Uso da técnica phage display para identificação e avaliação de peptídeos capazes de reduzir a taxa de infecção em macrófagos
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2023-11-17
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Universidade Estadual Paulista (Unesp)
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Diante da alta incidência e gravidade das leishmanioses, especialmente a leishmaniose visceral (LV), e reconhecendo as limitações dos métodos convencionais de controle e tratamento, nosso projeto de pesquisa buscou contribuir para o combate e prevenção dessas doenças. Tivemos como principal objetivo deste projeto a identificação de peptídeos capazes de reduzir a taxa de infecção de células THP-1 diferenciadas a macrófagos pelas espécies Leishmania amazonensis, Leishmania mexicana e Leishmania infantum. A identificação de tais peptídeos se deu via aplicação da técnica de phage display como um meio para selecionar fagos recombinantes que expressassem peptídeos com alta afinidade à superfície das diferentes espécies de Leishmania. O processo de seleção de sequências peptídicas de interesse se deu com base em três critérios i) análise da frequência de sequências identificadas por meio de sequenciamento, ii) análise qualitativa da interação fago-Leishmania via ensaios de fluorescência, e iii) ensaios de inibição da taxa de infecção em macrófagos utilizando fagos recombinantes. O fago selecionado a partir de ensaios de phage display frente à L. infantum, A9, reduziu a taxa de infecção de macrófagos em 51.5% e 53.6% para L. infantum e L. amazonensis, respectivamente. Já o fago, H1, selecionado via interação com L. amazonensis, apresentou reduções significativas da taxa de infecção, 47%, apenas da própria espécie. Partindo da sequência peptídica expressa por tais fagos, foram sintetizados os peptídeos La1 e Li1, derivados dos fagos H1 e A9, respectivamente. Análises dos ensaios de inibição de infecção com peptídeos sintéticos mostraram que o peptídeo La1 inibe em 45% a taxa de infecção de macrófagos por L. amazonensis, enquanto o peptídeo, Li1, reduziu a taxa de infecção de macrófagos tanto para L. infantum (42%) quanto para L. mexicana (39%). Estudos in vivo, utilizando o modelo murino BALB/c infectado com L. infantum, mostraram uma redução da carga parasitária em 84%, demonstrando o potencial terapêutico do peptídeo Li1. A descoberta de dois peptídeos, um capaz de inibir a infecção por duas espécies e um espécie-específico, abre a possibilidade de uma continuidade deste trabalho com capacidade de impacto em ciência básica. Futuros trabalhos, podem utilizar das sequências peptídicas e resultados apresentados nessa tese para identificar proteínas de superfície responsáveis pela interação parasita-hospedeiro das diferentes espécies. Além disso, a significativa redução da infecção in vivo também mostra a capacidade deste trabalho em contribuir com futuras estratégias de combate às leishmanioses.
Given the high incidence and severity of leishmaniases, especially visceral leishmaniasis (VL), and recognizing the limitations of conventional methods for control and treatment, our research aimed to contribute to the fighting and prevention of these diseases. The main objective of this project was to identify peptides capable of reducing the infection rate of Leishmania amazonensis, Leishmania mexicana, and Leishmania infantum species into THP-1 cells differentiated into macrophages. The identification of such peptides was achieved by applying the phage display technique to select recombinant phages expressing peptides with high affinity for the surface of the different Leishmania species. The selection process of peptide sequences of interest was based on three criteria: i) analysis of the frequency of predicted peptide sequence through sequencing, ii) qualitative analysis of the phage-Leishmania interaction via fluorescence assays, and iii) inhibition assays to evaluate the infection rate in macrophages using recombinant phages. The phage selected from phage display assays against L. infantum, A9, reduced the infection rate of macrophages by 51.5% and 53.6% for L. infantum and L. amazonensis, respectively. On the other hand, the phage H1, selected through interaction with L. amazonensis, showed significant reductions in the infection rate, 47%, only for L. amazonensis. We also assessed the synthetic peptides La1 and Li1 that were synthesized based on the peptide sequence expressed by H1 and A9 phages, respectively. Analyses of the synthetic peptides showed that peptide La1 inhibited the infection rate of macrophages by L. amazonensis by 45%, while peptide Li1 reduced the infection rate of macrophages for both L. infantum (42%) and L. mexicana (39%). In vivo studies using the BALB/c murine model infected with L. infantum showed an 84% reduction in parasite load, demonstrating the therapeutic potential of peptide Li1. The discovery of two peptides, one capable of inhibiting infection by two species and one specific to a species, opens the possibility of continuing this work with an impact on basic science. Future studies can use the peptide sequences and results presented in this thesis to identify surface proteins responsible for the parasite-host interaction among distinct species. Moreover, the significant reduction for the in vivo infection also demonstrates the ability of this work to contribute to future strategies for combating leishmaniases.
Given the high incidence and severity of leishmaniases, especially visceral leishmaniasis (VL), and recognizing the limitations of conventional methods for control and treatment, our research aimed to contribute to the fighting and prevention of these diseases. The main objective of this project was to identify peptides capable of reducing the infection rate of Leishmania amazonensis, Leishmania mexicana, and Leishmania infantum species into THP-1 cells differentiated into macrophages. The identification of such peptides was achieved by applying the phage display technique to select recombinant phages expressing peptides with high affinity for the surface of the different Leishmania species. The selection process of peptide sequences of interest was based on three criteria: i) analysis of the frequency of predicted peptide sequence through sequencing, ii) qualitative analysis of the phage-Leishmania interaction via fluorescence assays, and iii) inhibition assays to evaluate the infection rate in macrophages using recombinant phages. The phage selected from phage display assays against L. infantum, A9, reduced the infection rate of macrophages by 51.5% and 53.6% for L. infantum and L. amazonensis, respectively. On the other hand, the phage H1, selected through interaction with L. amazonensis, showed significant reductions in the infection rate, 47%, only for L. amazonensis. We also assessed the synthetic peptides La1 and Li1 that were synthesized based on the peptide sequence expressed by H1 and A9 phages, respectively. Analyses of the synthetic peptides showed that peptide La1 inhibited the infection rate of macrophages by L. amazonensis by 45%, while peptide Li1 reduced the infection rate of macrophages for both L. infantum (42%) and L. mexicana (39%). In vivo studies using the BALB/c murine model infected with L. infantum showed an 84% reduction in parasite load, demonstrating the therapeutic potential of peptide Li1. The discovery of two peptides, one capable of inhibiting infection by two species and one specific to a species, opens the possibility of continuing this work with an impact on basic science. Future studies can use the peptide sequences and results presented in this thesis to identify surface proteins responsible for the parasite-host interaction among distinct species. Moreover, the significant reduction for the in vivo infection also demonstrates the ability of this work to contribute to future strategies for combating leishmaniases.
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Inibição de infecção, Phage Display, Leishmanioses