ABSTRACT
In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.
Subject(s)
Malaria, Vivax , Plasmodium vivax , Brazil , Catalytic Domain , Genetic Variation/genetics , Humans , Plasmodium vivax/genetics , Protozoan Proteins/geneticsABSTRACT
BACKGROUND: The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. METHODS: We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. RESULTS: The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. CONCLUSIONS: Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.
Subject(s)
Cell Proliferation/drug effects , Diphosphonates/pharmacology , Geranyltranstransferase/chemistry , Giardia/enzymology , Giardia/ultrastructure , Leishmania/enzymology , Leishmania/ultrastructure , Amino Acids/genetics , Cell Death/drug effects , Cell Survival/drug effects , Geranyltranstransferase/antagonists & inhibitors , Giardia/drug effects , Inhibitory Concentration 50 , Leishmania/drug effects , Microscopy, Electron, Transmission , Phylogeny , Sequence Alignment , Structure-Activity RelationshipABSTRACT
A leishmaniose é um problema de saúde pública em diversas partes do mundo, devido a sua ampla distribuição e alta prevalência. Os principais fatores de risco resultantes de processos sociais, econômicos e ambientais facilitam a transmissão e dificultam seu controle. A infecção causada por parasitas do gênero Leishmania pode causar no ser humano um conjunto de sintomas. O tratamento atualmente empregado é extremamente tóxico, pode produzir resistência e possui alto custo para o paciente, o que limita a sua utilização em áreas endêmicas. Desta forma, é de grande relevância a identificação de novos alvos terapêuticos com importância crítica na sobrevivência do parasito visando ao desenvolvimento de novos fármacos mais eficazes e menos agressivos para os seres humanos. Neste contexto, a enzima fumarato hidratase (FH) surge como um alvo molecular promissor, uma vez que a FH de L. major e de H. sapiens são consideradas análogas funcionais
Estudos recentes mostram a importância desta enzima para a viabilidade dos parasitos, o que a torna um alvo potencial para o planejamento de compostos com ação leishmanicida. O mecanismo de ação dessa enzima no parasito é diferente da atividade no organismo humano e, sua inibição prejudicaria os processos essenciais para a sobrevivência do parasito. A comparação entre as sequências da fumarato hidratase de classe I entre algumas espécies do gênero Leishmania apontou que os resíduos do sítio catalítico permanecem totalmente conservados, o que sugere a possível inibição da enzima para várias espécies desse gênero. A análise da estrutura da enzima do parasita e do hospedeiro mostrou diferenças nos resíduos catalíticos envolvidos na reação, reforçando a ideia de uma possível inibição específica. (AU)
