Estudos sobre o efeito do nocaute e da superexpressão do componente RNA da telomerase (LeishTER) nos telômeros, no desenvolvimento e na sobrevida de Leishmania major
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2024-01-25
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Universidade Estadual Paulista (Unesp)
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Parasitos do gênero Leishmania causam leishmaniose, uma doença tropical negligenciada que afeta pessoas em todo o mundo. A doença apresenta diferentes manifestações clínicas, alta taxa de incidência e vai desde lesões auto curáveis até a forma letal, conhecida como leishmaniose visceral. Apesar de muitos esforços, até agora não existem vacinas e protocolos de tratamento eficientes disponíveis e, portanto, encontrar novos alvos terapêuticos é crucial. Os telômeros, extremidades físicas dos cromossomos de eucariotos, são essenciais para manter a estabilidade do genoma e têm sido considerados alvos potenciais contra parasitos. Em Leishmania spp. os telômeros são compostos por repetições conservadas do tipo TTAGGG, mantidas pela telomerase, uma ribonucleoproteína minimamente composta pelos componentes TERT (transcriptase reversa da telomerase) e TER (RNA da telomerase). Na maioria dos eucariotos o TER é uma molécula única e contém a sequência molde copiada pela TERT durante a elongação dos telômeros. Sua estrutura secundária contém domínios com funções de controle da inserção de nucleotídeos pela TERT, reconhecimento da sequência telomérica e de ligação com proteínas acessórias. Em 2014 nosso grupo identificou e caracterizou parcialmente o componente TER da telomerase de Leishmania (LeishTER). O presente trabalho visa desvendar os papéis biológicos desempenhados pelo componente TER de Leishmania major (LeishTER) na homeostase do parasito. Para isso obtivemos uma linhagem celular knockout (LmTER-/-) que apresentou alterações no perfil de proliferação do parasito, divisão celular e replicação do DNA, principalmente nas primeiras passagens in vitro. Durante o crescimento contínuo, LmTER-/- apresentou arrest parcial do ciclo celular G0/G1, encurtamento progressivo dos telômeros e maior porcentagem de fosforilação da histona γH2A. Além disso, alterações ultraestruturais características da autofagia foram detectadas sem que as células apresentassem danos no DNA ou fenótipos de apoptose. LmTER-/- promastigotas diferenciam-se em metacíclicos apresentando índice de infectividade in vitro menor que o controle Lm007. Curiosamente, o addback de LeishTER não retornou o comprimento dos telômeros aos níveis do tipo selvagem, corroborando resultados preliminares que mostram que a expressão epissomal de TER, semelhante à sua ablação, tem um efeito negativo dominante na vida do parasito, o que pode ser um potencial alvo terapêutico contra a leishmaniose. Além disso, resultados preliminares que visam acessar a estrutura secundária de LeishTER através da acilação seletiva de 2'-hidroxila analisada por extensão de primer e perfil mutacional (SHAPE MaP) com o uso de 2-metilnicotínico imidazolide (NAI) confirmaram algumas estruturas já descritas pelo nosso grupo, incluindo a Hélice II (TBE), o molde e a hélice proximal ao modelo (TPH). A estrutura inicial de ~100 nt de LeishTER se assemelhou à de TbTR (TER de Trypanosoma brucei), com um motivo 5' CCGUCA 3' na extremidade 3' proximal a TBE, similar a TBE de Tetrahymena thermophila. Nucleotídeos na base de TPH mostraram baixa reatividade, indicando rigidez, enquanto o ápice apresentou reatividade. O molde de LeishTER compreende principalmente elementos de cadeia simples, exceto por alguns nucleotídeos que sugerem envolvimento em interações de longo alcance ou transitórias. Entretanto, experimentos adicionais são necessários para confirmação e uma compreensão completa da estrutura de LeishTER.
Parasites of the genus Leishmania cause leishmaniasis, a neglected tropical disease that affects people worldwide. The disease has different clinical manifestations, a high incidence rate and ranges from self-healing lesions to the lethal form known as visceral leishmaniasis. Despite many efforts, until now there are no vaccines and efficient treatment protocols available and therefore finding new therapeutic targets is crucial. Telomeres, the physical ends of eukaryotic chromosomes, are essential for maintaining genome stability and have been considered potential targets against parasites. In Leishmania spp. telomeres are composed of conserved TTAGGG-like repeats maintained by telomerase, a ribonucleoprotein minimally composed of TERT (telomerase reverse transcriptase) and TER (telomerase RNA) components. In most eukaryotes, TER is a single molecule and contains the template sequence copied by TERT during telomere elongation. Its secondary structure contains domains with functions to control the insertion of nucleotides by TERT, recognition of the telomeric sequence and binding with accessory proteins. In 2014 our group identified and partially characterized the TER component of Leishmania telomerase (LeishTER). The present work aims to unravel the biological roles played by the TER component of Leishmania major (LeishTER) in the parasite's homeostasis. For this, we obtained a knockout cell line (LmTER-/-) that showed alterations in the profile of parasite proliferation, cell division and DNA replication, mainly in the first passages in vitro. During continuous growth, LmTER-/- showed partial arrest of the G0/G1 cell cycle, progressive telomere shortening and a higher percentage of histone γH2A phosphorylation. Furthermore, ultrastructural alterations characteristic of autophagy were detected without the cells showing DNA damage or apoptosis phenotypes. LmTER-/- promastigotes differentiate into metacyclics with an in vitro infectivity index lower than the Lm007 control. Interestingly, the LeishTER addback did not return telomere length to wild-type levels, corroborating preliminary results showing that episomal TER expression, similar to its ablation, has a dominant negative effect on parasite lifespan, which may be a potential therapeutic target against leishmaniasis. Additionally, preliminary results aiming to access the secondary structure of LeishTER through selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE MaP) using 2-methyl nicotinic imidazolide (NAI) confirmed some structures previously described by our group, including Helix II (TBE), the template, and the template proximal helix (TPH). The initial structure of ~100 nt of LeishTER closely resembled that of TbTR (Trypanosoma brucei TER), with a 5' CCGUCA 3' motif at the 3' end proximal to TBE, similar to TBE in Tetrahymena thermophila. Nucleotides at the TPH base showed low reactivity, indicating rigidity, while the apex exhibited reactivity. The LeishTER template mainly comprised single-stranded elements, except for a few nucleotides suggesting involvement in long-range or transient interactions. However, additional experiments are needed for confirmation and a comprehensive understanding of the LeishTER structure.
Parasites of the genus Leishmania cause leishmaniasis, a neglected tropical disease that affects people worldwide. The disease has different clinical manifestations, a high incidence rate and ranges from self-healing lesions to the lethal form known as visceral leishmaniasis. Despite many efforts, until now there are no vaccines and efficient treatment protocols available and therefore finding new therapeutic targets is crucial. Telomeres, the physical ends of eukaryotic chromosomes, are essential for maintaining genome stability and have been considered potential targets against parasites. In Leishmania spp. telomeres are composed of conserved TTAGGG-like repeats maintained by telomerase, a ribonucleoprotein minimally composed of TERT (telomerase reverse transcriptase) and TER (telomerase RNA) components. In most eukaryotes, TER is a single molecule and contains the template sequence copied by TERT during telomere elongation. Its secondary structure contains domains with functions to control the insertion of nucleotides by TERT, recognition of the telomeric sequence and binding with accessory proteins. In 2014 our group identified and partially characterized the TER component of Leishmania telomerase (LeishTER). The present work aims to unravel the biological roles played by the TER component of Leishmania major (LeishTER) in the parasite's homeostasis. For this, we obtained a knockout cell line (LmTER-/-) that showed alterations in the profile of parasite proliferation, cell division and DNA replication, mainly in the first passages in vitro. During continuous growth, LmTER-/- showed partial arrest of the G0/G1 cell cycle, progressive telomere shortening and a higher percentage of histone γH2A phosphorylation. Furthermore, ultrastructural alterations characteristic of autophagy were detected without the cells showing DNA damage or apoptosis phenotypes. LmTER-/- promastigotes differentiate into metacyclics with an in vitro infectivity index lower than the Lm007 control. Interestingly, the LeishTER addback did not return telomere length to wild-type levels, corroborating preliminary results showing that episomal TER expression, similar to its ablation, has a dominant negative effect on parasite lifespan, which may be a potential therapeutic target against leishmaniasis. Additionally, preliminary results aiming to access the secondary structure of LeishTER through selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE MaP) using 2-methyl nicotinic imidazolide (NAI) confirmed some structures previously described by our group, including Helix II (TBE), the template, and the template proximal helix (TPH). The initial structure of ~100 nt of LeishTER closely resembled that of TbTR (Trypanosoma brucei TER), with a 5' CCGUCA 3' motif at the 3' end proximal to TBE, similar to TBE in Tetrahymena thermophila. Nucleotides at the TPH base showed low reactivity, indicating rigidity, while the apex exhibited reactivity. The LeishTER template mainly comprised single-stranded elements, except for a few nucleotides suggesting involvement in long-range or transient interactions. However, additional experiments are needed for confirmation and a comprehensive understanding of the LeishTER structure.
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Palavras-chave
L. major TER nocaute, Alteração da proliferação, Encurtamento dos telômeros, Sinalização de danos ao DNA, Autofagia, Baixo índice de infectividade, CRISPR/Cas9, SHAPE-MaP, L. major TER knockout, Alteration of proliferation, Telomere shortening, DNA damage signaling, Autophagy, Low infectivity rate