Estudos in vitro da inibição da atividade da telomerase de Leishmania major
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Data
2018-10-26
Autores
Gentil, Whisnayder
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Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são doenças tropicais negligenciadas, causadas por protozoários do gênero Leishmania. Elas são consideradas pela Organização Mundial da Saúde como uma doença endêmica em muitos países, incluindo o Brasil. Assim, a busca por novas metodologias de combate e controle dessas doenças parasitárias se faz necessária. Uma das abordagens refere-se ao estudo da biologia molecular dos parasitos causadores da doença, como o estudo dos telômeros, os quais são repetições em tandem associadas a proteínas e localizadas nas extremidades dos cromossomos da maioria dos eucariontes. Sua função é diferenciar os terminais de cromossomos das quebras de DNA em dupla fita e assim proteger tais extremidades da degradação e dos mecanismos de reparo das células, que em muitos casos resulta na perda de material genético codificante ao fim de cada ciclo celular. Os estudos da biologia telomérica de Leishmania têm auxiliado bastante no entendimento deste organismo, no qual o complexo telomerase é composto minimamente por um componente proteico, TERT, e por um RNA, TER, sendo que a caracterização funcional destes componentes é uma das metas de nosso grupo de pesquisa. Tal caracterização tende a ser feita, principalmente, por meios de compostos inibitórios específicos da função desses componentes ou por meio de manipulação gênica, tal qual a tecnologia CRISPR-Cas, que tem possibilitado alterações genômicas precisas e pontuais bem como o silenciamento rápido e específico de genes em diferentes organismos, incluindo Leishmania. Assim, buscamos observar os efeitos da remoção da atividade do componente proteico da telomerase (TERT) em L. major, sendo para tal utilizado um inibidor específico da TERT, denominado BIBR1532, e a tecnologia CRISPR-Cas9, para alvejar especificamente o gene que codifica esse componente. Essa empreitada resultou em linhagens de L. major expressando de forma estável a endonuclease Cas9 e clones de L. major com nocaute em um dos alelos do gene da TERT cuja remoção não resultou em alterações na taxa de crescimento e nem gerou efeitos morfológicos aparentes, de acordo com os padrões de análises usados, embora a realização de Southern blot de telômeros sugira prováveis alterações nessas estruturas decorrente da ausência de um dos alelos do gene LmTERT. Além disso, o uso dessa tecnologia em nosso laboratório provou ser um grande sucesso, e uma formidável aliada, tornando-se uma ferramenta indispensável em nossos estudos futuros. A inibição da TERT, por meio do BIBR1532, gerou uma gama de resultados, os quais abrangem atraso no crescimento das culturas, parasitos com perda de flagelo e formação de vacúolos, e, dependendo da dose do inibidor usada e do tempo de tratamento, observou-se morte celular, sendo que tais efeitos podem ser ainda mais amplos tendo em vista que nossas análises foram extremamente superficiais e que estudos mais elaborados podem fornecer informações mais precisas e refinadas do efeito desse inibidor em L. major. Temos também, trabalhado na padronização de uma ferramenta de bloqueio pós-transcricional em L. major, denominada oligonucleotídeo morpholino antisenso, com o intuito de observar futuramente o efeito de knockdown de alguns componentes teloméricos de L. major. Usando essas abordagens, esperamos que uma nova luz possa ser lançada sobre a função de componentes da maquinaria telomérica de Leishmania, na homeostase celular do parasito, e que as informações aqui obtidas colaborem futuramente no desenvolvimento de um método de controle e/ou erradicação das leishmanioses.
Leishmaniasis are neglected tropical disease, caused by protozoa of the genus Leishmania, which according to the World Health Organization is endemic in many countries, including Brazil. Thus, the search for new alternatives to combat and control these parasitic diseases, are necessary. One of the current potential approaches are to study the biology of telomeres, which are tandemly repeated sequences associated with proteins located at the ends of the chromosomes of most eukaryotes. The main function of telomeres is to differentiate the chromosome end termini from double-stranded DNA breaks and thus, to protect the ends from degradation and damage repair mechanisms, which, at the end of each cell cycle, can result in loss of genetic material. The telomere biology studies of Leishmania have greatly improved our knowledge about this organism, in which the telomerase complex is composed minimally by a protein component, TERT, and by an RNA, TER. The functional characterization of these components is one of the goals of our research group. Such characterization is usually done by using inhibitory compounds specific to the function of these components or by gene manipulation, such as the CRISPR-Cas technology, which has enabled precise genomic alterations as well as the rapid and specific silencing of genes in different organisms, including Leishmania. Thus, we sought to observe the effects of inhibiting the L. major telomerase activity, using a specific TERT inhibitor, called BIBR1532, and to induce gene knockout using CRISPR-Cas9 technology, to specifically target the gene encoding the protein component (TERT). This work resulted in L. major strains stabling expressing the Cas9 endonuclease and heminockout L. major strains for one of the TERT alleles, whose removal did not result in changes in parasite growth rate and did not generate apparent morphological effects according to our analysis. However, the Southern blotting of telomeres suggests probable changes in these structures due to the absence of one of the homologues of the LmTERT gene. In addition, the use of this technology in our laboratory has proved a great success, and a formidable ally, becoming an indispensable tool in our future studies. The inhibition of TERT, through BIBR1532 generated a range of results which included, cell growth arrest, loss of flagella, formation of intracellular vacuoles, and depending on the dose of inhibitor used and the time of treatment, we could also observe cell death. In our view such effects may be even broader because our analyses were superficial and need to be more elaborate to provide more accurate and refined information about the effect of this inhibitor in L. major. We have also been working on the standardization of a post-transcriptional blocking tool for L. major, called morpholino antisense oligonucleotide, with the aim to induce in the future knockdown effects of some L. major telomeric components. Using these approaches, we hope that a new light can be launched about the function of components of the Leishmania telomeric machinery in the parasite cellular homeostasis and that the information obtained here will collaborate in the future to the development of a methods to control and/or eradicate leishmaniasis.
Leishmaniasis are neglected tropical disease, caused by protozoa of the genus Leishmania, which according to the World Health Organization is endemic in many countries, including Brazil. Thus, the search for new alternatives to combat and control these parasitic diseases, are necessary. One of the current potential approaches are to study the biology of telomeres, which are tandemly repeated sequences associated with proteins located at the ends of the chromosomes of most eukaryotes. The main function of telomeres is to differentiate the chromosome end termini from double-stranded DNA breaks and thus, to protect the ends from degradation and damage repair mechanisms, which, at the end of each cell cycle, can result in loss of genetic material. The telomere biology studies of Leishmania have greatly improved our knowledge about this organism, in which the telomerase complex is composed minimally by a protein component, TERT, and by an RNA, TER. The functional characterization of these components is one of the goals of our research group. Such characterization is usually done by using inhibitory compounds specific to the function of these components or by gene manipulation, such as the CRISPR-Cas technology, which has enabled precise genomic alterations as well as the rapid and specific silencing of genes in different organisms, including Leishmania. Thus, we sought to observe the effects of inhibiting the L. major telomerase activity, using a specific TERT inhibitor, called BIBR1532, and to induce gene knockout using CRISPR-Cas9 technology, to specifically target the gene encoding the protein component (TERT). This work resulted in L. major strains stabling expressing the Cas9 endonuclease and heminockout L. major strains for one of the TERT alleles, whose removal did not result in changes in parasite growth rate and did not generate apparent morphological effects according to our analysis. However, the Southern blotting of telomeres suggests probable changes in these structures due to the absence of one of the homologues of the LmTERT gene. In addition, the use of this technology in our laboratory has proved a great success, and a formidable ally, becoming an indispensable tool in our future studies. The inhibition of TERT, through BIBR1532 generated a range of results which included, cell growth arrest, loss of flagella, formation of intracellular vacuoles, and depending on the dose of inhibitor used and the time of treatment, we could also observe cell death. In our view such effects may be even broader because our analyses were superficial and need to be more elaborate to provide more accurate and refined information about the effect of this inhibitor in L. major. We have also been working on the standardization of a post-transcriptional blocking tool for L. major, called morpholino antisense oligonucleotide, with the aim to induce in the future knockdown effects of some L. major telomeric components. Using these approaches, we hope that a new light can be launched about the function of components of the Leishmania telomeric machinery in the parasite cellular homeostasis and that the information obtained here will collaborate in the future to the development of a methods to control and/or eradicate leishmaniasis.
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Palavras-chave
Leishmania, Telomerase, BIBR1532, CRISPR-Cas, Leishmania, Telomerase, BIBR1532, CRISPR-Cas