

RESUMO

In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated.


Assuntos
Endossomos/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte Vesicular/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Endocitose , Complexo de Golgi/metabolismo , Humanos , Cinética , Lipossomos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteínas Recombinantes de Fusão/química , Proteínas SNARE , Fatores de Tempo , Leveduras/metabolismo
18.
Proc Natl Acad Sci U S A ; 101(10): 3376-80, 2004 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-14981247

RESUMO

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core of the fusion machinery, and isolated SNAREs fuse membranes with exquisite specificity by cognate pairing. Most SNAREs have a membrane-spanning region, an N-terminal domain, and a membrane proximal SNARE motif domain. Although the SNARE motif is critical for SNARE complex formation, is it the sole determinant of the specificity of SNARE-dependent fusion? To test this, we make use of a SNARE complex functioning in the late endosomal compartment in yeast. Studying this complex and the previously identified early endosomal SNARE complex, we find that the specificity of fusion resides in the SNARE motifs.


Assuntos
Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Motivos de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Endocitose , Proteínas de Membrana/química , Estrutura Terciária de Proteína , Proteínas R-SNARE , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE
19.
J Cell Biol ; 164(1): 79-88, 2004 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-14699088

RESUMO

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.


Assuntos
Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Sítios de Ligação/fisiologia , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , Proteínas SNARE , Transdução de Sinais/fisiologia , Rede trans-Golgi/fisiologia
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