Effect of the anti-microtubule compound tubulozole on Leishmania protozoan parasites in vitro

Abstract For the cis and trans stereoisomers of the synthetic anti-microtubule compound tubulozole, at micromolar concentrations, tubulozole-C is cytotoxic to mammalian cells whereas tubulozole-T is not. The effect of tubulozoles on the parasitic protozoan Leishmania was tested. For the promastigote stage of L. mexicana amazonensis , both isomers inhibited parasite growth. For the amastigote stage of L. mexicana amazonensis and L. major , within murine J774 macrophage line as host cells in vitro, tubulozole-T reduced the infective index. Despite the observation of macrophage cytotoxicity of tubulozole-T, this compound may be a potentially useful and novel anti-leishmanial drug.     

Effect of the microtubule stabilising agent taxol on leishmanial protozoan parasites in vitro

Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.     

Prostaglandin derivatives inhibit the growth of malarial parasites in mice

New prostaglandin oligomeric derivatives, termed MR-256 and MR-356, were found to inhibit the growth of murine malarial parasites, P. chabaudi and P. vinckei, within red blood cells in vivo. When mice were infected with P. chabaudi, both MR-256 and MR-356 suppressed the growth of parasites, but MR-356 had a greater inhibitory effect than MR-256. With P. vinckei, MR-356 also inhibited the growth of parasites, and improved the survival rate. The effect of MR-256 was much less. A possible inhibitory mechanism of action of these drugs is discussed.     

Phosphofructokinase of Leishmania donovani and Leishmania braziliensis and its Role in Glycolysis*

SYNOPSIS. It was shown in an investigation of the phosphofructokinases of Leishmania donovani and Leishmania braziliensis that both enzymes are similar to that of Crithidia fasciculata. Although the enzymes are allosteric with respect to their substrates and require AMP for activation, there is no influence by other heterotropic modifiers. The Mg²⁺-ATP chelate activates these enzymes in a first order process and they can be inhibited by free ATP. The inhibition is reversed by the activator, AMP, in a competitive manner. The requirement for the nucleotide in L. donovani can be eliminated by decreasing the pH. The data indicate that phosphofructokinase, a pivotal enzyme in glycolysis for most organisms, probably does not play an important role in glycolysis in Leishmania.     

Complex mutations in a high proportion of microsatellite loci from the protozoan parasite Plasmodium falciparum

Microsatellite loci are generally assumed to evolve via a stepwise mutational process and a battery of statistical techniques has been developed in recent years based on this or related mutation models. It is therefore important to investigate the appropriateness of these models in a wide variety of taxa. We used two approaches to examine mutation patterns in the malaria parasite Plasmodium falciparum: (i) we examined sequence variation at 12 tri-nucleotide repeat loci; and (ii) we analysed patterns of repeat structure and heterozygosity at 114 loci using data from 12 laboratory parasite lines. The sequencing study revealed complex patterns of mutation in five of the 12 loci studied. Alleles at two loci contain indels of 24 bp and 57 bp in flanking regions, while in the other three loci, blocks of imperfect microsatellites appear to be duplicated or inserted; these loci essentially consist of minisatellite repeats, with each repeat unit containing four to eight microsatellites. The survey of heterozygosity revealed a positive relationship between repeat number and microsatellite variability for both di- and trinucleotides, indicating a higher mutation rate in loci with longer repeat arrays. Comparisons of levels of variation in different repeat types indicate that the mutation rate of dinucleotide-bearing loci is 1.6-2.1 times faster than trinucleotides, consistent with the lower mean number of repeats in trinucleotide-bearing loci. However, despite the evidence that microsatellite arrays themselves are evolving in a manner consistent with stepwise mutation model in P. falciparum, the high frequency of complex mutations precludes the use of analytical tools based on this mutation model for many microsatellite-bearing loci in this protozoan. The results call into question the generality of models based on stepwise mutation for analysing microsatellite data, but also demonstrate the ease with which loci that violate model assumptions can be detected using minimal sequencing effort.     

Imaging ion flux and ion homeostasis in blood stage malaria parasites

The steady-state regulation of intracellular levels of essential ions and ionic gradients is critical for almost all functions within a cell. Thus, it is not surprising to find that ions have been shown to play an important role in numerous parasitic processes, such as invasion, development and possibly drug resistance mechanisms. Live cell imaging has become a widespread technique to visualize and quantify several of these processes, including pH and Ca²⁺ homeostasis, in an effort to better understand the biology and physiology of cells. This is now also the case for many human pathogens. The aim of this review is to emphasize the importance of this technique and provide an overview of what we have learned so far, using the malaria parasite Plasmodium falciparum as a paradigm.     

Lipid synthesis in protozoan parasites: A comparison between kinetoplastids and apicomplexans

Lipid metabolism is of crucial importance for pathogens. Lipids serve as cellular building blocks, signalling molecules, energy stores, posttranslational modifiers, and pathogenesis factors. Parasites rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Here we discuss the tremendous recent advances that have been made in the understanding of the synthesis and uptake pathways for fatty acids and phospholipids in apicomplexan and kinetoplastid parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania. Lipid synthesis differs in significant ways between parasites from both phyla and the human host. Parasites have acquired novel pathways through endosymbiosis, as in the case of the apicoplast, have dramatically reshaped substrate and product profiles, and have evolved specialized lipids to interact with or manipulate the host. These differences potentially provide opportunities for drug development. We outline the lipid pathways for key species in detail as they progress through the developmental cycle and highlight those that are of particular importance to the biology of the pathogens and/or are the most promising targets for parasite-specific treatment.     

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodiumfalciparumblood forms (W2 clone, chloroquine-resistant) oftamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl(ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 humanhepatoma cells. Surprisingly with these series, results indicate that the biologicalactivity of ruthenocifens is better than that of ferrocifens and other tamoxifen-likecompounds. The synthesis of a new metal-based compound is also described. It wasshown, for the first time, that ruthenocifens are good antiplasmodial prototypes.Further studies will be conducted aiming at a better understanding of their mechanismof action and at obtaining new compounds with better therapeutic profile.     

Evolution of gametocyte sex ratios in malaria and related apicomplexan (protozoan) parasites

'Survival of the fittest' is usually interpreted to mean that natural selection favours genes that maximize their transmission to the next generation. Here, we discuss recent applications of this principle to the study of gametocyte sex ratios in malaria and other apicomplexan parasites. Sex ratios matter because they are an important determinant of fitness and transmission success -- and hence of disease epidemiology and evolution. Moreover, inbreeding rates can be estimated from gametocyte sex ratios. The sex ratio is also an excellent model trait for testing the validity of important components of what is being marketed as 'Darwinian medicine'.     

Coumarins inhibit η-class carbonic anhydrase from Plasmodium falciparum

Coumarins were discovered to act as inhibitors of α-carbonic anhydrases (CAs, EC 4.2.1.1) after undergoing hydrolysis mediated by the esterase activity of the enzyme to the corresponding 2-hydroxycinnamic acids. Other classes of CAs among the eight currently known do not possess esterase activity or this activity was poorly investigated. Hence, we decided to look at the potential of coumarins as inhibitors of the η-CA from the malaria-producing protozoan Plasmodium falciparum, PfaCA. A panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system acted as low to medium micromolar PfaCA inhibitors, whereas their affinities for the cytosolic off-target human isoforms hCA I and II were in a much higher range. Thus, we confirm that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective PfaCA inhibitors.     

Single-genome sequencing reveals within-host evolution of human malaria parasites

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PERMANENT GENETIC RESOURCES: A set of 12 microsatellite loci for genetic studies of Leishmania braziliensis

Twelve microsatellite loci of Leishmania braziliensis were examined, nine of which were developed in this work. Fifty‐six Leishmania braziliensis were genotyped with these microsatellite loci. The 12 loci studied were polymorphic with the number of alleles ranging from five to 19, with a mean of 9.7 ± 4.1 and the observed heterozygosity averaging 0.425 ± 0.202. The important heterozygote deficits we observed (F_IS = 0.41, P value = 0.004) appear incompatible with the heterozygote excess expected in clonal diploids. This last result could revive the clonality/sexuality debate regarding Leishmania. This work validates the potential use of these microsatellites for population genetics analysis.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.     

Development of a conditional localization approach to control apicoplast protein trafficking in malaria parasites

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2‐HCS1, we were able to control protein biotinylation in the apicoplast in a ligand‐dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites.     

Interaction of sitamaquine with membrane lipids of Leishmania donovani promastigotes

Sitamaquine is an 8-aminoquinoline which is active by the oral route for the treatment of life-threatening visceral leishmaniasis caused by Leishmania donovani, with an IC₅₀ of 29.2 μM against the promastigote form in vitro. At high concentration (100 μM), sitamaquine affected parasite motility, morphology and growth in a way that was only partially reversible. As a first approach to determine its mechanism of action, we describe the interaction of sitamaquine with parasite membrane components, representing the first barrier to be crossed by the drug. Analysis of the physicochemical interactions of sitamaquine with monolayers of phospholipids and sterols at the air-water interface showed that these interactions only occurred in the presence of anionic phospholipids. Thus, electrostatic interactions between positively charged sitamaquine and the negative polar headgroups are a pre-requisite for subsequent hydrophobic interactions between the sitamaquine aromatic ring and the alkyl chains of phospholipids leading to drug insertion into the monolayer.     

Interactions of human malaria parasites, Plasmodium wVaxand P.falciparum, with the midgut of Anopheles mosquitoes

Abstract Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and P.falciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement. The passage of the P. vivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in P.falciparum requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for P.vivax and P.falciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector-parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.     

Iso-mukaadial acetate and ursolic acid acetate inhibit the chaperone activity of Plasmodium falciparum heat shock protein 70-1

Plasmodium falciparum is the most lethal malaria parasite. The present study investigates the interaction capabilities of select plant derivatives, iso-mukaadial acetate (IMA) and ursolic acid acetate (UAA), against P. falciparum Hsp70-1 (PfHsp70-1) using in vitro approaches. PfHsp70-1 facilitates protein folding in the parasite and is deemed a prospective antimalarial drug target. Recombinant PfHsp70-1 protein was expressed in E. coli BL21 cells and homogeneously purified by affinity chromatography. The interaction between the compounds and PfHsp70-1 was evaluated using malate dehydrogenase (MDH), and luciferase aggregation assay, ATPase activity assay, and Fourier transform infrared (FTIR). PfHsp70-1 prevented the heat-induced aggregation of MDH and luciferase. However, the PfHsp70-1 chaperone role was inhibited by IMA or UAA, leading to both MDH and luciferase’s thermal aggregation. The basal ATPase activity of PfHsp70-1 (0.121 nmol/min/mg) was closer to UAA (0.131 nmol/min/mg) (p = 0.0675) at 5 mM compound concentration, suggesting that UAA has no effect on PfHsp70-1 ATPase activity. However, ATPase activity inhibition was similar between IMA (0.068 nmol/min/mg) (p < 0.0001) and polymyxin B (0.083 nmol/min/mg) (p < 0.0001). The lesser the Pi values, the lesser ATP hydrolysis observed due to compound binding to the ATPase domain. FTIR spectra analysis of IMA and UAA resulted in PfHsp70-1 structural alteration for β-sheets shifting the amide I band from 1637 cm⁻¹ to 1639 cm⁻¹, and for α-helix from 1650 cm⁻¹ to 1652 cm⁻¹, therefore depicting secondary structural changes with an increase in secondary structure percentage suggesting that these compounds interact with PfHsp70-1.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01212-6.     

Trisubstituted thiazoles as potent and selective inhibitors of Plasmodium falciparum protein kinase G (PfPKG)

A series of trisubstituted thiazoles have been identified as potent inhibitors of Plasmodium falciparum (Pf) cGMP-dependent protein kinase (PfPKG) through template hopping from known Eimeria PKG (EtPKG) inhibitors. The thiazole series has yielded compounds with improved potency, kinase selectivity and good in vitro ADME properties. These compounds could be useful tools in the development of new anti-malarial drugs in the fight against drug resistant malaria.