Autophagy-related protein PfATG18 participates in food vacuole dynamics and autophagy-like pathway in Plasmodium falciparum.

Plasmodium falciparum has a limited repertoire of autophagy-related genes (ATGs), and the functions of various proteins of the autophagy-like pathway are not fully established in this protozoan parasite. Studies suggest that some of the autophagy proteins are crucial for parasite growth. PfATG18, for example, is essential for parasite replication and has a noncanonical role in apicoplast biogenesis. In this study, we demonstrate the conserved functions of PfATG18 in food vacuole (FV) dynamics and autophagy. Intriguingly, the P. falciparum FV is found to undergo fission and fusion and PfATG18 gets enriched at the interfaces of the newly generated multilobed FV during the process. In addition, expression of PfATG18 is induced upon starvation, both at the mRNA and protein level indicating its participation in the autophagy-like pathway, which is independent of its role in apicoplast biogenesis. The study also shows that PfATG18 is transported to the FV via the haemoglobin trafficking pathway. Overall, this study establishes the conserved functions of Atg18 in this important apicomplexan.

Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii.

The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of 'tethered' daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division.

Centromere and its associated proteins-what we know about them in Plasmodium falciparum.

The complex life cycle of intracellular parasitic protozoans entails multiple rounds of DNA replication and mitosis followed by cytokinesis to release daughter parasites. To gain insights into mitotic events it is imperative to identify the biomarkers that constitute the chromosome segregation machinery in the parasite. Chromosomal loci called centromeres and their associated proteins play an essential role in accurate chromosome segregation. Although new information on the centromere-kinetochore proteins has been added to the existing pool of knowledge, a paucity of biomarkers for nuclear division prevents a global view of chromosome segregation mechanism in the malaria parasite. In Plasmodium falciparum, except CENH3 and CENP-C homologues, other centromere associated proteins responsible for centromere functions and kinetochore assembly are not known. The focus of this review is to summarize the current understanding on the centromere organization and its associated proteins in eukaryotes with the emerging information in P. falciparum. © 2018 IUBMB Life, 70(8):732-742, 2018.

Drug resistance genes: pvcrt-o and pvmdr-1 polymorphism in patients from malaria endemic South Western Coastal Region of India.

BACKGROUND: Malaria is highly prevalent in many parts of India and is mostly caused by the parasite species Plasmodium vivax followed by Plasmodium falciparum. Chloroquine (CQ) is the first-line treatment for blood stage P. vivax parasites, but cases of drug resistance to CQ have been reported from India. One of the surveillance strategies which is used to monitor CQ drug resistance, is the analysis of single nucleotide polymorphisms (SNPs) of the associated gene markers. Susceptibility to CQ can also be determined by copy number assessment of multidrug resistant gene (mdr-1). The current study has examined the prevalence of SNPs in P. vivax orthologs of P. falciparum chloroquine resistant and multi-drug resistant genes (pvcrt-o and pvmdr-1, respectively) and pvmdr-1 copy number variations in isolates from the highly endemic Mangaluru city near the South Western Coastal region of India. METHODS: A total of 140 blood samples were collected from P. vivax infected patients attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of these 140 samples, sequencing was carried out for 54 (38.5%) and 85 (60.7%) isolates for pvcrt-o and pvmdr-1, respectively. Single nucleotide polymorphisms (SNPs) in the pvcrt-o and pvmdr-1 genes were analysed by direct sequencing method, while copy number variations of 60 isolates (42. 8%) were determined by real time PCR. RESULTS: Out of 54 clinical isolates analysed for pvcrt-o, three (5.6%) showed K10 insertion and the rest had wild type sequence. This is the first report to show K10 insertion in P. vivax isolates from India. Further, out of 85 clinical isolates of P. vivax analysed for mutations in pvmdr-1 gene, only one isolate had wild type sequence (~ 1%) while the remaining (99%) carried mutant alleles. Seven non-synonymous mutations with two novel mutations (I946V and Y1028C) were observed. Of all the observed mutations in pvmdr-1 gene, T958M was most highly prevalent (present in 90% of samples) followed by F1076L (76%), and Y976F (7%). Amplification of pvmdr-1 gene was observed in 31.6% of the isolates, out of 60 amplified. CONCLUSION: The observed variations both in pvmdr-1 and pvcrt-o genes indicate a trend towards parasite acquiring CQ resistance in this endemic area.

Presence of novel triple mutations in the pvdhfr from Plasmodium vivax in Mangaluru city area in the southwestern coastal region of India.

BACKGROUND: Genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) are the targets of sulfadoxine-pyrimethamine (SP) present in artemisinin based combination therapy (ACT; artesunate + sulfadoxine pyrimethamine) for Plasmodium falciparum. Although SP is generally not used to treat vivax infection, mutations in dhfr and dhps that confer antifolate resistance in Plasmodium vivax are common; which may be attributed to its sympatric existence with P. falciparum. Current study was aimed to determine the pattern of mutations in dhfr and dhps in P. vivax isolates from Mangaluru region. METHODS: A total of 140 blood samples were collected from P. vivax-infected people attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of 140 isolates, 25 (18%) and 50 (36%) isolates were selected randomly for sequence analysis of pvdhfr and pvdhps genes respectively. Fragment of pvdhps and full length pvdhfr were amplified, sequenced and analysed for single nucleotide polymorphisms. dhps was analysed by PCR-RFLP also, to detect the two specific mutations (A383G and A553G). RESULTS: Analysis of pvdhps sequences from 50 isolates revealed single and double mutants at 38 and 46% respectively. Three non-synonymous mutations (K55R, S58R and S117N) were identified for pvdhfr. Among these, K55R was detected for the first time. CONCLUSIONS: The current study indicates that P. vivax dhps and dhfr mutant alleles are prevalent in this area, suggesting significant SP pressure.

The dimerization domain of PfCENP-C is required for its functions as a centromere protein in human malaria parasite Plasmodium falciparum.

BACKGROUND: The conserved centromere-associated proteins, CENH3 (or CENP-A) and CENP-C are indispensable for the functional centromere-kinetochore assembly, chromosome segregation, cell cycle progression, and viability. The presence and functions of centromere proteins in Plasmodium falciparum are not well studied. Identification of PfCENP-C, an inner kinetochore protein (the homologue of human CENP-C) and its co-localization with PfCENH3 was recently reported. This study aims to decipher the functions of inner kinetochore protein, PfCENP-C as a centromere protein in P. falciparum. METHODS: Bio-informatic tools were employed to demarcate the two conserved domains of PfCENP-C, and the functions of PfCENP-C domains were demonstrated by functional complementation assays in the temperature sensitive (TS) mutant strains (mif2-3 and mif2-2) of Saccharomyces cerevisiae with MIF2p (the yeast homologue of CENP-C) loss-of-function. By site-directed mutagenesis, the key residues essential for PfCENP-C functions were determined. The chromatin immunoprecipitation was carried out to determine the in vivo binding of PfCENP-C to the Plasmodium centromeres and the in vivo interactions of PfCENP-C with PfCENH3, and mitotic spindles were shown by co-immunopreciptation experiments. RESULTS: The studies demonstrate that the motif and the dimerization domain of PfCENP-C is able to functionally complement MIF2p functions. The essential role of some of the key residues: F1993, F1996 and Y2069 within the PfCENP-C dimerization domain in mediating its functions and maintenance of mitotic spindle integrity is evident from this study. The pull-down assays show the association of PfCENP-C with PfCENH3 and mitotic spindles. The ChIP-PCR experiments confirm PfCENP-C-enriched Plasmodium centromeres. These studies thus provide an insight into the roles of this inner kinetochore protein and establish that the centromere proteins are evolutionary conserved in the parasite. CONCLUSIONS: PfCENP-C is a true CENP-C homologue in P. falciparum which binds to the centromeric DNA and its dimerization domain is essential for its in vivo functions as a centromere protein. The identification and functional characterization of the P. falciparum centromeric proteins will provide mechanistic insights into some of the mitotic events that occur during the chromosome segregation in human malaria parasite, P. falciparum.

The many paths to artemisinin resistance in Plasmodium falciparum.

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity.

15-Deoxyspergualin hinders physical interaction between basic residues of transit peptide in PfENR and Hsp70-1.

The apicoplast of Plasmodium harbors several metabolic pathways. The enzymes required to perform these reactions are all nuclearly encoded and apicoplast targeted (NEAT) proteins. Plasmodium falciparum Enoyl-ACP Reductase (PfENR) is one such NEAT protein. The NEAT proteins have a transit peptide which is required for crossing the membranes of apicoplast. We studied the importance of basic residues like Arginine and Lysine within the transit peptide. Previous studies have suggested that all basic residues are essential for apicoplast trafficking. In this study, we demonstrate that only some of these residues are essential (K44, R48, K51, and R52), whereas others are dispensable (R40, K42, and K49). On mutating these specific residues, PfENR is not imported into the apicoplast and is mislocalized to the cytoplasm. We also demonstrate that these residues are also crucial for interaction with Hsp70-1, implying that interactions of Lysine 44, Arginine 48, Lysine 51, and Arginine 52 of the transit peptide with PfHsp70-1 are required for apicoplast trafficking. 15-Deoxyspergualin, which has earlier been proposed to interact with EEVD motif of PfHsp70-1 hinders the physical interaction between these cationic residues of PfENR and Hsp70-1. Hence, we propose that in the transport competent state of NEAT proteins some specific positively charged amino acids in the transit peptide interact with PfHsp70-1, and this interaction is essential for apicoplast targeting.

Autophagy Underlies the Proteostasis Mechanisms of Artemisinin Resistance in P. falciparum Malaria.

Emerging resistance to artemisinin (ART) has become a challenge for reducing worldwide malaria mortality and morbidity. The C580Y mutation in Plasmodium falciparum Kelch13 has been identified as the major determinant for ART resistance in the background of other mutations, which include the T38I mutation in autophagy-related protein PfATG18. Increased endoplasmic reticulum phosphatidylinositol-3-phosphate (ER-PI3P) vesiculation, unfolded protein response (UPR), and oxidative stress are the proteostasis mechanisms proposed to cause ART resistance. While UPR and PI3P are known to stimulate autophagy in higher organisms to clear misfolded proteins, participation of the parasite autophagy machinery in these mechanisms of ART resistance has not yet been experimentally demonstrated. Our study establishes that ART-induced ER stress leads to increased expression of P. falciparum autophagy proteins through induction of the UPR. Furthermore, the ART-resistant K13C580Y isolate shows higher basal expression levels of autophagy proteins than those of its isogenic counterpart, and this magnifies under starvation conditions. The copresence of PfK13 with PfATG18 and PI3P on parasite hemoglobin-trafficking vesicles demonstrate interactions between the autophagy and hemoglobin endocytosis pathways proposed to be involved in ART resistance. Analysis of PfK13 mutations in 2,517 field isolates, revealing an impressive >85% coassociation between PfK13 C580Y and PfATG18 T38I, together with our experimental studies with an ART-resistant P. falciparum strain establishes that parasite autophagy underpins various mechanisms of ART resistance and is a starting point to further explore this pathway for developing antimalarials. IMPORTANCE There is an urgent need to clearly understand the mechanisms of ART resistance as it is emerging in the Greater Mekong Subregion (GMS) and other parts of the world, such as Africa. Deciphering the mechanisms of the parasite's stress response pathways of ART resistance will provide insights to identify novel drug targets for developing new antimalarial regimens.

Structural basis for the functional and inhibitory mechanisms of ß-hydroxyacyl-acyl carrier protein dehydratase (FabZ) of Plasmodium falciparum.

The ß-hydroxyacyl-acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ is available in hexameric (active) and dimeric (inactive) forms. However, PfFabZ has not been crystallized with any bound inhibitors until now. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of four of these complexes. This is the first report on any FabZ enzyme with active site inhibitors that interact directly with the catalytic residues. Inhibitor binding not only stabilized the substrate binding loop but also revealed that the substrate binding tunnel has an overall shape of "U". In the crystal structures, residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed. Thus, Phe169, merely by changing its side chain conformation, appears to be controlling the length of the tunnel to make it suitable for accommodating longer substrates. The volume of the substrate binding tunnel is determined by the sequence as well as by the conformation of the substrate binding loop region and varies between organisms for accommodating fatty acids of different chain lengths. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms.

Benzothiophene carboxamide derivatives as novel antimalarials.

Bromo-benzothiophene carboxamide derivatives have been shown in the preceding article to inhibit Plasmodium falciparum Enoyl-ACP reductase. Here, we report bromo-benzothiophene carboxamide derivatives as potent inhibitors of Plasmodium asexual blood-stages in vitro as well as in vivo in the mouse model. These compounds specifically impair the development of metabolically active trophozoite stage of intraerythrocytic cycle and the intravenous administration of 3-bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) enhances the longevity of P. berghei infected mice by 2 weeks compared to disease control animals thereby preventing the onset of ataxia and convulsions in treated mice. These compounds thus hold promise for the development of potent antimalarials.

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum.

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.

Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl-ACP reductase.

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (Ki) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.

SAR and pharmacophore models for the rhodanine inhibitors of Plasmodium falciparum enoyl-acyl carrier protein reductase.

Significance of type II fatty acid synthase pathway in the life cycle of malarial parasite has long been established. Enoyl acyl carrier protein (ACP) reductase of Plasmodium falciparum (PfENR) is the rate determining enzyme of its elongation module. Hence, PfENR has been a target for the development of antimalarials as well as vaccines. Towards this endeavour, we had recently identified rhodanine class of compounds as inhibitors of PfENR. Here, we report a number of new inhibitors belonging to this class. These inhibitors have been divided into two broad subclasses: rhodanine-furans and rhodanine-phenyls. The inhibitory activity of all compounds was determined against purified PfENR. IC(50) of these compounds were found to be in nanomolar to low-micromolar range. The structure-activity relationship of both the classes has been explored in detail for the first time. Separate 3D pharmacophore models for this enzyme have been generated for both rhodanine furans and phenyls. The pharmacophore model for rhodanine furan has a Hydrogen bond donor, two Hydrogen bond acceptors, two metal ligators, three hydrophobic, and two aromatic ring features, whereas the pharmacophore model for the phenyl subclass has two hydrogen bond donors, two hydrogen bond acceptor, a metal ligator, two hydrophobic, and two aromatic ring features. These models could be used for in silico screening of compound libraries for PfENR inhibitors.

X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition.

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors.

Partial molar volumes of acyl carrier proteins are related to their states of acylation.

Acyl carrier protein (ACP), an abundant protein in every cell, plays a central role in a number of metabolic processes requiring acyl group transfer. Conformational flexibility while crucial for its function remains substantially unaddressed. By dual polarization interferometry we establish correlation between the chain length of aliphatic groups covalently linked to Escherichia coli and Plasmodium falciparum ACP and their respective partial molar volumes in solution which helps to subserve the aforesaid goal.

Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

BACKGROUND: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. RESULTS: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. CONCLUSION: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Triclosan inhibit the growth of the late liver-stage of Plasmodium.

Annually, approximately two million human deaths are caused worldwide by malaria, most of them being children. Plasmodium falciparum is the leading cause of cerebral malaria, the most severe and fatal form of disease. Moreover, the emergence of resistant strains to the existing drugs has worsened the situation. Currently, primaquine is the only drug available for eliminating liver-stage parasites. Because of the emergence of resistant parasite strains, it becomes necessary to find new targets unique to the malaria parasites. In the Plasmodium species, the discovery of a distinct Type-II fatty-acid synthesis pathway has created an opportunity to target this pathway for the development of new inhibitors of malaria parasite growth. The present study explored the growth inhibition potential of triclosan in the case of liver-stage parasites. Liver-stage of Plasmodium is an excellent target for intervention due to very small parasite load as well as possibility of eliminating parasites before it can cause blood-stage infection. Here we report that triclosan inhibits the development of the Plasmodium liver-stage parasites.

Evolutionary significance of self-acylation property in acyl carrier proteins.

Acyl carrier protein is an integral component of many cellular metabolic processes. A number of studies have reported self-acylation behavior in acyl carrier proteins. Although ACPs exhibit high levels of similarity in their primary and tertiary structures, self-acylation behavior is restricted to only some ACPs that can be classified into two major families based on their function. The first family of ACPs is involved in polyketide biosynthesis, whereas the second family participates in fatty acid synthesis. Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were used in these studies to uncover as to how self-acylation behavior of acyl carrier proteins is linked with the evolution of metabolic pathways in organisms. These studies show that self-acylation behavior in acyl carrier proteins was lost during the course of evolution, with certain organisms and organelles viz. plastids, retaining it for specified functions.

Beta-ketoacyl-ACP synthase I/II from Plasmodium falciparum (PfFabB/F)--is it B or F?

Condensing enzymes play an important and decisive role in terms of fatty acid composition of any organism. They can be classified as condensing enzymes involved in initiating the cycle and enzymes involved in elongating the initiated fatty acyl chain. In E. coli, two isoforms for the elongation condensing enzymes (FabB and FabF) exists whereas Plasmodium genome contains only one isoform. By in vitro complementation studies in E. coli CY244 cells, we show that PfFabB/F functions like E. coli FabF as the growth of the mutant cells could be rescued only in the presence of oleic acid. But unlike bacterial enzyme, PfFabB/F does not increase the cis-vaccenic acid content in the mutant cells upon lowering the growth temperature. This study thus highlights the distinct properties of P. falciparum FabF which sets it apart from E. coli and most other enzymes of this family described so far.