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Identification of proteases as diagnostic and drug targets in bovine babesiosis
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A babesiose bovina é uma doença transmitida por carraças, que causa elevada morbidade e mortalidade, e provoca consideráveis perdas económicas devido aos esforços para controlar esta doença. As medidas de controlo da babesiose bovina incluem a erradicação ou redução de carraças, correcto diagnóstico, assim como tratamento e vacinação apropriados. Este trabalho tem como objectivo contribuir para um melhor diagnóstico da infecção e a consequente melhoria de algumas das medidas de controlo, bem como identificar e caracterizar genes de proteases utilizados para o desenvolvimento de um método de diagnóstico e estudados como potenciais alvos para fármacos.
Para este estudo, foi realizado um trabalho de colaboração em Moçambique, onde foram colhidas amostras de sangue de bovinos naturalmente infectados, em cinco explorações situadas na província de Maputo, no sul do país. Um novo método de detecção molecular por PCR foi desenvolvido e testado utilizando DNA genómico e amostras de campo aleatórias colhidas numa das explorações. Os iniciadores de PCR foram desenhados com base no gene putativo da protease aspártica babesipsina-1 identificado nos genomas de Babesia bigemina e de B. bovis. O novo seminested hot-start PCR foi desenvolvido utilizando a combinação de iniciadores longos de 30 pb de comprimento e uma hot-start polimerase, que permitem teoricamente a utilização de temperaturas de emparelhamento acima da temperatura de melting, impedindo assim a formação de amplificações não específicas, o que aumenta a especificidade do método.
O novo seminested hot-start PCR foi avaliado utilizando 117 amostras de campo, e em paralelo com um método amplamente utilizado, o nested PCR. O seminested hot-start PCR neste estudo foi mais sensível que o nested PCR. Com o seminested hot-start PCR, 90% das amostras foram positivas para B. bigemina, e 82% foram positivas para B. bovis. Os resultados sugeriram que a babesiose bovina é comum e endémica em Moçambique, e que a doença se encontra numa situação de estabilidade endémica.
O estudo do estado da babesiose bovina em Moçambique, foi então aprofundado, através da análise de amostras de campo aleatórias de mais quatro explorações utilizando o seminested hot-start PCR. Todas as amostras das cinco explorações foram também analisadas utilizando o RLB, e os resultados deste método foram comparados com os dados obtidos pelo seminested hot-start PCR. A detecção de Babesia spp. diferiu significativamente entre os métodos utilizados e os locais de recolha. Com o seminested hot-start PCR, a detecção de B. bigemina nas várias explorações, variou entre 30% e 89%, com uma detecção total de 61%, e a detecção de B. bovis variou entre 27% e 83% com uma frequência global de 53%. Utilizando o RLB, não foi detectado B. bigemina e a detecção de B. bovis variou entre 0% e 17% com uma frequência total de 5,1%. A análise de novas sequências do gene 18S rRNA, revelou que a actual sonda do RLB para B. bigemina não é adequada para a detecção de todos os isolados desta espécie identificados em Moçambique. O seminested hot-start PCR foi portanto mais sensível que o RLB. No entanto, dez espécies diferentes dos quatro Géneros Anaplasma, Babesia, Ehrlichia e Theileria foram detectadas pelo ensaio RLB, e isso demonstra que as infecções múltiplas são comuns em Moçambique.
Os resultados deste estudo mostram que a babesiose bovina é comum na província de Maputo, e também que existem alguns locais com baixa prevalência de infecções, e portanto, os resultados sugerem que esta doença não está numa situação de estabilidade endémica na província de Maputo. São agora necessários novos estudos epidemiológicos para confirmar estes resultados.
Tem sido demonstrado que as proteases têm papéis essenciais em parasitas protozoários e estão sob estudo como promissores alvos de fármacos. Algumas proteases cisteínicas de parasitas protozoários, são já reconhecidos alvos de fármacos, e encontram-se em validação inibidores específicos para a quimioterapia da leishmaniose, da malária e da tripanossomíase. Neste estudo, o nosso principal interesse na identificação e caracterização de proteases como alvos de fármacos foi portanto nesta classe de proteases.
Foram identificados no banco de dados do projecto em curso de sequenciação do genoma de B. bigemina, os genes putativos de proteases cisteínicas em pesquisas por similaridade de sequência, que posteriormente foram comparados com os genes anotados nos genomas completos das espécies de piroplasmas bovinos B. bovis, Theileria annulata e T. parva. Para avaliar os eventos da evolução molecular que ocorreram na família C1 de proteases cisteínicas, foram feitos alinhamentos múltiplos entre os genomas e análises das sequências obtidas destes piroplasmas de importância veterinária. Existem assim, cinco grupos distintos de genes de proteases cisteínicas da família C1 em B. bigemina (5 genes), quatro grupos em B. bovis (4 genes) e seis grupos em Theileria spp. (13 genes). No Género Theileria a evolução molecular ocorreu através da duplicação de genes e da diversificação da sequência das proteínas codificadas por estes genes. Estas importantes diferenças observadas entre os Géneros Babesia e Theileria na família das proteases cisteínicas, podem parcialmente explicar os diferentes mecanismos de infecção destas espécies, em que parasitas Babesia não invadem linfócitos e parasitas Theileria invadem primeiro os linfócitos no hospedeiro vertebrado.
A babesipaína-1, uma das proteases cisteínicas identificadas no genoma de B. bigemina, foi expressa como uma proteína de fusão com a glutationa S-transferase (GST) e a respectiva fracção solúvel foi purificada por cromatografia de afinidade. A babesipaína-1 recombinante apresentou actividade contra certos péptidos que são substratos típicos de proteases cisteínicas, e foi inibida por um inibidor geral da sua classe, mas o baixo rendimento da purificação da fracção solúvel impediu a sua caracterização adicional.
A babesipaína-1 foi então purificada a partir da fracção insolúvel, e a proteína desnaturada foi re-enrolada e activada para produzir uma enzima activa. A análise da actividade da babesipaína-1 revelou propriedades típicas de uma protease cisteínica da família da papaína, incluindo a hidrólise de alguns péptidos que são substratos típicos da família da papaína, um pH ácido óptimo (5.5-6.0), o requisito de um ambiente redutor para ter actividade máxima, e a inibição por inibidores de proteases cisteínicas como o E-64, a leupeptina, o ALLN e a cistatina. Os resultados sugerem que a babesipaína-1 tem um papel no citosol, já que a babesipaína-1 manteve elevada actividade contra substratos a pH 7,5 (83% do máximo), uma característica incomum das proteases cisteínicas de parasitas protozoários.
Assim, os resultados deste estudo demonstram que a babesiose bovina é uma infecção comum na província de Maputo em Moçambique, embora a doença não esteja numa situação de estabilidade endémica. Os resultados também sugerem que as proteases cisteínicas de Babesia spp. são alvos promissores para fármacos e consequentemente para o desenvolvimento de um tratamento eficaz para a babesiose bovina. Na sequência destes resultados foi associado um plano de trabalho futuro. Alguns pormenores e resultados deste trabalho podem ser transferidos para outros países, inclusivamente Portugal.
Bovine babesiosis is a tick–borne disease with significant morbidity and mortality, and the economic losses associated to this disease can be considerable. Control measures of bovine babesiosis include the eradication or reduction of ticks, good diagnosis, use of vaccination and correct treatment. The aim of this work was to contribute to a better diagnosis of the infection aiming at the improvement of some of the control measures, as well as to identify and characterize protease genes for the development of a diagnostic method and studied as potential drug targets. For this study, collaborative work was carried out in Mozambique, from where blood samples from naturally infected cattle were collected in five farms located in the Maputo province, south of the country. A new molecular detection PCR method was then developed and tested using genomic DNA and random field samples collected from one farm. Primers were designed based in the babesial aspartic protease putative babesipsin gene identified in the genomes of Babesia bigemina and B. bovis. The new seminested hot-start PCR method was developed using the combination of 30 bp long primers and a hot start polymerase that theoretically allows the use of annealing temperatures above the melting temperatures of the primers and prevents the formation of unspecific amplifications and therefore increases the specificity. The new seminested hot-start PCR method was assayed using 117 field samples in parallel with the widely used nested PCR method. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. With the seminested hot-start PCR, 90% of the samples were positive for B. bigemina and 82% were positive for B. bovis. The results suggested that bovine babesiosis is common and endemic in Mozambique and that the disease was in an endemically stable situation. The status of bovine babesiosis in Mozambique was then further studied, by testing random field samples from four more farms using the seminested hot-start PCR. All the samples from the five farms were also analysed using the reverse line blot (RLB) assay, and the results were compared with the data obtained by the seminested hot-start PCR. The detection of Babesia spp. differed considerably between methods and locations. Using the seminested hot-start PCR, detection of B. bigemina between farms varied from 30% to 89% with an overall detection of 61%, and detection of B. bovis ranged between 27% and 83% with an overall frequency of 53%. Using the RLB assay B. bigemina was not detected and detection of B. bovis ranged between 0% and 17% with a total frequency of 5.1%. Analysis of new sequences of the 18S rRNA gene revealed that the current B. bigemina RLB probe is unspecific for the detection of all the identified isolates from Mozambique. The seminested hot-start PCR was therefore more sensitive than the RLB assay. Nevertheless, ten different species of the four genera Anaplasma, Babesia, Ehrlichia and Theileria were detected by the RLB assay, and this illustrates that multiple infections are widespread in Mozambique. The results of this study show that bovine babesiosis is common in Maputo province, but there are some locations with low prevalence of infections and therefore the results suggest that this disease is not in an endemically stable situation in Maputo province. Further epidemiological studies are now needed to corroborate these findings. Proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Some cysteine proteases of protozoan parasites are now recognized drug targets and specific inhibitors are in validation for chemotherapy of leishmaniasis, malaria, and trypanosomiasis. In this study our focus on the identification and characterization of proteases as drug targets was therefore in this class of proteases. Cysteine protease putative genes were identified by sequence similarity search in the ongoing B. bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasms genomes of B. bovis, Theileria annulata, and T. parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. There are five distinct groups of cysteine protease genes of C1 family in B. bigemina (5 genes), four groups in B. bovis (4 genes) and six groups in Theileria spp. (13 genes). Molecular evolution in Theileria occurred through the duplication of genes and sequence diversity. These considerable differences observed in the cysteine protease family between Babesia and Theileria genera, may partially explain why Babesia cannot infect lymphocytes and Theileria infect first lymphocytes in the vertebrate host. One of the identified cysteine proteases in the B. bigemina genome, babesipain-1, was expressed as a fusion protein with gluthatione S-transferase (GST) and the soluble protein was purified by affinity chromatography. The recombinant babesipain-1 showed activity against typical peptide substrates of cysteine proteases, and was inhibited by a general inhibitor of its class, but the low yield of the soluble purification prevented additional characterization. Babesipain-1 was then purified from the insoluble fraction, and the denatured protein was refolded and activated to produce an active mature enzyme. Analysis of the activity of babesipain-1 revealed typical properties of a papain-family cysteine protease, including hydrolysis of typical papain-family peptide substrate, an acidic pH optimum (5.5-6.0), requirement for a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors as E-64, leupeptin, ALLN and cystatin. The results suggest that babesipain-1 has a role in cytosol environment, since babesipain-1 retained high activity against peptide substrates at pH 7.5 (83% of maximum), an uncommon feature of cysteine proteases of parasitic protozoa. Thus the results of this study demonstrate that bovine babesiosis is widespread in Maputo province in Mozambique, although the disease is not in an endemically stable situation. The results also suggest that cysteine proteases of Babesia spp. are promising drug targets for the development of an effective treatment of bovine babesiosis. In face of these results a plan for future work is associated. Some aspects and results from this work can be adjusted to other countries, including Portugal.
Bovine babesiosis is a tick–borne disease with significant morbidity and mortality, and the economic losses associated to this disease can be considerable. Control measures of bovine babesiosis include the eradication or reduction of ticks, good diagnosis, use of vaccination and correct treatment. The aim of this work was to contribute to a better diagnosis of the infection aiming at the improvement of some of the control measures, as well as to identify and characterize protease genes for the development of a diagnostic method and studied as potential drug targets. For this study, collaborative work was carried out in Mozambique, from where blood samples from naturally infected cattle were collected in five farms located in the Maputo province, south of the country. A new molecular detection PCR method was then developed and tested using genomic DNA and random field samples collected from one farm. Primers were designed based in the babesial aspartic protease putative babesipsin gene identified in the genomes of Babesia bigemina and B. bovis. The new seminested hot-start PCR method was developed using the combination of 30 bp long primers and a hot start polymerase that theoretically allows the use of annealing temperatures above the melting temperatures of the primers and prevents the formation of unspecific amplifications and therefore increases the specificity. The new seminested hot-start PCR method was assayed using 117 field samples in parallel with the widely used nested PCR method. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. With the seminested hot-start PCR, 90% of the samples were positive for B. bigemina and 82% were positive for B. bovis. The results suggested that bovine babesiosis is common and endemic in Mozambique and that the disease was in an endemically stable situation. The status of bovine babesiosis in Mozambique was then further studied, by testing random field samples from four more farms using the seminested hot-start PCR. All the samples from the five farms were also analysed using the reverse line blot (RLB) assay, and the results were compared with the data obtained by the seminested hot-start PCR. The detection of Babesia spp. differed considerably between methods and locations. Using the seminested hot-start PCR, detection of B. bigemina between farms varied from 30% to 89% with an overall detection of 61%, and detection of B. bovis ranged between 27% and 83% with an overall frequency of 53%. Using the RLB assay B. bigemina was not detected and detection of B. bovis ranged between 0% and 17% with a total frequency of 5.1%. Analysis of new sequences of the 18S rRNA gene revealed that the current B. bigemina RLB probe is unspecific for the detection of all the identified isolates from Mozambique. The seminested hot-start PCR was therefore more sensitive than the RLB assay. Nevertheless, ten different species of the four genera Anaplasma, Babesia, Ehrlichia and Theileria were detected by the RLB assay, and this illustrates that multiple infections are widespread in Mozambique. The results of this study show that bovine babesiosis is common in Maputo province, but there are some locations with low prevalence of infections and therefore the results suggest that this disease is not in an endemically stable situation in Maputo province. Further epidemiological studies are now needed to corroborate these findings. Proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Some cysteine proteases of protozoan parasites are now recognized drug targets and specific inhibitors are in validation for chemotherapy of leishmaniasis, malaria, and trypanosomiasis. In this study our focus on the identification and characterization of proteases as drug targets was therefore in this class of proteases. Cysteine protease putative genes were identified by sequence similarity search in the ongoing B. bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasms genomes of B. bovis, Theileria annulata, and T. parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. There are five distinct groups of cysteine protease genes of C1 family in B. bigemina (5 genes), four groups in B. bovis (4 genes) and six groups in Theileria spp. (13 genes). Molecular evolution in Theileria occurred through the duplication of genes and sequence diversity. These considerable differences observed in the cysteine protease family between Babesia and Theileria genera, may partially explain why Babesia cannot infect lymphocytes and Theileria infect first lymphocytes in the vertebrate host. One of the identified cysteine proteases in the B. bigemina genome, babesipain-1, was expressed as a fusion protein with gluthatione S-transferase (GST) and the soluble protein was purified by affinity chromatography. The recombinant babesipain-1 showed activity against typical peptide substrates of cysteine proteases, and was inhibited by a general inhibitor of its class, but the low yield of the soluble purification prevented additional characterization. Babesipain-1 was then purified from the insoluble fraction, and the denatured protein was refolded and activated to produce an active mature enzyme. Analysis of the activity of babesipain-1 revealed typical properties of a papain-family cysteine protease, including hydrolysis of typical papain-family peptide substrate, an acidic pH optimum (5.5-6.0), requirement for a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors as E-64, leupeptin, ALLN and cystatin. The results suggest that babesipain-1 has a role in cytosol environment, since babesipain-1 retained high activity against peptide substrates at pH 7.5 (83% of maximum), an uncommon feature of cysteine proteases of parasitic protozoa. Thus the results of this study demonstrate that bovine babesiosis is widespread in Maputo province in Mozambique, although the disease is not in an endemically stable situation. The results also suggest that cysteine proteases of Babesia spp. are promising drug targets for the development of an effective treatment of bovine babesiosis. In face of these results a plan for future work is associated. Some aspects and results from this work can be adjusted to other countries, including Portugal.
Descrição
Palavras-chave
Parasitologia Médica Babesíase Babesiose bovina Proteases
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical