RESUMEN

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, ß-xylosidases, ß-galactosidases, cellulolytic enzymes like ß-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Asunto(s)

Aspergillus/enzimología , Fibras de la Dieta , Proteínas Fúngicas/análisis , Glicósido Hidrolasas/análisis , Triticum/metabolismo , Zea mays/metabolismo , Aspergillus/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Glicósido Hidrolasas/aislamiento & purificación

RESUMEN

Mycobacterium bovis is the causative agent of tuberculosis in farms, wildlife and causes sporadic disease in humans. Despite the high similitude in genome sequence between M. bovis strains, some strains like the wild boar 04-303 isolate show a highly virulent phenotype in animal models. Comparative studies will contribute to link protein expression with the virulence phenotype. In vitro, the 04-303 strain was more phagocytized by J774A.1 macrophages in comparison with 444 strain (a cow isolate with the same genotype) and BCG. The secretome of these strains showed a significant proportion of shared proteins (368 spots). Among the proteins only visualized in the secretome of the 04-303 strain, we identify the nine most abundant proteins by LC-MS/MS. The most relevant were EsxA and EsxB proteins, which are encoded in the RD1 region, deleted in BCG strains. These proteins are the major virulence factor of M. tuberculosis. The other proteins identified belong to functional categories of virulence, detoxification, and adaptation; lipid metabolism; and cell wall and cell processes. The relatively high proportion of proteins involved in the cell wall and cell process is consistent with the previously described variation among M. bovis genomes.

Asunto(s)

Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Mycobacterium bovis/química , Mycobacterium bovis/metabolismo , Proteoma/análisis , Factores de Virulencia/análisis , Animales , Cromatografía Liquida , Macrófagos/inmunología , Macrófagos/microbiología , Mycobacterium bovis/inmunología , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis , Fagocitosis , Sus scrofa , Espectrometría de Masas en Tándem

RESUMEN

Trichinella spiralis is an ubiquitous parasitic nematode that lives in muscle tissue of many hosts and causes trichinellosis in humans. Numerous efforts have been directed at specific detection of this infection and strategies for its control. TSL-1 and other antigens, mainly from muscle larvae (ML), have been used to induce partial protection in rodents. An improvement in protective immunity may be achieved by using antigens from other parasite stages. Further, identification of other parasite antigens may provide insights into their role in the host-parasite interaction. In this study, T. spiralis antigens from early developmental parasite stages, namely ML and pre-adult (PA) obtained at 6h, 18h and 30h post-infection, were identified by proteomic and mass spectrometry analyses. Our findings showed a differential expression of several proteins with molecular weights in the range of 13-224kDa and pI range of 4.54-9.89. Bioinformatic analyses revealed a wide diversity of functions in the identified proteins, which include structural, antioxidant, actin binding, peptidyl prolyl cis-trans isomerase, motor, hydrolase, ATP binding, magnesium and calcium binding, isomerase and translation elongation factor. This, together with the differential recognition of antigens from these parasite stages by antibodies present in intestinal fluid, in supernatants from intestinal explants, and in serum samples from mice infected with T. spiralis or re-infected with this parasite, provides information that may lead to alternatives in the design of vaccines against this parasite or for modulation of immune responses.

Asunto(s)

Antígenos Helmínticos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Helminto/metabolismo , Inmunoensayo/métodos , Trichinella spiralis/crecimiento & desarrollo , Trichinella spiralis/metabolismo , Animales , Antígenos Helmínticos/genética , Proteínas del Helminto/genética , Ratones , Ratones Endogámicos BALB C , Proteómica

RESUMEN

Epidemiological information and animal models have shown various Mycobacterium tuberculosis phenotypes ranging from hyper- to hypovirulent forms. Recent genomic and proteomic studies suggest that the outcome of infection depends on the M. tuberculosis fitness, which is a direct consequence of its phenotype. However, little is known about the molecular and cellular mechanisms used by mycobacteria to survive, replicate and persist during infection. The aim of this study was to perform a comprehensive proteomic analysis of culture filtrate from hypo- (CPT23) and hypervirulent (CPT31) M. tuberculosis isolates. Using two-dimensional electrophoresis we observed that 70 proteins were unique, or more abundant in culture filtrate of CPT31, and 15 of these were identified by mass spectrometry. Our analysis of protein expression showed that most of the proteins identified are involved in lipid metabolism (FadA3, FbpB and EchA3), detoxification and adaptation (GroEL2, SodB and HspX) and cell wall processes (LprA, Tig and EsxB). These results suggest that overrepresented proteins in M. tuberculosis CPT31 secretome could facilitate mycobacterial infection and persistence.

Asunto(s)

Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteoma/metabolismo , Tuberculosis/microbiología , Biología Computacional/métodos , Genotipo , Mycobacterium tuberculosis/genética , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteómica/métodos , Virulencia

RESUMEN

Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

Asunto(s)

Aedes/metabolismo , Virus del Dengue/fisiología , Interacciones Huésped-Patógeno/fisiología , Ácido N-Acetilneuramínico/biosíntesis , Aedes/genética , Aedes/virología , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Ácido N-Acetilneuramínico/genética , Saliva/enzimología , Saliva/virología , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , beta-D-Galactósido alfa 2-6-Sialiltransferasa

RESUMEN

Latex allergy is a health problem that mainly affects medical environments, causing anaphylactic shocks in extreme cases. Sensitization and reactions to this material is closely linked to the use of latex gloves. The objective of this study was to purify two of the major allergens from latex surgical gloves to study the biochemical and structural changes that could be generated during the product manufacture and to compare their IgE recognition with the non-processed allergens. Glycosylated allergen Hev b 2 (ß-1,3-glucanase) and Hev b 6.02 (hevein) were purified from glove extracts using affinity (Concanavalin A) and reversed-phase chromatographies, respectively. ELISA experiments were performed with both proteins and sera from allergic patients to assess the IgE recognition, which was heterogeneous. Crystallographic methods were used to obtain the 3D structure of Hev b 6.02 from surgical gloves, which did not show evident modification when compared with the protein from the natural non-processed form. Despite having the same crystallographic structure, the IgE from some patients showed different recognition when the glove and the natural allergen were used in ELISA. Furthermore, using electrophoretic techniques, we identified three forms of Hev b 2: one corresponding to the complete polypeptide chain with posttranslational modifications, and two glycosylated fragments. The mixture of these three forms showed stronger recognition by IgE from latex-allergic patients than the pure non-processed allergen. In conclusion, IgE from subjects sensitized to latex products showed different recognition between the allergens obtained from a natural source and the processed material, even when the structure was maintained. This demonstrates the importance of using processed allergens in further investigations of diagnosis, prevalence, product allergenicity, and therapies.

Asunto(s)

Antígenos de Plantas , Péptidos Catiónicos Antimicrobianos , Guantes Quirúrgicos/efectos adversos , Inmunoglobulina E , Hipersensibilidad al Látex/inmunología , Látex , Lectinas de Plantas , Proteínas de Plantas , Antígenos de Plantas/química , Antígenos de Plantas/inmunología , Antígenos de Plantas/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Cristalografía por Rayos X , Femenino , Hevea/química , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Látex/efectos adversos , Látex/química , Masculino , Lectinas de Plantas/química , Lectinas de Plantas/inmunología , Lectinas de Plantas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Procesamiento Proteico-Postraduccional/inmunología , Relación Estructura-Actividad

RESUMEN

Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.

Asunto(s)

Proteínas Bacterianas , Gluconacetobacter/enzimología , Glucosa Deshidrogenasas , Proteínas de la Membrana , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Transporte de Electrón , Glucosa Deshidrogenasas/química , Glucosa Deshidrogenasas/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Especificidad por Sustrato

RESUMEN

Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.

Asunto(s)

Entamoeba histolytica/metabolismo , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Caveolina 1/inmunología , Membrana Celular/metabolismo , Embrión de Pollo , Colesterol/metabolismo , Reacciones Cruzadas , Citoplasma/metabolismo , Entamoeba histolytica/efectos de los fármacos , Entamoeba histolytica/genética , Filipina/farmacología , Datos de Secuencia Molecular , Nistatina/farmacología , Fosfatidilcolinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/inmunología , Proteínas de Transferencia de Fosfolípidos/aislamiento & purificación , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfoproteínas/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología

RESUMEN

The present study aimed to analyze sera proteins in females with cervical intraepithelial neoplasia, grade III (CIN III) and in healthy control females, in order to identify a potential biomarker which detects lesions that have a greater probability of cervical transformation. The present study investigated five sera samples from females who were Human Papilloma Virus (HPV) 16+ and who had been histopathologically diagnosed with CIN III, as well as five sera samples from healthy control females who were HPV-negative. Protein separation was performed using two-dimensional (2D) gel electrophoresis and the proteins were stained with Colloidal Coommassie Blue. Quantitative analysis was performed using ImageMaster 2D Platinum 6.0 software. Peptide sequence identification was performed using a nano-LC ESIMS/MS system. The proteins with the highest Mascot score were validated using western blot analysis in an additional 55 sera samples from the control and CIN III groups. The eight highest score spots that were found to be overexpressed in the CIN III sera group were identified as α-1-B glycoprotein (A1BG), complement component 3 (C3), a pro-apolipoprotein, two apolipoproteins and three haptoglobins. Only A1BG and C3 were validated using western blot analysis, and the bands were compared between the two groups using densitometry analysis. The relative density of the bands of A1BG and C3 was found to be greater in all of the serum samples from the females with CIN III, compared with those of the individuals in the control group. In summary, the present study identified two proteins whose expression was elevated in females with CIN III, suggesting that they could be used as biomarkers for CIN III. However, further investigations are required in order to assess the expression of A1BG and C3 in different pre-malignant lesions.

RESUMEN

The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

Asunto(s)

Cisticercosis/veterinaria , Proteínas/análisis , Enfermedades de los Porcinos/parasitología , Porcinos/sangre , Taenia solium/química , Vesículas Transportadoras/química , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Western Blotting , Encéfalo/parasitología , Cisticercosis/sangre , Cisticercosis/parasitología , Cysticercus/química , Electroforesis en Gel Bidimensional , Interacciones Huésped-Parásitos , Espectrometría de Masas , Músculo Esquelético/parasitología , Proteínas/química , Enfermedades de los Porcinos/sangre

RESUMEN

Actin cytoskeleton is an essential structure involved in cell migration and invasion in parasites. In Entamoeba histolytica, the protozoan parasite causing human amoebiasis, the mechanisms underlying the expression of migration-related genes are poorly understood. Here, we investigated the biological effects of ectopic overexpression of EhPC4 (positive coactivator 4) in cell migration of E. histolytica trophozoites. Using differential in gel two-dimensional electrophoresis, 33 modulated proteins were detected in EhPC4-overexpressing cells. By electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis, 16 of these proteins were identified. Interestingly, four up-regulated proteins involved in cytoskeleton organization and cell migration were identified. Particularly, we found the up-regulation of a 16-kDa actin-binding protein (EhABP16) which is a putative member of the cofilin/tropomyosin family involved in actin polymerization. EhPC4 overexpression induced a significant increase in migration of trophozoites and in the destruction of human SW480 colon cells. Consistently, silencing of gene expression by RNA interference of EhABP16 significantly impairs cell migration. These changes were associated to alterations in the organization of actin cytoskeleton, and suppression of uropod-like structure formation in EhABP16-deficient cells. In summary, we have uncovered novel proteins modulated by EhPC4, including EhABP16, with a potential role in cell migration, cytopathogenicity and virulence in E. histolytica. BIOLOGICAL SIGNIFICANCE: The human pathogen Entamoeba histolytica infects around 50million people worldwide resulting in 40,000-100,000 deaths annually. Cell motility is a complex trait that is critical for parasites adaptation, spread and invasion processes into host tissues; it has been associated with virulence. In this study, we used a differential proteomic approach to demonstrate that E. histolytica EhPC4 induces changes in the expression of actin cytoskeleton proteins, including EhABP16, promoting a significant increase in cell motility and destruction of intestinal human cells. Particularly, we demonstrated for the first time that abrogation of EhABP16 impairs cell migration by altering the actin cytoskeleton dynamics and uropod-like structure formation in trophozoites. These data contribute to the understanding of molecular mechanisms that regulate virulence properties in this neglected protozoan parasite.

Asunto(s)

Entamoeba histolytica/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteoma , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Actinas/metabolismo , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Línea Celular Tumoral , Movimiento Celular , Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Silenciador del Gen , Humanos , Masculino , Proteómica , Interferencia de ARN , Conejos , Espectrometría de Masa por Ionización de Electrospray , Trofozoítos/metabolismo

RESUMEN

Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a ß-pleaded sheet at the sequence region of residues 181-190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

Asunto(s)

Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Serina Endopeptidasas/química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

Asunto(s)

Aedes/genética , Virus del Dengue/metabolismo , Dengue/genética , Proteínas/aislamiento & purificación , Aedes/virología , Animales , Proteínas Portadoras , Dengue/transmisión , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Células Epiteliales/metabolismo , Humanos , Proteínas/química , Proteínas/metabolismo , Proteómica

RESUMEN

The initiation factor eIF5A in Trichomonas vaginalis (TveIF5A) is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TveIF5A have been reported. The most acidic isoform (pI 5.2) corresponds to the precursor TveIF5A, whereas the mature TveIF5A appears to be the most basic isoform (pI 5.5). In addition, the intermediary isoform (pI 5.3) is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TveIF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TveIF5A contains four phosphorylated residues (S3, T55, T78 and T82). Phosphorylation at S3 and T82 is also identified in the intermediary TveIF5A, while no phosphorylated residues are found in the precursor TveIF5A. It has been demonstrated that eIF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 (CK2). Interestingly, a gene encoding a protein highly similar to CK2 (TvCK2) is found in T. vaginalis, which might be involved in the phosphorylation of TveIF5A in T. vaginalis.

Asunto(s)

Factores de Iniciación de Péptidos/química , Proteínas de Unión al ARN/química , Trichomonas vaginalis/química , Secuencia de Aminoácidos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/metabolismo , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/metabolismo , Trichomonas vaginalis/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.

Asunto(s)

Cisticercosis/diagnóstico , Electroforesis en Gel Bidimensional/métodos , Immunoblotting/métodos , Enfermedades de los Porcinos/diagnóstico , Taenia solium/aislamiento & purificación , Animales , Antígenos de Protozoos/sangre , Cisticercosis/parasitología , Electroforesis en Gel Bidimensional/veterinaria , Immunoblotting/veterinaria , Proteómica/métodos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Porcinos , Enfermedades de los Porcinos/parasitología , Taenia solium/inmunología

RESUMEN

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.

Asunto(s)

Escherichia coli Enteropatógena/inmunología , Proteínas de Escherichia coli/inmunología , Inmunoglobulina A Secretora/inmunología , Leche Humana/inmunología , Células Hep G2 , Humanos , Factores de Virulencia/inmunología

RESUMEN

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.

Asunto(s)

Asteraceae/química , Metabolismo Energético/efectos de los fármacos , Entamoeba histolytica/efectos de los fármacos , Lactonas/farmacología , Sesquiterpenos/farmacología , Western Blotting , Regulación hacia Abajo , Disentería Amebiana/tratamiento farmacológico , Electroforesis en Gel Bidimensional , Entamoeba histolytica/metabolismo , Entamoeba histolytica/ultraestructura , Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucógeno/metabolismo , Humanos , Concentración 50 Inhibidora , Lactonas/aislamiento & purificación , Microscopía Electrónica de Transmisión , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Hojas de la Planta/química , Plantas Medicinales/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sesquiterpenos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem

RESUMEN

In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

Asunto(s)

Entamoeba histolytica/genética , Exorribonucleasas/genética , Poli A/genética , Subunidades de Proteína/genética , Proteínas de Unión al ARN/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Secuencia de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Entamoeba histolytica/metabolismo , Exorribonucleasas/metabolismo , Datos de Secuencia Molecular , Poli A/metabolismo , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo

RESUMEN

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural alternative agents, as resveratrol, may be an effective adjuvant in breast cancer therapy.

Asunto(s)

Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteómica , Estilbenos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Células MCF-7 , Proteoma , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Resveratrol , Espectrometría de Masas en Tándem

RESUMEN

In this study we performed proteomic and transcriptional analyses to identify and characterize genes differentially expressed in Giardia duodenalis clones resistant to albendazole. The expression of proteins and their corresponding mRNAs was analyzed in clones resistant in vitro to different concentrations of albendazole (1.35, 8.0 and 250 µM) and these were compared with albendazole-sensitive clones using two approaches: (1) two-dimensional protein electrophoresis to analyze the proteome by the LC-MS/MS technique, and (2) semi-quantitative RT-PCR to assess the mRNA levels of proteins with the highest levels of differential expression .This strategy allowed the identification of eight proteins differentially expressed in albendazole resistant clones with roles in: (a) the cytoskeletal system (alpha 2-giardin and RanBP1), (b) the antioxidant metabolism (NADH oxidase) and (c) energy metabolism (triosephosphate isomerase, phosphoglycerate kinase and ornithine carbamoyltransferase). Gene expression analyses of these genes correlated well with the proteomics results. These observations suggest that resistance to albendazole in Giardia encompasses a complex response involving an altered expression of genes regulated at the transcriptional level that might have an important role in maintaining cell structural stability, coping with oxidative stress and adapting energy supply to a new metabolic status. These molecules are indeed promising targets for drug development.

Asunto(s)

Resistencia a Medicamentos , Regulación de la Expresión Génica , Giardia lamblia/genética , Giardia lamblia/metabolismo , Proteoma , Transcriptoma , Albendazol/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Giardia lamblia/efectos de los fármacos , Humanos , Proteómica/métodos