Barbara Cristina de Albuquerque-Melo, Bernardo Acácio Santini Pereira, Vítor Ennes-Vidal, Maria Eduarda Pinto Gonçalves, Luzia Monteiro de Castro Côrtes, Léa Cysne-Finkelstein, Herbert Leonel de Matos Guedes, Geovane Dias-Lopes, Carlos Roberto Alves
{"title":"评估巨噬细胞感染期间巴西利什曼病（Viannia）Thor 株亚群中的蛋白酶和锥硫蛋白系统酶。","authors":"Barbara Cristina de Albuquerque-Melo, Bernardo Acácio Santini Pereira, Vítor Ennes-Vidal, Maria Eduarda Pinto Gonçalves, Luzia Monteiro de Castro Côrtes, Léa Cysne-Finkelstein, Herbert Leonel de Matos Guedes, Geovane Dias-Lopes, Carlos Roberto Alves","doi":"10.1590/0074-02760240038","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22.</p><p><strong>Objectives: </strong>Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations.</p><p><strong>Methods: </strong>Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study.</p><p><strong>Findings: </strong>Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03.</p><p><strong>Main conclusions: </strong>The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.</p>","PeriodicalId":18469,"journal":{"name":"Memorias do Instituto Oswaldo Cruz","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251415/pdf/","citationCount":"0","resultStr":"{\"title\":\"Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.\",\"authors\":\"Barbara Cristina de Albuquerque-Melo, Bernardo Acácio Santini Pereira, Vítor Ennes-Vidal, Maria Eduarda Pinto Gonçalves, Luzia Monteiro de Castro Côrtes, Léa Cysne-Finkelstein, Herbert Leonel de Matos Guedes, Geovane Dias-Lopes, Carlos Roberto Alves\",\"doi\":\"10.1590/0074-02760240038\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22.</p><p><strong>Objectives: </strong>Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations.</p><p><strong>Methods: </strong>Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study.</p><p><strong>Findings: </strong>Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03.</p><p><strong>Main conclusions: </strong>The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.</p>\",\"PeriodicalId\":18469,\"journal\":{\"name\":\"Memorias do Instituto Oswaldo Cruz\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2024-07-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11251415/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Memorias do Instituto Oswaldo Cruz\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1590/0074-02760240038\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"PARASITOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Memorias do Instituto Oswaldo Cruz","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1590/0074-02760240038","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"PARASITOLOGY","Score":null,"Total":0}
Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.
Background: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22.
Objectives: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations.
Methods: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study.
Findings: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03.
Main conclusions: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.
期刊介绍：
Memórias do Instituto Oswaldo Cruz is a journal specialized in microbes & their vectors causing human infections. This means that we accept manuscripts covering multidisciplinary approaches and findings in the basic aspects of infectious diseases, e.g. basic in research in prokariotes, eukaryotes, and/or virus. Articles must clearly show what is the main question to be answered, the hypothesis raised, and the contribution given by the study.
Priority is given to manuscripts reporting novel mechanisms and general findings concerning the biology of human infectious prokariotes, eukariotes or virus. Papers reporting innovative methods for diagnostics or that advance the basic research with these infectious agents are also welcome.
It is important to mention what we do not publish: veterinary infectious agents research, taxonomic analysis and re-description of species, epidemiological studies or surveys or case reports and data re-analysis. Manuscripts that fall in these cases or that are considered of low priority by the journal editorial board, will be returned to the author(s) for submission to another journal.
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