Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia

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 an...

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Published in:	Parasites & vectors Vol. 13; no. 1; p. 168
Main Authors:	Gadelha, Ana Paula R, Brigagao, Claudia Maia, da Silva, Martha Barros, Rodrigues, Aline Beatriz Mello, Guimarães, Ana Carolina Ramos, Paiva, Fernando, de Souza, Wanderley, Henriques, Cristina
Format:	Journal Article
Language:	English
Published:	England BioMed Central Ltd 05-04-2020 BioMed Central BMC
Subjects:	Alendronate Alendronic acid Alignment Amino acids Analysis Apoptosis Aspartate Biosynthesis Bisphosphonates Cell death Cell viability Cholesterol Electron microscopy Enzymes Ergosterol Eukaryotes Evaluation Farnesyl diphosphate synthase Flow cytometry FPPS Giardia Ibandronic acid Leishmania Mammals Maximum likelihood method Membrane potential Membranes Metabolism Metabolites Mitochondria Modelling Myelin Nitrogen Nucleotide sequence Parasitic diseases Phylogenetics Phylogeny Physiological aspects Phytosterols Plant biochemistry Proliferation Promastigotes Proteins Protozoan Risedronic acid Sequencing Sterols Terpenes Three dimensional models Transmission electron microscopy Trophozoites Tropical diseases Ultrastructure Brazil Ethiopia Sudan Ergosterol Leishmania Farnesyl diphosphate synthase Protozoan Giardia FPPS Isoprenoid Bisphosphonates Isoprenylation Sterol
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Summary:	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. 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 IC via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC of 13.8 µM, followed by ibandronate and alendronate with IC values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC 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. 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1756-3305 1756-3305
DOI:	10.1186/s13071-020-04019-z