RESUMO
We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30min of incubation in lysis buffer at 4°C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS(E) approach. ProteinLynx Global Server (version 2.5) with Expression(E) analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.
Assuntos
Cromatografia Líquida de Alta Pressão , Proteoma/análise , Proteômica , Espectrometria de Massas em Tandem , Trypanosoma cruzi/metabolismo , Citometria de Fluxo , Microscopia de Interferência , Pressão Osmótica , Propídio/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , SonicaçãoRESUMO
INTRODUCTION: This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS: Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS: Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS: These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.
Assuntos
Leishmania braziliensis/genética , Leishmania guyanensis/genética , Leishmaniose Cutânea/diagnóstico , Adolescente , Adulto , Biópsia , Brasil/epidemiologia , Criança , Pré-Escolar , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Doenças Endêmicas , Feminino , Humanos , Lactente , Recém-Nascido , Leishmaniose Cutânea/epidemiologia , Masculino , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Abstract INTRODUCTION This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.