Caracterización del transcriptoma y proteoma intestinal de Ornithodoros moubata. Utilidad para la búsqueda de antígenos vacunales

[ES] La garrapata Ornithodoros moubata es un argásido africano distribuido por los países del este, centro y sur del continente (Vial, 2009; Quembo et al., 2016), donde transmite la Fiebre recurrente humana causada por Borrelia duttoni y la Peste porcina africana (PPA). La presencia de O. moubata en el entorno doméstico y peridoméstico contribuye a la persistencia de ambas enfermedades en las zonas endémicas y, además, representa un constante riesgo de propagación de dichas enfermedades hacia otras regiones (Cutler, 2010; Sánchez-Vizcaíno et al., 2015; Quembo et al., 2016). La prevención y control de dichas enfermedades requiere la eliminación de las poblaciones sinantrópicas de O. moubata. La aplicación de acaricidas como método de lucha frente a este argásido resulta ineficaz y, por tanto, se necesitan métodos alternativos de control, como podrían ser las vacunas anti-garrapata. Con ese objetivo, nuestro grupo inició el desarrollo de una vacuna anti-O. moubata evaluando la capacidad protectora de dos tipos de antígenos: salivales e intestinales. Los estudios con antígenos salivales nos permitieron identificar tres componentes antihemostáticos protectores que, administrados conjuntamente, proporcionan más de un 50% de eficacia protectora, lo cual los convierte en interesantes candidatos vacunales. Pero, pese a ello, aún no se ha podido obtener una vacuna que sea completamente eficaz frente a O. moubata y que esté basada únicamente en antígenos salivales (Díaz-Martín et al., 2015). Por su parte, la posibilidad de obtener respuestas protectoras más eficaces utilizando antígenos intestinales de O. moubata parecía más probable al ser este tipo de antígenos los utilizados en las dos únicas vacunas anti-garrapata eficaces comercializadas hasta la fecha, TickGARD© y GAVAC©. En nuestros estudios previos con O. moubata y con la especie ibérica Ornithodoros erraticus, la inmunización de animales con extractos de membrana del intestino indujo respuestas protectoras que redujeron significativamente la alimentación y fecundidad de las hembras (García Varas, 2004; Manzano-Román et al., 2006). En esos mismos estudios se comprobó que los antígenos responsables del efecto protector eran proteínas de la membrana luminal de los enterocitos y que su expresión aumentaba significativamente tras la ingestión de la sangre, alcanzando un máximo en torno a las 48 horas post-alimentación (Manzano-Román et al., 2007). Estas proteínas aún no han sido identificadas, pero los resultados anteriores confirman la capacidad protectora de los antígenos de membrana del intestino de ambas especies, convirtiendo al intestino medio de los ornithodoros en objetivo prioritario de estudio en la búsqueda e identificación de candidatos vacunales. El intestino medio de las garrapatas es el órgano encargado de digerir la sangre del hospedador y de absorber los nutrientes imprescindibles para su supervivencia y reproducción (Sojka et al., 2013). Además constituye la primera barrera defensiva a la que se enfrentan los microorganismos patógenos ingeridos con la sangre, la cual debe ser eficientemente superada por éstos para invadir y multiplicarse en otros órganos y, desde ellos, transmitirse a la siguiente fase evolutiva, a la descendencia y a otros hospedadores (Kocan et al., 2004). En definitiva, el intestino medio forma parte de la interfase hospedador-garrapata-patógeno y en él se expresan numerosas proteínas que intervienen en procesos fisiológicos vitales para la garrapata y/o en los mecanismos de invasión, multiplicación y transmisión de patógenos. Por tanto, dichas proteínas son de gran interés como candidatos antigénicos para el desarrollo de vacunas anti-garrapata y vacunas bloqueantes de la transmisión de enfermedades. Cabe esperar que ese conjunto de proteínas intestinales experimenten una expresión diferencial en respuesta al estímulo proporcionado por la fijación al hospedador y la ingestión de la sangre. Y de acuerdo con nuestras observaciones previas (Manzano-Román et al., 2007) y lo descrito por otros autores (Akov, 1982; Sojka et al., 2013), cabría esperar que las proteínas sobre-expresadas en torno a las 48 horas post-alimentación muestren un alto potencial protector. Estas proteínas, al ser antígenos ocultos no habrán sufrido la presión selectiva de la respuesta inmunitaria del hospedador, por lo que: (i) mantendrán una alta inmunogenicidad y es previsible que induzcan fuertes repuestas humorales si se administran artificialmente con adyuvantes; y (ii) es probable que estén conservadas en otros argásidos, e incluso en ixódidos, lo que permitiría utilizarlas en una vacuna de amplio espectro frente a estos vectores hematófagos. De dichas proteínas, las expresadas en la membrana plasmática luminal del enterocito (junto con las secretadas a la luz intestinal) son las más fácilmente accesibles a los efectores inmunes ingeridos con la sangre del hospedador (esencialmente, los anticuerpos y el complemento), por lo que serían las de primera elección como dianas vacunales. A la vista de lo anterior, y dado que en O. moubata se desconoce casi por completo la composición proteica de su tubo digestivo, en la presente tesis doctoral nos propusimos los siguientes OBJETIVOS GENERALES: (i) La obtención del mialoma (transcriptoma + proteoma) del intestino medio de O. moubata. (ii) El análisis comparado de dicho mialoma antes y después de la alimentación para identificar genes/proteínas expresados diferencialmente en respuesta a la digestión de la sangre. (iii) La selección de antígenos potencialmente protectores entre las proteínas de membrana sobre-expresadas tras la alimentación aplicando una estrategia de vacunología reversa e incluyendo la validación experimental de los candidatos en pruebas de inmunización de animales. La consecución de dichos objetivos generales se planteó a través de los siguientes OBJETIVOS ESPECÍFICOS: 1. Obtención del transcriptoma del intestino de hembras de O. moubata en ayunas y a las 48 horas post-alimentación. Anotación y comparación entre los dos estados fisiológicos. 2. Obtención del proteoma del intestino de hembras de O. moubata en ayunas y a las 48 horas post-alimentación. Anotación y comparación entre ambos estados fisiológicos. 3. Selección in silico de antígenos potencialmente protectores a partir de la información transcriptómica y proteómica basada, esencialmente, en criterios de inmunogenicidad, localización celular y sobreexpresión tras la alimentación. 4. Producción de formas recombinantes de los antígenos seleccionados en el objetivo anterior o bien diseño y síntesis química de péptidos antigénicos a partir de dichos candidatos. 5. Evaluación del efecto protector de las proteínas recombinantes y péptidos sintéticos frente a O. moubata y O. erraticus mediante experimentos de vacunación de conejos y validación, en su caso, como candidatos vacunales

Midgut proteome of an argasid tick, Ornithodoros erraticus: a comparison between unfed and engorged females

16 páginas, 5 figuras y 2 tablas. -- Agradecimientos: Asistencia técnica de Rocío Vizcaíno Marín y María González Sánchez, del Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC) y de la Dra. Luz Valero, de la Unidad Proteómica de la Universidad de Valencia. -- The electronic version of this article is the complete one and can be found online at: http://www.parasitesandvectors.com/content/8/1/525The argasid tick Ornithodoros erraticus is the vector of African swine fever virus and of several Borrelia species that cause human relapsing fever in the Iberian Peninsula. The tick midgut is part of the ectoparasite-host interface and expresses proteins that are vital for the survival of the tick. Midgut proteins are therefore potential targets for drug and/or vaccine design aimed at the development of new strategies for tick control. Thus, the aim of this work was the characterization of the proteome of the O. erraticus midgut before and after a blood meal trying to elucidate the induced changes upon blood feeding. Midgut tissues from unfed and engorged O. erraticus females were dissected and proteins were fractionated by centrifugation and SDS-PAGE, and the corresponding gel pieces analysed by LC–MS/MS. The identified proteins were classified according to their Protein Class and Molecular Function and the differences between fed and unfed specimens were analysed. Overall 555 tick proteins were identified: 414 in the midgut of the unfed specimens and 376 in the fed specimens, of which 235 were present in both groups. The proteins with catalytic, binding and structural functions were the most numerous and abundant, consistent with their role in the intracellular processing of the blood meal. The analysis of some groups of proteins putatively involved directly in blood meal digestion, including protein digestion (peptidase activity), iron metabolism, enzymes involved in oxidative stress and detoxification and membrane traffic and transport proteins, detected some differences between the fed and unfed ticks. This work reports for the first time the collection and analysis of the midgut proteome of an argasid tick species and provides molecular information about the argasid machinery involved in blood digestion. This information represents a starting point for the identification and selection of new targets for the development of alternative control strategies.This research was funded by project AGL2013-42745-P granted by the Spanish Ministry of Economy and Competitiveness.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

A proteomic insight into the midgut proteome of Ornithodoros moubata females reveals novel information on blood digestion in argasid ticks

[Background]: The argasid tick Ornithodoros moubata is the main African vector of the human relapsing fever agent Borrelia duttoni and the African swine fever virus. Together with saliva, the tick midgut forms part of the host-tick-pathogen interface, and numerous midgut proteins play key functions in the blood digestion-related process and the infection and transmission of pathogens. This work explores the composition of the midgut proteome of unfed and fed O. moubata females with the aim to complete the biological information already obtained from the midgut transcriptome and provide a more robust and comprehensive perspective of this biological system.[Methods]: Midgut tissues taken from females before feeding and 48 h after feeding were subjected to LC/MS-MS analysis. After functional characterization and classification of the proteins identified, the differences in the proteome between unfed and fed females were analysed and discussed. Additionally, a detailed analysis of particular groups of proteins that are involved in the processes of nutrient digestion and responses to the oxidative stress was carried out.[Results]: 1491 non-redundant tick proteins were identified: 1132 of them in the midgut of unfed ticks, 1138 in the midgut of fed ticks, and up to 779 shared by both physiological conditions. Overall, the comparative analysis of the midgut proteomes of O. moubata females before and after feeding did not reveal great differences in the number or class of proteins expressed, enzymatic composition or functional classification.[Conclusions]: The hemoglobinolytic system in ixodids and argasids is very similar in spite of the fact that they display very different feeding and reproductive strategies. Although the main source of nutrients in ticks are proteins, lipids and carbohydrates also constitute significant nutritional sources and play an important part in the process of blood digestion. The genes and proteins involved in intracellular transport mechanisms, defensive responses, detoxifying responses and stress responses seem to be closely regulated, highlighting the complexity and importance of these processes in tick biology, which in turn assigns them a great interest as targets for therapeutic and immunological interventions.We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).This research was funded by project AGL2013–42745-P granted by the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Midgut proteome of an argasid tick, Ornithodoros erraticus: a comparison between unfed and engorged females

16 páginas, 5 figuras y 2 tablas. -- Agradecimientos: Asistencia técnica de Rocío Vizcaíno Marín y María González Sánchez, del Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC) y de la Dra. Luz Valero, de la Unidad Proteómica de la Universidad de Valencia. -- The electronic version of this article is the complete one and can be found online at: http://www.parasitesandvectors.com/content/8/1/525The argasid tick Ornithodoros erraticus is the vector of African swine fever virus and of several Borrelia species that cause human relapsing fever in the Iberian Peninsula. The tick midgut is part of the ectoparasite-host interface and expresses proteins that are vital for the survival of the tick. Midgut proteins are therefore potential targets for drug and/or vaccine design aimed at the development of new strategies for tick control. Thus, the aim of this work was the characterization of the proteome of the O. erraticus midgut before and after a blood meal trying to elucidate the induced changes upon blood feeding. Midgut tissues from unfed and engorged O. erraticus females were dissected and proteins were fractionated by centrifugation and SDS-PAGE, and the corresponding gel pieces analysed by LC–MS/MS. The identified proteins were classified according to their Protein Class and Molecular Function and the differences between fed and unfed specimens were analysed. Overall 555 tick proteins were identified: 414 in the midgut of the unfed specimens and 376 in the fed specimens, of which 235 were present in both groups. The proteins with catalytic, binding and structural functions were the most numerous and abundant, consistent with their role in the intracellular processing of the blood meal. The analysis of some groups of proteins putatively involved directly in blood meal digestion, including protein digestion (peptidase activity), iron metabolism, enzymes involved in oxidative stress and detoxification and membrane traffic and transport proteins, detected some differences between the fed and unfed ticks. This work reports for the first time the collection and analysis of the midgut proteome of an argasid tick species and provides molecular information about the argasid machinery involved in blood digestion. This information represents a starting point for the identification and selection of new targets for the development of alternative control strategies.This research was funded by project AGL2013-42745-P granted by the Spanish Ministry of Economy and Competitiveness.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Function-guided selection of midgut antigens from Ornithodoros erraticus ticks and an evaluation of their protective efficacy in rabbits.

57 páginas, 11 figuras y 4 tablas .-- The final version is available at http://www.elsevier.comThe identification of candidate protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that play key biological functions. Tick midgut proteins that play essential functions in tick survival and disease transmission are upregulated in response to blood feeding and digestion. In this study, Ornithodoros erraticus midgut transcriptomic and proteomic data upon feeding were inspected to select functionally relevant antigens to be assessed as vaccine candidate antigens. For this, we primarily focused on proteins with relevant biological functions in key physiological processes for ticks and tick-host-pathogen interactions. Later, we used additional criteria based on overexpression after feeding, predicted antigenicity and cellular localisation, resulting in the selection of four theoretical candidates, two aquaporins (OeAQP, OeAQP1), one ABC transporter (OeABC) and one selenoprotein T (OeSEL). Rabbit vaccination with synthetic immunogenic peptides designed from the extracellular antigenic regions of the selected candidates induced humoral responses that reduced tick feeding and reproduction performance. Both AQPs and OeSEL demonstrated significant protection efficacy against the homologous species O. erraticus, but lower non-significant cross-species protection against Ornithodoros moubata. Conversely, OeABC showed no protection against the homologous species O. erraticus, but significant cross-species protection against O. moubata. These results are the first demonstration of the protective potential of argasid aquaporins, suggesting that they might be included in vaccines for the control of multiple tick species. Additionally, these results also unveiled two novel protective antigens from argasid ticks, OeABC and OeSEL, belonging to functional protein families that have never been explored as a source of vaccine candidates and are deserving of further studies. Finally, our data add value to the midgut as a protective candidate antigen source in argasids for the control of tick infestations.This work was supported by project AGL2013–42745-P, funded by a grant from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

A proteomic insight into the midgut proteome of Ornithodoros moubata females reveals novel information on blood digestion in argasid ticks

Abstract Background The argasid tick Ornithodoros moubata is the main African vector of the human relapsing fever agent Borrelia duttoni and the African swine fever virus. Together with saliva, the tick midgut forms part of the host-tick-pathogen interface, and numerous midgut proteins play key functions in the blood digestion-related process and the infection and transmission of pathogens. This work explores the composition of the midgut proteome of unfed and fed O. moubata females with the aim to complete the biological information already obtained from the midgut transcriptome and provide a more robust and comprehensive perspective of this biological system. Methods Midgut tissues taken from females before feeding and 48 h after feeding were subjected to LC/MS-MS analysis. After functional characterization and classification of the proteins identified, the differences in the proteome between unfed and fed females were analysed and discussed. Additionally, a detailed analysis of particular groups of proteins that are involved in the processes of nutrient digestion and responses to the oxidative stress was carried out. Results 1491 non-redundant tick proteins were identified: 1132 of them in the midgut of unfed ticks, 1138 in the midgut of fed ticks, and up to 779 shared by both physiological conditions. Overall, the comparative analysis of the midgut proteomes of O. moubata females before and after feeding did not reveal great differences in the number or class of proteins expressed, enzymatic composition or functional classification. Conclusions The hemoglobinolytic system in ixodids and argasids is very similar in spite of the fact that they display very different feeding and reproductive strategies. Although the main source of nutrients in ticks are proteins, lipids and carbohydrates also constitute significant nutritional sources and play an important part in the process of blood digestion. The genes and proteins involved in intracellular transport mechanisms, defensive responses, detoxifying responses and stress responses seem to be closely regulated, highlighting the complexity and importance of these processes in tick biology, which in turn assigns them a great interest as targets for therapeutic and immunological interventions

In silico selection of functionally important proteins from the mialome of Ornithodoros erraticus ticks and assessment of their protective efficacy as vaccine targets

[Background] New candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens.[Methods] Transcripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4).[Results] Rabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata.[Conclusions] These results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.The authors also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work was supported by project AGL2013-42745-P, funded by a grant from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Evaluation of the protective efficacy of Ornithodoros moubata midgut membrane antigens selected using omics and in silico prediction algorithms

64 páginas, 7 figuras, 5 tablas, 4 figuras suplementarias, 3 tablas suplementarias. -- The definitve version is available at: http://www.elsevier.comThe African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni, the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund’s adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus. All candidates were highly immunogenic, but provided low protection against the O. moubata infestations (ranging from 7% to 39%). Interestingly, all candidates except one also protected against infestations by O. erraticus, achieving higher efficacies against this species (from 20% to 66%). According to their protective potential, three of the five antigens tested (Om17, Om86 and OM99) were considered little suitable for use in tick vaccines, while the other two (OM85 and OM03) were considered useful antigens for tick vaccine development, deserving further studies.This work was supported by project AGL2013–42745-P funded by a grant from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

De novo assembly and analysis of midgut transcriptome of the argasid tick Ornithodoros erraticus and identification of genes differentially expressed after blood feeding

65 páginas, 9 gráficas, 7 tablas . -- The definitive version is available at http://www.elsevier.comTicks are hematophagous vectors of great medical and veterinary importance because they transmit numerous pathogenic microorganisms to humans and animals. The argasid Ornithodoros erraticus is the main vector of tickborne human relapsing fever and African swine fever in the Mediterranean Basin. Tick enterocytes express bioactive molecules that perform key functions in blood digestion, feeding, toxic waste processing and pathogen transmission. To explore new strategies for tick control, in this work we have obtained and compared the midgut transcriptomes of O. erraticus female ticks before and after a blood meal and identified the genes whose expression is differentially regulated after feeding. The transcript sequences were annotated, functionally and structurally characterised and their expression levels compared between both physiological conditions (unfed females and fed females at 2 days postengorgement). Up to 29,025 transcripts were assembled, and 9,290 of them corresponded to differentially expressed genes (DEGs) after feeding. Of these, 4,656 genes were upregulated and nearly the same number of genes was downregulated in fed females compared to unfed females. BLASTN and BLASTX analyses of the 29,025 transcripts allowed the annotation of 9,072 transcripts/proteins. Among them, the most numerous were those with catalytic and binding activities and those involved in diverse metabolic pathways and cellular processes. The analyses of functional groups of upregulated DEGs potentially related to the digestion of proteins, carbohydrates and lipids, and the genes involved in the defence response and response to oxidative stress, confirm that these processes are narrowly regulated in ticks, highlighting their complexity and importance in tick biology. The expression patterns of six genes throughout the blood digestion period revealed significant differences between these patterns, strongly suggesting that the transcriptome composition is highly dynamic and subjected to important variation along the trophogonic cycle. This may guide future studies aimed at improving the understanding of the molecular physiology of tick digestion and digestion-related processes. The current work provides a more robust and comprehensive understanding of the argasid tick digestive system.This research was funded by project AGL2013-42745-P granted by the Spanish Ministry of Economy and Competitiveness.Peer reviewe