Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development

<i>Plasmodium falciparum</i>, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for par...

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Autores principales:	Carolina C. Hoff, Mauro F. Azevedo, Adriana B. Thurler, Sarah El Chamy Maluf, Pollyana M. S. Melo, Maday Alonso del Rivero, Jorge González-Bacerio, Adriana K. Carmona, Alexandre Budu, Marcos L. Gazarini
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Plasmodium falciparum</i> malaria M1 alanyl-aminopeptidase antimalarial PfA-M1 overexpression Medicine R
Acceso en línea:	https://doaj.org/article/a0d20a6310e044c2a2d8837dadf331db
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spelling	oai:doaj.org-article:a0d20a6310e044c2a2d8837dadf331db2021-11-25T18:38:22ZOverexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development10.3390/pathogens101114522076-0817https://doaj.org/article/a0d20a6310e044c2a2d8837dadf331db2021-11-01T00:00:00Zhttps://www.mdpi.com/2076-0817/10/11/1452https://doaj.org/toc/2076-0817<i>Plasmodium falciparum</i>, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of <i>P. falciparum</i> overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in <i>P. falciparum</i>.Carolina C. HoffMauro F. AzevedoAdriana B. ThurlerSarah El Chamy MalufPollyana M. S. MeloMaday Alonso del RiveroJorge González-BacerioAdriana K. CarmonaAlexandre BuduMarcos L. GazariniMDPI AGarticle<i>Plasmodium falciparum</i>malariaM1 alanyl-aminopeptidaseantimalarialPfA-M1 overexpressionMedicineRENPathogens, Vol 10, Iss 1452, p 1452 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Plasmodium falciparum</i> malaria M1 alanyl-aminopeptidase antimalarial PfA-M1 overexpression Medicine R
spellingShingle	<i>Plasmodium falciparum</i> malaria M1 alanyl-aminopeptidase antimalarial PfA-M1 overexpression Medicine R Carolina C. Hoff Mauro F. Azevedo Adriana B. Thurler Sarah El Chamy Maluf Pollyana M. S. Melo Maday Alonso del Rivero Jorge González-Bacerio Adriana K. Carmona Alexandre Budu Marcos L. Gazarini Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
description	<i>Plasmodium falciparum</i>, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of <i>P. falciparum</i> overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in <i>P. falciparum</i>.
format	article
author	Carolina C. Hoff Mauro F. Azevedo Adriana B. Thurler Sarah El Chamy Maluf Pollyana M. S. Melo Maday Alonso del Rivero Jorge González-Bacerio Adriana K. Carmona Alexandre Budu Marcos L. Gazarini
author_facet	Carolina C. Hoff Mauro F. Azevedo Adriana B. Thurler Sarah El Chamy Maluf Pollyana M. S. Melo Maday Alonso del Rivero Jorge González-Bacerio Adriana K. Carmona Alexandre Budu Marcos L. Gazarini
author_sort	Carolina C. Hoff
title	Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
title_short	Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
title_full	Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
title_fullStr	Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
title_full_unstemmed	Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development
title_sort	overexpression of <i>plasmodium falciparum</i> m1 aminopeptidase promotes an increase in intracellular proteolysis and modifies the asexual erythrocytic cycle development
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/a0d20a6310e044c2a2d8837dadf331db
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Overexpression of <i>Plasmodium falciparum</i> M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development

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