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1.
PLoS Pathog ; 19(2): e1010777, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36800397

RESUMO

Brugia malayi, a parasitic roundworm of humans, is colonized by the obligate intracellular bacterium, Wolbachia pipientis. The symbiosis between this nematode and bacterium is essential for nematode reproduction and long-term survival in a human host. Therefore, identifying molecular mechanisms required by Wolbachia to persist in and colonize B. malayi tissues will provide new essential information regarding the basic biology of this endosymbiosis. Wolbachia utilize a Type IV secretion system to translocate so-called "effector" proteins into the cytosol of B. malayi cells to promote colonization of the eukaryotic host. However, the characterization of these Wolbachia secreted proteins has remained elusive due to the genetic intractability of both organisms. Strikingly, expression of the candidate Wolbachia Type IV-secreted effector protein, Wbm0076, in the surrogate eukaryotic cell model, Saccharomyces cerevisiae, resulted in the disruption of the yeast actin cytoskeleton and inhibition of endocytosis. Genetic analyses show that Wbm0076 is a member of the family of Wiskott-Aldrich syndrome proteins (WAS [p]), a well-conserved eukaryotic protein family required for the organization of actin skeletal structures. Thus, Wbm0076 likely plays a central role in the active cell-to-cell movement of Wolbachia throughout B. malayi tissues during nematode development. As most Wolbachia isolates sequenced to date encode at least partial orthologs of wBm0076, we find it likely that the ability of Wolbachia to directly manipulate host actin dynamics is an essential requirement of all Wolbachia endosymbioses, independent of host cell species.


Assuntos
Brugia Malayi , Wolbachia , Animais , Humanos , Actinas/metabolismo , Brugia Malayi/genética , Células Eucarióticas , Saccharomyces cerevisiae/genética , Simbiose/genética , Wolbachia/fisiologia , Proteínas de Bactérias
2.
J Bacteriol ; 205(9): e0011023, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37655916

RESUMO

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.


Assuntos
Helicobacter pylori , Proteínas Monoméricas de Ligação ao GTP , Humanos , Proteínas de Bactérias/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Flagelos/genética , Flagelos/metabolismo
3.
Traffic ; 22(8): 284-302, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34184807

RESUMO

Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires' disease. Legionella actively alters host organelle trafficking through the activities of "effector" proteins secreted via a type-IVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is created with membrane derived from host endoplasmic reticulum (ER), secretory vesicles and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology and induces aberrant ER:endosome interactions, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by driving ER:endosome contacts, potentially through interaction with host membrane tethering complexes and/or cargo adapters.


Assuntos
Legionella pneumophila , Proteínas de Bactérias/genética , Retículo Endoplasmático , Endossomos , Saccharomyces cerevisiae , Vacúolos
4.
J Biol Chem ; 293(49): 19101-19112, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30315104

RESUMO

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.


Assuntos
Proteínas Fúngicas/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Oócitos/metabolismo , Domínios Proteicos , Proteínas de Protozoários/genética , Saccharomyces cerevisiae , Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Trypanosoma brucei brucei , Xenopus laevis
5.
Mol Microbiol ; 107(3): 428-444, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29205554

RESUMO

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi-containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA32-189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host-derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA32-189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.


Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Fosfatídicos/metabolismo , Rhodococcus equi/metabolismo , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Lipídeos , Macrófagos/microbiologia , Fagossomos/metabolismo , Rhodococcus equi/genética , Proteína Estafilocócica A , Virulência/genética , Fatores de Virulência/metabolismo
6.
Proc Natl Acad Sci U S A ; 112(1): 100-5, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25453092

RESUMO

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.


Assuntos
Proteínas de Bactérias/metabolismo , Fusão de Membrana , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vibrio parahaemolyticus/metabolismo , Ácidos/metabolismo , Eletroquímica , Concentração de Íons de Hidrogênio , Canais Iônicos/metabolismo , Lipídeos/química , Proteínas Mutantes/metabolismo , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
7.
Proc Natl Acad Sci U S A ; 110(28): 11559-64, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23798441

RESUMO

Defects in normal autophagic pathways are implicated in numerous human diseases--such as neurodegenerative diseases, cancer, and cardiomyopathy--highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved Vo domain of the vacuolar-type H(+)-ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ∼18 Šin diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H(+)-ATPase-containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation.


Assuntos
Autofagia , Proteínas de Bactérias/fisiologia , Homeostase/fisiologia , Ativação do Canal Iônico , Lisossomos/fisiologia , Vibrio parahaemolyticus/fisiologia , Íons
8.
Cells ; 13(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39273010

RESUMO

Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, where it can cause a variety of diseases. H. pylori uses a cluster of sheathed flagella for motility, which is required for host colonization in animal models. The flagellar sheath is continuous with the outer membrane and is found in most Helicobacter species identified to date. HP0018 is a predicted lipoprotein of unknown function that is conserved in Helicobacter species that have flagellar sheaths but is absent in Helicobacter species that have sheath-less flagella. Deletion of hp0018 in H. pylori B128 resulted in the formation of long chains of outer membrane vesicles, which were most evident in an aflagellated variant of the Δhp0018 mutant that had a frameshift mutation in fliP. Flagellated cells of the Δhp0018 mutant possessed what appeared to be a normal flagellar sheath, suggesting that HP0018 is not required for sheath formation. Cells of the Δhp0018 mutant were also less helical in shape compared to wild-type cells. A HP0018-superfolder green fluorescent fusion protein expressed in the H. pylori Δhp0018 mutant formed fluorescent foci at the cell poles and lateral sites. Co-immunoprecipitation assays with HP0018 identified two enzymes involved in the modification of the cell wall peptidoglycan, AmiA and MltD, as potential HP0018 interaction partners. HP0018 may modulate the activity of AmiA or MltD, and in the absence of HP0018, the unregulated activity of these enzymes may alter the peptidoglycan layer in a manner that results in an altered cell shape and hypervesiculation.


Assuntos
Flagelos , Helicobacter pylori , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/fisiologia , Flagelos/metabolismo , Membrana Celular/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Humanos , Lipoproteínas/metabolismo , Lipoproteínas/genética
9.
PLoS One ; 18(11): e0287514, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37976320

RESUMO

Flagella-driven motility is essential for Helicobacter pylori to colonize the human stomach, where it causes a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. H. pylori has evolved a high-torque-generating flagellar motor that possesses several accessories not found in the archetypical Escherichia coli motor. FlgV was one of the first flagellar accessory proteins identified in Campylobacter jejuni, but its structure and function remain poorly understood. Here, we confirm that deletion of flgV in H. pylori B128 and a highly motile variant of H. pylori G27 (G27M) results in reduced motility in soft agar medium. Comparative analyses of in-situ flagellar motor structures of wild-type, ΔflgV, and a strain expressing FlgV-YFP showed that FlgV forms a ring-like structure closely associated with the junction of two highly conserved flagellar components: the MS and C rings. The results of our studies suggest that the FlgV ring has adapted specifically in Campylobacterota to support the assembly and efficient function of the high-torque-generating motors.


Assuntos
Helicobacter pylori , Humanos , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Bactérias/química , Estômago , Meios de Cultura/metabolismo , Flagelos/metabolismo
10.
Nat Struct Mol Biol ; 27(6): 589-597, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424347

RESUMO

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vibrio parahaemolyticus/metabolismo , Proteínas de Bactérias/genética , Membrana Celular , Microscopia Crioeletrônica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , ATPases Vacuolares Próton-Translocadoras/genética
11.
Cell Rep ; 27(7): 2132-2146.e7, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091451

RESUMO

Vacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways.


Assuntos
Membrana Celular/enzimologia , Proteínas de Protozoários/metabolismo , Vesículas Secretórias/metabolismo , Toxoplasma/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio
12.
PLoS One ; 13(9): e0204736, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30261054

RESUMO

Wolbachia is an unculturable, intracellular bacterium that persists within an extremely broad range of arthropod and parasitic nematode hosts, where it is transmitted maternally to offspring via vertical transmission. In the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis, Wolbachia is an endosymbiont, and its presence is essential for proper nematode development, survival, and pathogenesis. While the elucidation of Wolbachia:nematode interactions that promote the bacterium's intracellular persistence is of great importance, research has been hampered due to the fact that Wolbachia cannot be cultured in the absence of host cells. The Wolbachia endosymbiont of B. malayi (wBm) has an active Type IV secretion system (T4SS). Here, we have screened 47 putative T4SS effector proteins of wBm for their ability to modulate growth or the cell biology of a typical eukaryotic cell, Saccharomyces cerevisiae. Five candidates strongly inhibited yeast growth upon expression, and 6 additional proteins showed toxicity in the presence of zinc and caffeine. Studies on the uptake of an endocytic vacuole-specific fluorescent marker, FM4-64, identified 4 proteins (wBm0076 wBm00114, wBm0447 and wBm0152) involved in vacuole membrane dynamics. The WAS(p)-family protein, wBm0076, was found to colocalize with yeast cortical actin patches and disrupted actin cytoskeleton dynamics upon expression. Deletion of the Arp2/3-activating protein, Abp1p, provided resistance to wBm0076 expression, suggesting a role for wBm0076 in regulating eukaryotic actin dynamics and cortical actin patch formation. Furthermore, wBm0152 was found to strongly disrupt endosome:vacuole cargo trafficking in yeast. This study provides molecular insight into the potential role of the T4SS in the Wolbachia endosymbiont:nematode relationship.


Assuntos
Proteínas de Bactérias , Brugia Malayi/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Simbiose , Sistemas de Secreção Tipo IV , Wolbachia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Wolbachia/genética , Wolbachia/metabolismo
13.
J Mol Biol ; 340(5): 1005-12, 2004 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-15236963

RESUMO

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD(+)-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S.enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes.


Assuntos
Acetato-CoA Ligase/química , Acetato-CoA Ligase/metabolismo , Acetiltransferases/metabolismo , Salmonella enterica/enzimologia , Acetato-CoA Ligase/genética , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Difosfato de Adenosina/metabolismo , Lisina/metabolismo , Fases de Leitura Aberta/genética , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Salmonella enterica/isolamento & purificação , Sirtuínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Genetics ; 163(2): 545-55, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12618394

RESUMO

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.


Assuntos
Coenzima A Ligases/metabolismo , Proteínas de Escherichia coli , Ácidos Graxos/metabolismo , Histona Desacetilases/metabolismo , Saccharomyces cerevisiae/enzimologia , Salmonella enterica/enzimologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuínas/metabolismo , Acetato Quinase/metabolismo , Acetatos/metabolismo , Mutação , Fosfato Acetiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila) , Propionatos/metabolismo , Sirtuína 2 , Sirtuínas/genética
15.
PLoS One ; 10(2): e0116824, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25643265

RESUMO

The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.


Assuntos
Proteínas de Bactérias/metabolismo , Endocitose , Endossomos/metabolismo , Legionella pneumophila , Saccharomyces cerevisiae/citologia , Proteínas de Bactérias/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/deficiência , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Deleção de Genes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo
16.
Autophagy ; 9(12): 2169-70, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24145145

RESUMO

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.


Assuntos
Autofagia , Proteínas de Bactérias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Vibrio parahaemolyticus/patogenicidade , Fatores de Virulência/metabolismo , Proteínas de Bactérias/farmacologia , Células HeLa , Humanos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Vibrio parahaemolyticus/metabolismo , Fatores de Virulência/farmacologia
17.
PLoS One ; 8(2): e56798, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437241

RESUMO

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Fusão de Membrana/fisiologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Legionella pneumophila/genética , Lisossomos/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/genética
18.
Mol Biol Cell ; 19(6): 2500-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18385512

RESUMO

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.


Assuntos
Complexos Multiproteicos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Trifosfato de Adenosina/farmacologia , Motivos de Aminoácidos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas SNARE/química , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Vacúolos/efeitos dos fármacos
19.
Proc Natl Acad Sci U S A ; 104(34): 13551-8, 2007 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-17699614

RESUMO

Although concentrated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive liposome fusion and lysis, the fusion of intracellular membranes also requires Rab GTPases, Rab effectors, SM proteins, and specific regulatory lipids and is accompanied by little or no lysis. To rationalize these findings, we generated yeast strains that overexpress all four vacuolar SNAREs (4SNARE(++)). Although vacuoles with physiological levels of Rab, Rab effector/SM complex, and SNAREs support rapid fusion without Rab- and SNARE-dependent lysis, vacuoles from 4SNARE(++) strains show extensive lysis and a reduced need for the Rab Ypt7p or regulatory lipids for fusion. SNARE overexpression and the addition of pure homotypic fusion and vacuole protein sorting complex (HOPS), which bears the vacuolar SM protein, enables ypt7Delta vacuoles to fuse, allowing direct comparison of Rab-dependent and Rab-independent fusion. Because 3- to 40-fold more of each of the five components that form the SNARE/HOPS fusion complex are required for vacuoles from ypt7Delta strains to fuse at the same rate as vacuoles from wild-type strains, the apparent forward rate constant of 4SNARE/HOPS complex assembly is enhanced many thousand-fold by Ypt7p. Rabs function in normal membrane fusion by concentrating SNAREs, other proteins (e.g., SM), and key lipids at a fusion site and activating them for fusion without lysis.


Assuntos
Fusão de Membrana , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Cinética , Ligação Proteica , Proteínas SNARE/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Proteínas rab de Ligação ao GTP/genética
20.
EMBO J ; 25(22): 5260-9, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17082764

RESUMO

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion.


Assuntos
Proteínas Q-SNARE/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Vacúolos/fisiologia , Catálise , Membranas Intracelulares/fisiologia , Ligantes , Fusão de Membrana , Proteínas Q-SNARE/antagonistas & inibidores , Proteínas Q-SNARE/genética , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/fisiologia , Proteínas Qb-SNARE/antagonistas & inibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/fisiologia , Proteínas Qc-SNARE/antagonistas & inibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteína 25 Associada a Sinaptossoma
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