RESUMEN
Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.
Asunto(s)
Ancylostoma , Glutatión Transferasa/metabolismo , Ancylostoma/genética , Ancylostoma/inmunología , Ancylostoma/metabolismo , Anquilostomiasis , Animales , Anticuerpos Antihelmínticos , Antígenos Helmínticos/genética , Antígenos Helmínticos/metabolismo , Clonación Molecular , ADN de Helmintos/genética , ADN de Helmintos/aislamiento & purificación , Escherichia coli/genética , Glutatión Transferasa/genética , Inmunohistoquímica , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
Ancylostoma ceylanicum is a zoonotic hookworm, which mainly causes iron deficiency anemia (IDA) in humans and animals. Hookworm platelet inhibitor (HPI) has been isolated from adult Ancylostoma caninum and linked to the pathogenesis of hookworm associated intestinal hemorrhage and IDA. However, there is no available data about HPI from A. ceylanicum. To study the molecular characteristics of A. ceylanicum HPI (Ace-HPI), its corresponding cDNA was amplified from adult A. ceylanicum mRNA using the primers designed based on the Ac-HPI gene sequence, and its sequence homology and phylogenetic relationship were analyzed. The differential expression of Ace-hpi mRNA in the adult and third larval (L3) stages was compared using the quantitative real-time PCR. Ace-HPI reactivity and tissue localization were studied by Western blot and immunofluorescence, respectively. Platelet aggregation activity was monitored in a 96-well microplate reader. The results showed that the Ace-HPI encoding gene was 603â¯bp in length. Ace-HPI showed 91% homology to Ac-HPI, was closely related to Ac-ASP3, and belonged to the CAP superfamily. Ace-hpi transcripts were most abundant in the adult stage, followed by serum-stimulated infective larvae (ssL3), and finally in L3 stage, with a significant difference. Escherichia coli-expressed recombinant protein had good reactivity with the positive serum of A. ceylanicum-infected dogs. Immunolocalization indicated that Ace-HPI was located in the esophagus and cephalic glands of the adult. As well as, recombinant Ace-HPI inhibited the platelet aggregation in-vitro. HPI overexpression, anatomical location in adults, antigenicity and its in-vitro activity indicate its possible role in adult worm blood-feeding and as a valuable target for hookworm vaccine and drug development.
Asunto(s)
Ancylostoma/crecimiento & desarrollo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Análisis de Secuencia de ADN/métodos , Ancylostoma/genética , Ancylostoma/metabolismo , Animales , Clonación Molecular , Esófago/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/química , Larva/crecimiento & desarrollo , Larva/metabolismo , Modelos Moleculares , Filogenia , Estructura Secundaria de Proteína , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
Parasitic nematode infections are treated using anthelmintic drugs, some of which target nicotinic acetylcholine receptors (nAChRs) located in different parasite tissues. The limited arsenal of anthelmintic agents and the prevalence of drug resistance imply that future defense against parasitic infections will depend on the discovery of novel targets and therapeutics. Previous studies have suggested that Ascaris suum ACR-16 nAChRs are a suitable target for the development of antinematodal drugs. In this study, we characterized the pharmacology of the Ancylostoma caninum ACR-16 receptor using two-electrode voltage-clamp electrophysiology. This technique allowed us to study the effects of cholinergic agonists and antagonists on the nematode nAChRs expressed in Xenopus laevis oocytes. Aca-ACR-16 was not sensitive to many of the existing cholinomimetic anthelmintics (levamisole, oxantel, pyrantel, and tribendimidine). 3-Bromocytisine was the most potent agonist (> 130% of the control acetylcholine current) on the Aca-ACR-16 nAChR but, unlike Asu-ACR-16, oxantel did not activate the receptor. The mean time constants of desensitization for agonists on Aca-ACR-16 were longer than the rates observed in Asu-ACR-16. In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DHßE and moderately inhibited by α-BTX. In conclusion, we have successfully reconstituted a fully functional homomeric nAChR, ACR-16, from A. caninum, a model for human hookworm infections. The pharmacology of the receptor is distinct from levamisole-sensitive nematode receptors. The ACR-16 homologue also displayed some pharmacological differences from Asu-ACR-16. Hence, A. caninum ACR-16 may be a valid target site for the development of anthelmintics against hookworm infections.
Asunto(s)
Ancylostoma/metabolismo , Antihelmínticos/farmacología , Proteínas del Helminto/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Anquilostomiasis , Animales , Colinérgicos/farmacología , Proteínas del Helminto/análisis , Proteínas del Helminto/metabolismo , Receptores Nicotínicos/análisis , Receptores Nicotínicos/metabolismoRESUMEN
Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.
Asunto(s)
Ancylostoma/química , Antiinflamatorios/administración & dosificación , Colitis/terapia , Citocinas/inmunología , Leucocitos Mononucleares/inmunología , Ancylostoma/metabolismo , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Terapia Biológica , Colitis/genética , Colitis/inmunología , Citocinas/genética , Modelos Animales de Enfermedad , Ácidos Grasos/administración & dosificación , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Different reports have highlighted the potential use of helminths and their secretions in the treatment of inflammatory bowel disease (IBD) conditions; however, no reports have investigated their effects at a proteome level. Herein, we characterise the protein expression changes that occur in lamina propria (LP) and the intestinal epithelial cells (IEC) of mice with dextran sulfate sodium (DSS)-induced colitis treated with Ancylostoma caninum excretory/secretory (ES) products using a quantitative proteomic approach. We have shown how parasite products can significantly alter the expression of proteins involved in immune responses, cell death and with an antioxidant activity. Interestingly, significant changes in the expression levels of different mucins were observed in this study. MUC13, a mucin implicated in gastrointestinal homeostasis, was upregulated in the LP of mice with DSS-induced colitis treated with ES, while MUC2, a major component of mucus, was upregulated in the IEC. In addition, A. caninum proteins have an important effect on proteins with antioxidant functions and proteins involved in intestinal homeostasis and tissue integrity and regeneration. Understanding how parasites can ameliorate IBD pathogenesis can help us design novel treatments for autoimmune diseases.
Asunto(s)
Ancylostoma/química , Antiinflamatorios/farmacología , Productos Biológicos/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Proteínas del Helminto/farmacología , Mucosa Intestinal/efectos de los fármacos , Ancylostoma/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Productos Biológicos/uso terapéutico , Colitis Ulcerosa/etiología , Sulfato de Dextran/toxicidad , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Proteínas del Helminto/uso terapéutico , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucina 2/genética , Mucina 2/metabolismo , Regulación hacia ArribaRESUMEN
Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.
Asunto(s)
Ancylostoma/metabolismo , Anquilostomiasis/veterinaria , Enfermedades de los Perros/parasitología , Metaboloma/fisiología , Óvulo/fisiología , Ancylostoma/fisiología , Anquilostomiasis/parasitología , Anquilostomiasis/patología , Animales , Perros , Heces/parasitología , Cromatografía de Gases y Espectrometría de Masas , Humanos , Ácidos Láuricos/metabolismo , Ácido Mirístico/metabolismo , Prostaglandinas/metabolismoRESUMEN
When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.
Asunto(s)
Ancylostoma/genética , Proteínas HSP90 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Factores de Transcripción/genética , Ancylostoma/efectos de los fármacos , Ancylostoma/crecimiento & desarrollo , Ancylostoma/metabolismo , Animales , Compuestos de Bencidrilo/farmacología , Proteínas de Caenorhabditis elegans/genética , Clonación Molecular , Cricetinae , Perros , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/biosíntesis , Larva/genética , Larva/metabolismo , Masculino , Mesocricetus , Triterpenos Pentacíclicos , Pirrolidinonas/farmacología , Análisis de Secuencia de ADN , Factores de Transcripción/biosíntesis , Transcriptoma , Triterpenos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbß3 and α2ß1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.
Asunto(s)
Ancylostoma/química , Plaquetas/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Proteínas del Helminto/química , Secuencias de Aminoácidos , Ancylostoma/metabolismo , Animales , Plaquetas/química , Dominio Catalítico , Colágeno/química , Colágeno/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrinógeno/química , Fibrinógeno/metabolismo , Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas del Helminto/farmacología , Humanos , Enlace de Hidrógeno , Integrina alfa2beta1/química , Integrina alfa2beta1/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
Mutations in the ß-tubulin isotype 1 gene at codons 167 (F167Y), 198 (E198A) and 200 (F200Y) have been associated with benzimidazole resistance in helminths. The F200Y polymorphism has previously been described for Ancylostom caninum; however, the F167Y polymorphism has not been investigated in members of the Ancylostomatidae family. The aim of this study was to screen for the F167Y polymorphism in A. caninum isolates recovered from naturally infected dogs in two Brazilian states. No mutation was observed at codon 167 in the 230 analyzed samples from the two populations; however, it is possible that this change may be present at a low frequency in other populations of the same species. These results highlight the importance of monitoring the genetic basis involved in the drug resistance process.
Asunto(s)
Ancylostoma/genética , Ancylostoma/metabolismo , Genotipo , Polimorfismo de Nucleótido Simple , Tubulina (Proteína)/genética , Animales , Regulación de la Expresión Génica , Tubulina (Proteína)/metabolismoRESUMEN
Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12-C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.
Asunto(s)
Ancylostoma/genética , Antígenos Helmínticos/genética , Clonación Molecular , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Ancylostoma/metabolismo , Anquilostomiasis/parasitología , Anquilostomiasis/veterinaria , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/metabolismo , Cricetinae , ADN Complementario/genética , Ácidos Grasos/metabolismo , Femenino , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Evidence from human studies and mouse models shows that infection with parasitic helminths has a suppressive effect on the pathogenesis of some inflammatory diseases. Recently, we and others have shown that some of the suppressive effects of hookworms reside in their excretory/secretory (ES) products. Here, we demonstrate that ES products of the hookworm Ancylostoma caninum (AcES) suppress intestinal pathology in a model of chemically induced colitis. This suppression was associated with potent induction of a type 2 cytokine response characterized by coexpression of interleukin-4 (IL-4) and IL-10 by CD4(+) T cells, downregulation of proinflammatory cytokine expression in the draining lymph nodes and the colon, and recruitment of alternatively activated (M2) macrophages and eosinophils to the site of ES administration. Protease digestion and heat denaturation of AcES resulted in impaired induction of CD4(+) IL-4(+) IL-10(+) cell responses and diminished ability to suppress colitis, indicating that protein component(s) are responsible for some of the immunosuppressive effects of AcES. Identification of the specific parasite-derived molecules responsible for reducing pathology during chemically induced colitis could lead to the development of novel therapeutics for the treatment of human inflammatory bowel disease.
Asunto(s)
Ancylostoma/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Colitis/patología , Proteínas del Helminto/farmacología , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Ancylostoma/inmunología , Animales , Linfocitos T CD4-Positivos/clasificación , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colon/inmunología , Colon/patología , Citocinas/metabolismo , Sulfato de Dextran/toxicidad , Eosinófilos/citología , Femenino , Proteínas del Helminto/uso terapéutico , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Cavidad Peritoneal/citologíaRESUMEN
Ancylostoma ceylanicum belongs to a group of soil-transmitted helminths, which infect almost 576 mln people worldwide and are a major cause of anaemia and malnutrition. Upon contact with a permissive host, third-stage larvae (L3) residing in the environment become activated larvae (ssL3), a process associated with changes in the profile of gene expression. Ancylostoma secreted proteins (ASPs) are the major proteins secreted during larvae activation and play a crucial role in hookworm adaptation to parasitism. Here we report the cloning using RACE-PCR technique of three novel ASPs from the hookworm A. ceylanicum (Ace-asp-3, Ace-asp-4, and Ace-asp-5) and computational analysis of the protein sequences. All three proteins contain SCP (Sperm Coating Protein) domain characteristic for previously described ASP proteins. Real-time PCR analysis shows significant up-regulation of Ace-asp-3 and Ace-asp-5 expression in adult worms and correlated down-regulation in ssL3 larvae. On the other hand, expression of Ace-asp-4 was increased in ssL3 stages and decreased in adult parasites.
Asunto(s)
Ancylostoma/genética , Ancylostoma/metabolismo , Clonación Molecular , ADN Complementario/genética , Proteínas del Helminto/metabolismo , Animales , ADN de Helmintos/química , ADN de Helmintos/genética , Proteínas del Helminto/genética , Datos de Secuencia Molecular , FilogeniaRESUMEN
Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction. Recently, several anticoagulant serine protease inhibitors have been identified from adult Ancylostoma caninum hookworms. One of them, A. caninum anticoagulant peptide c2 (AcAPc2), can inhibit the activity of factor VIIa/tissue factor complex to exert its antithrombotic effect. However, it is difficult to adopt traditional expression and purification systems to yield high-purity recombinant AcAPc2 (rAcAPc2). Here, we employed a simple method to produce high-yield and high-purity rAcAPc2. We obtained the full-length double-stranded cDNA encoding AcAPc2 by overlapping PCR and cloned it into an intein-based expression vector. The AcAPc2 cDNA was expressed in Escherichia coli and comprised 30% of the total bacterial proteins. The expressed rAcAPc2 was purified by cleaving the fused chitin-binding domain at pH 7.2. Finally, we produced a high yield of rAcAPc2 at a purity of greater than 98%. Importantly, the generated rAcAPc2 prolonged the prothrombin time (PT) and activated partial thromboplastin time (aPTT) of human plasma in vitro in a dose-dependent manner. Therefore, this method to generate the high-purity and bioactive rAcAPc2 may contribute to the scientific research on its biological function and the treatment of thrombotic diseases.
Asunto(s)
Ancylostoma/metabolismo , Proteínas del Helminto/farmacología , Proteínas Recombinantes de Fusión/farmacología , Inhibidores de Serina Proteinasa/farmacología , Ancylostoma/genética , Animales , Coagulación Sanguínea/efectos de los fármacos , Western Blotting , Quitina , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Humanos , Inteínas , Tiempo de Tromboplastina Parcial , Empalme de Proteína , Tiempo de Protrombina , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.
Asunto(s)
Ancylostoma/química , Ácidos y Sales Biliares/química , Colestenos/química , Proteínas del Helminto/química , Receptores Citoplasmáticos y Nucleares/química , Ancylostoma/genética , Ancylostoma/metabolismo , Anquilostomiasis/metabolismo , Anquilostomiasis/terapia , Animales , Ácidos y Sales Biliares/genética , Ácidos y Sales Biliares/metabolismo , Colestenos/metabolismo , Cristalografía por Rayos X , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Mamíferos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología Estructural de ProteínaRESUMEN
SCP/TAPS proteins are a diverse family of molecules in eukaryotes, including parasites. Despite their abundant occurrence in parasite secretomes, very little is known about their functions in parasitic nematodes, including blood-feeding hookworms. Current information indicates that SCP/TAPS proteins (called Ancylostoma-secreted proteins, ASPs) of the canine hookworm, Ancylostoma caninum, represent at least three distinct groups of proteins. This information, combined with comparative modelling, indicates that all known ASPs have an equatorial groove that binds extended structures, such as peptides or glycans. To elucidate structure-function relationships, we explored the three-dimensional crystal structure of an ASP (called Ac-ASP-7), which is highly up-regulated in expression in the transition of A. caninum larvae from a free-living to a parasitic stage. The topology of the N-terminal domain is consistent with pathogenesis-related proteins, and the C-terminal extension that resembles the fold of the Hinge domain. By anomalous diffraction, we identified a new metal binding site in the C-terminal extension of the protein. Ac-ASP-7 is in a monomer-dimer equilibrium, and crystal-packing analysis identified a dimeric structure which might resemble the homo-dimer in solution. The dimer interaction interface includes a novel binding site for divalent metal ions, and is proposed to serve as a binding site for proteins involved in the parasite-host interplay at the molecular level. Understanding this interplay and the integration of structural and functional data could lead to the design of new approaches for the control of parasitic diseases, with biotechnological outcomes.
Asunto(s)
Ancylostoma/genética , Ancylostoma/patogenicidad , Proteínas del Helminto/química , Caballos/parasitología , Interacciones Huésped-Parásitos/genética , Ancylostoma/metabolismo , Animales , Sitios de Unión , Proteínas del Helminto/metabolismo , Péptidos/química , Polisacáridos/química , Conformación Proteica , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.
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Ancylostoma/metabolismo , Membrana Celular/metabolismo , Proteínas del Helminto/metabolismo , Saposinas/metabolismo , Animales , Cristalografía por Rayos X , Perros , Proteínas del Helminto/química , Humanos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Saposinas/química , Homología Estructural de ProteínaRESUMEN
The evolution of drug resistant bacteria is a severe public health problem, both in hospitals and in the community. Currently, some countries aim at concentrating highly specialized services in large hospitals in order to improve patient outcomes. Emergent resistant strains often originate in health care facilities, but it is unknown to what extent hospital size affects resistance evolution and the resulting spillover of hospital-associated pathogens to the community. We used two published datasets from the US and Ireland to investigate the effects of hospital size and controlled for several confounders such as antimicrobial usage, sampling frequency, mortality, disinfection and length of stay. The proportion of patients acquiring both sensitive and resistant infections in a hospital strongly correlated with hospital size. Moreover, we observe the same pattern for both the percentage of resistant infections and the increase of hospital-acquired infections over time. One interpretation of this pattern is that chance effects in small hospitals impede the spread of drug-resistance. To investigate to what extent the size distribution of hospitals can directly affect the prevalence of antibiotic resistance, we use a stochastic epidemiological model describing the spread of drug resistance in a hospital setting as well as the interaction between one or several hospitals and the community. We show that the level of drug resistance typically increases with population size: In small hospitals chance effects cause large fluctuations in pathogen population size or even extinctions, both of which impede the acquisition and spread of drug resistance. Finally, we show that indirect transmission via environmental reservoirs can reduce the effect of hospital size because the slow turnover in the environment can prevent extinction of resistant strains. This implies that reducing environmental transmission is especially important in small hospitals, because such a reduction not only reduces overall transmission but might also facilitate the extinction of resistant strains. Overall, our study shows that the distribution of hospital sizes is a crucial factor for the spread of drug resistance.
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Ancylostoma/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Depsipéptidos/farmacología , Haemonchus/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Actividad Motora/genética , Mutación , Ancylostoma/genética , Anquilostomiasis/tratamiento farmacológico , Anquilostomiasis/genética , Anquilostomiasis/metabolismo , Animales , Antihelmínticos/farmacología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Depsipéptidos/antagonistas & inhibidores , Antagonismo de Drogas , Evaluación Preclínica de Medicamentos/métodos , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hemoncosis/tratamiento farmacológico , Hemoncosis/genética , Hemoncosis/metabolismo , Haemonchus/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Actividad Motora/efectos de los fármacos , Micotoxinas/farmacología , Especificidad de la EspecieRESUMEN
Immunoscreening an Ancylostoma caninum cDNA library with canine hookworm-infected dog serum resulted in the isolation of a 461 bp cDNA encoding Ac-AP-12, a new 9.1 kDa anticoagulant peptide (100 amino acids) with 43-69% amino acid homology to other nematode anticoagulant peptides (NAPs) from Ancylostoma hookworms. Messenger RNA transcription and expression of Ac-AP-12 was unique to the adult stage of A. caninum. The yeast expressed recombinant Ac-AP-12 demonstrated potent anticoagulant activity on human blood plasma in a concentration dependent manner, and was shown to specifically inhibit human factor Xa activity. Immunolocalization with specific rabbit antiserum showed that Ac-AP-12 was exclusively located in the esophageal glands of adult hookworm. Ac-AP-12 is hypothesized to facilitate both parasite blood feeding and digestion.
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Ancylostoma/metabolismo , Anticoagulantes/metabolismo , Esófago/metabolismo , Inhibidores del Factor Xa , Péptidos/metabolismo , Secuencia de Aminoácidos , Ancylostoma/química , Ancylostoma/clasificación , Ancylostoma/genética , Anquilostomiasis/sangre , Anquilostomiasis/parasitología , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Clonación Molecular , Esófago/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/farmacología , Filogenia , Alineación de SecuenciaRESUMEN
BACKGROUND: Ancylostoma caninum third-stage larvae are the non-feeding infective stage of this parasite and are able to infect potential hosts via different infection routes. Since percutaneous infection is one of the most important routes and skin penetration is the first step into parasitic life, an existing in vitro model for percutaneous migration was modified and evaluated. The main parameter used to evaluate migration was the migration ratio (migrated larvae as a percentage of total number of larvae recovered). Additionally, the skin lag was calculated, expressing the percentage of larvae remaining in the skin and therefore not being recovered. Since initiation of feeding is proposed to be an important step in the transition from free-living to parasitic A. caninum larvae, feeding assays were performed with in vitro percutaneously migrated larvae. Additionally, infective larvae of A. caninum were activated via serum-stimulation and feeding behaviour was analysed and compared between percutaneously migrated and serum-stimulated larvae. RESULTS: Maximum skin migration levels of infective larvae were observed at temperatures above 32°C when larvae were placed on the epidermal side of skin for more than 12 hours. The medium beneath the skin had no effect on migration ratio, and no significant difference between the migration ratios through fresh and frozen/thawed skin was observed. Maximum feeding levels of 93.2% were observed for percutaneously migrated larvae after 48 h incubation, whereas serum-stimulated larvae reached the maximum of 91.0% feeding larvae after 24 h. CONCLUSIONS: The PERL chamber system was optimised and standardised as an in vitro model for percutaneous migration. The larvae recovered after percutaneous migration showed characteristic signs of activation similar to that of serum-stimulated larvae. The observed difference in time course of resumption of feeding indicates that percutaneously migrated larvae are not identical to serum-stimulated larvae, which are currently representing the model for early parasitic stages.
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Ancylostoma/patogenicidad , Ancylostoma/crecimiento & desarrollo , Ancylostoma/metabolismo , Animales , Modelos Animales de Enfermedad , Perros , Conducta Alimentaria , Técnicas In Vitro , Larva/crecimiento & desarrollo , Larva/metabolismo , Larva/fisiología , LocomociónRESUMEN
Protease inhibitors play important roles in the parasitic nematodes' survival within their host, in the development and reproduction of the parasites. The present study described the isolation, identification, and characterization of a novel member of the Ascaris family of serine protease inhibitors, designated AduTIL-1, from the human hookworm Ancylostoma duodenale. AduTIL-1 is composed of a signal sequence and two trypsin inhibitor-like (TIL) domains, which showed the highest similarity with OdmCRP, a putative serine protease inhibitor with two TIL domains in Oesophagostomum dentatum. Each TIL domain of the AduTIL-1 was expressed in Escherichia coli, and their inhibitory activities against serine proteases from animals and human were characterized, respectively. Both of the two TIL domains inhibited human neutrophil elastase and pancreatic trypsin, but different in effectiveness. Although the first TIL domain of AduTIL-1 inhibited bovine pancreatic chymotrypsin (Ki=18.0 nM), both of the two domains showed no inhibitory activity against the human pancreatic chymotrypsin. Immunohistochemical studies demonstrated that AduTIL-1 was localized in esophagus, intestine, and cuticular surface of the adult worms. These results suggested that AduTIL-1 may be involved in the survival of A. duodenale in host by targeting related digestive enzymes and neutrophil elastase.