Abstract
Plasmodium falciparum, 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 P. falciparum 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 P. falciparum.
Highlights
Malaria is a disease caused by parasites of the genus Plasmodium, which affects millions of people worldwide, being responsible for hundreds of thousands of deaths each year [1]
Some metalloaminopeptidase inhibitors have been tested in vitro and precluded parasite growth, highlighting the importance of these proteases as targets for the development of antimalarials [7,26,27,28,29] The cellular localization of PfA-M1 has been reported in different subcellular spaces such as cytosol, nucleus, and food vacuole [11,30,31,32]. As both calcium and aminopeptidases play crucial roles in Plasmodium, and calcium can be stored in the acidic compartment, where hemoglobin is hydrolyzed, we aimed to investigate the interplay between calcium and PfA-M1 in the P. falciparum intraerythrocytic cycle progression
E64d, a cysteine protease inhibitor [49] led to an increase in Ala-AMC and Met-AMC hydrolysis (Figure 6). These results suggest that the aminopeptidase activity in P. falciparum is altered by changes in the intracellular substrate pool after the treatment with cysteine proteases or calmodulin inhibitors, which is completely different for untreated parasites, allowing a favorable hydrolysis of non-native substrates, which were not normally available to PfA-M1
Summary
Malaria is a disease caused by parasites of the genus Plasmodium, which affects millions of people worldwide, being responsible for hundreds of thousands of deaths each year [1]. Five protozoan species are known to infect humans (reviewed in [2]); the majority of malaria-related deaths are caused by P. falciparum. The major feature that contributes to the severity of the disease caused by these parasite species is the cytoadherence to the endothelial cells of capillaries, which in turn leads to the clogging of brain microvasculature (reviewed in [3]). The infected female mosquito inoculates sporozoites in the dermis of the human host [5], which reach the bloodstream and infect liver cells. The resulting liver merozoites inside a merosome enter the bloodstream and infect erythrocytes inside which they undergo differentiation into ring, trophozoite, and schizont forms. The resulting merozoites burst the red blood cell and reinvade new erythrocytes, repeating the intraerythrocytic cycle (reviewed in [3]).
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