A Plasmodium apicoplast-targeted unique exonuclease/FEN exhibits interspecies functional differences attributable to an insertion that alters DNA-binding.

The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.

Plasmodium falciparum ZIP1 Is a Zinc-Selective Transporter with Stage-Dependent Targeting to the Apicoplast and Plasma Membrane in Erythrocytic Parasites.

Replication of the malarial parasite in human erythrocytes requires massive zinc fluxes, necessitating the action of zinc transporters across the parasite plasma and organellar membranes. Although genetic knockout studies have been conducted on a few "orphan" zinc transporters in Plasmodium spp., none of them have been functionally characterized. We used the recombinant Plasmodium falciparum Zrt-/Irt-like protein (PfZIP1) and specific antibodies generated against it to explore the subcellular localization, function, metal-ion selectivity, and response to cellular zinc levels. PfZIP1 expression was enhanced upon the depletion of cytosolic Zn2+. The protein transitioned from the processed to unprocessed form through blood stages, localizing to the apicoplast in trophozoites and to the parasite plasma membrane in schizonts and gametocytes, indicating stage-specific functional role. The PfZIP1 dimer mediated Zn2+ influx in proteoliposomes. It exhibited preferential binding to Zn2+ compared to Fe2+, with the selectivity for zinc being driven by a C-terminal histidine-rich region conserved only in primate-infecting Plasmodium species.

Novel Baicalein-Derived Inhibitors of Plasmodium falciparum.

Malaria, a life-threatening mosquito-borne disease caused by Plasmodium parasites, continues to pose a significant global health burden. Despite notable progress in combating the disease in recent years, malaria remains prevalent in many regions, particularly in Southeast Asia and most of sub-Saharan Africa, where it claims hundreds of thousands of lives annually. Flavonoids, such as the baicalein class of compounds, are known to have antimalarial properties. In this study, we rationally designed and synthesized a series of baicalein derivatives and identified a lead compound, FNDR-10132, that displayed potent in vitro antimalarial activity against Plasmodium falciparum (P. falciparum), both chloroquine-sensitive (60 nM) and chloroquine-resistant (177 nM) parasites. FNDR-10132 was evaluated for its antimalarial activity in vivo against the chloroquine-resistant strain Plasmodium yoelii N67 in Swiss mice. The oral administration of 100 mg/kg of FNDR-10132 showed 44% parasite suppression on day 4, with a mean survival time of 13.5 ± 2.3 days vs. 8.4 ± 2.3 days of control. Also, FNDR-10132 displayed equivalent activity against the resistant strains of P. falciparum in the 200-300 nM range. This study offers a novel series of antimalarial compounds that could be developed into potent drugs against chloroquine-resistant malarial parasites through further chemistry and DMPK optimization.

Isolation and characterization of novel lytic bacteriophages that infect multi drug resistant clinical strains of Escherichia coli.

The pathogenic strains of Escherichia coli (E. coli) are frequent cause of urinary tract infections including catheter-associated, soft tissue infections and sepsis. The growing antibiotic resistance in E. coli is a major health concern. Bacteriophages are specific for their bacterial host, thus providing a novel and effective alternatives. This study focuses on isolation of bacteriophages from urban sewage treatment plants. Initially 50 different bacteriophages have been isolated against non-resistant reference E. coli strain and fifty multidrug resistant clinical isolates of extraintestinal infections. Out of which only thirty-one lytic phages which gave clear plaques were further analysed for different physico-chemical aspects such as thermal inactivation, pH, effect of organic solvents and detergents. Two bacteriophages, ASEC2201 and ASEC2202, were selected for their ability to withstand temperature fluctuation from -20 to 62 °C and a pH range from 4 to 10. They also showed good survival (40-94%) in the presence of organic solvents like ethanol, acetone, DMSO and chloroform or ability to form plaques even after the treatment with detergents like SDS, CTAB and sarkosyl. Both efficiently killed reference strain and 40-44% of multidrug resistant clinical isolates of E. coli. Later ASEC2201 and ASEC2202 were subjected to morphological characterisation through transmission electron microscopy, which revealed them to be tailed phages. The genomic analysis confirmed them to be Escherichia phages which belonged to family Drexlerviridae of Caudovirales.

Prediction of Putative Epitope Peptides against BaeR Associated with TCS Adaptation in Acinetobacter baumannii Using an In Silico Approach.

Background and Objectives: The BaeR protein is involved in the adaptation system of A. baumannii and is associated with virulence factors responsible for systemic infections in hospitalized patients. This study was conducted to characterize putative epitope peptides for the design of vaccines against BaeR protein, using an immune-informatic approach. Materials and Methods: FASTA sequences of BaeR from five different strains of A. baumannii were retrieved from the UNIPROT database and evaluated for their antigenicity, allergenicity and vaccine properties using BepiPred, Vaxijen, AlgPred, AntigenPro and SolPro. Their physio-chemical properties were assessed using the Expasy Protparam server. Immuno-dominant B-cell and T-cell epitope peptides were predicted using the IEDB database and MHC cluster server with a final assessment of their interactions with TLR-2. Results: A final selection of two peptide sequences (36aa and 22aa) was made from the 38 antigenic peptides. E1 was considered a soluble, non-allergenic antigen, and possessed negative GRAVY values, substantiating the hydrophilic nature of the proteins. Further analysis on the T-cell epitopes, class I immunogenicity and HLA allele frequencies yielded T-cell immuno-dominant peptides. The protein-peptide interactions of the TLR-2 receptor showed good similarity scores in terms of the high number of hydrogen bonds compared to other protein-peptide interactions. Conclusions: The two epitopes predicted from BaeR in the present investigation are promising vaccine candidates for targeting the TCS of A. baumannii in systemic and nosocomial infections. This study also demonstrates an alternative strategy to tackling and mitigating MDR strains of A. baumannii and provides a useful reference for the design and construction of novel vaccine candidates against this bacteria.

Prevalence of Bacterial Pathogens among Symptomatic-SARS-CoV-2 PCR-Negative Patients.

The epidemiological and clinical aspects of coronavirus disease-2019 (COVID-19) have been subjected to several investigations, but little is known about symptomatic patients with negative SARS-CoV-2 PCR results. The current study investigated patients who presented to the hospital with respiratory symptoms (but negative SARS-CoV-2 RT-PCR results) to determine the prevalence of bacterial pathogens among these patients. A total of 1246 different samples were collected and 453 species of bacterial pathogens were identified by culture. Antibiotic susceptibility testing was performed via the Kirby Bauer disc diffusion test. Patients showed symptoms, such as fever (100%), cough (83%), tiredness (77%), loss of taste and smell (23%), rigors (93%), sweating (62%), and nausea (81%), but all tested negative for COVID-19 by PCR tests. Further examinations revealed additional and severe symptoms, such as sore throats (27%), body aches and pain (83%), diarrhea (11%), skin rashes (5%), eye irritation (21%), vomiting (42%), difficulty breathing (32%), and chest pain (67%). The sum of n = 1246 included the following: males, 289 were between 5 and 14 years, 183 (15-24 years), 157 (25-34 years), 113 (35-49 years), and 43 were 50+ years. Females: 138 were between 5 and 14 years, 93 (15-24 years), 72 (25-34 years), 89 (35-49 years), and 68 were 50+ years. The Gram-positive organisms isolated were Staphylococcus aureus (n = 111, 80.43%, MRSA 16.6%), E. faecalis (n = 20, 14.49%, VRE: 9.4%), and Streptococcus agalactiae (n = 7, 5.07%), while, Gram-negative organisms, such as E. coli (n = 135, 42.85%, CRE: 3.49%), K. pneumoniae (n = 93, 29.52%, CRE: 1.58%), P. aeruginosa (n = 43, 13.65%), C. freundii (n = 21, 6.66%), Serratia spp. (n = 8, 2.53%), and Proteus spp. (n = 15, 4.76%) were identified.

Spermine-Conjugated Short Proline-Rich Lipopeptides as Broad-Spectrum Intracellular Targeting Antibacterial Agents.

Toward the design of new proline-rich peptidomimetics, a short peptide segment, present in several proline-rich antimicrobial peptides (AMPs), was selected. Fatty acids of varying lengths and spermine were conjugated at the N- and C-terminals of the peptide, respectively. Spermine-conjugated lipopeptides, C10-PR-Spn and C12-PR-Spn, exhibited minimum inhibitory concentrations within 1.5-6.2 µM against the tested pathogens including resistant bacteria and insignificant hemolytic activity against human red blood cells up to 100 µM concentrations and demonstrated resistance against trypsin digestion. C10-PR-Spn and C12-PR-Spn showed synergistic antimicrobial activity against multidrug-resistant methicillin-resistant Staphylococcus aureus with several tested antibiotics. These lipopeptides did not permeabilize bacterial membrane-mimetic lipid vesicles or damage the Escherichia coli membrane like the nonmembrane-lytic AMP, buforin-II. The results suggested that C10-PR-Spn and C12-PR-Spn could interact with the 70S ribosome of E. coli and inhibit its protein synthesis. C10-PR-Spn and C12-PR-Spn demonstrated superior clearance of bacteria from the spleen, liver, and kidneys of mice, infected with S. aureus ATCC 25923 compared to levofloxacin.

[Fe-S] biogenesis and unusual assembly of the ISC scaffold complex in the Plasmodium falciparum mitochondrion.

The malaria parasite harbors two [Fe-S] biogenesis pathways of prokaryotic origin-the SUF and ISC systems in the apicoplast and mitochondrion, respectively. While the SUF machinery has been delineated, there is little experimental evidence on the ISC pathway. We confirmed mitochondrial targeting of Plasmodium falciparum ISC proteins followed by analyses of cysteine desulfurase, scaffold, and [Fe-S]-carrier components. PfIscU functioned as the scaffold in complex with the PfIscS-PfIsd11 cysteine desulfurase and could directly assemble [4Fe-4S] without prior [2Fe-2S] formation seen in other homologs. Small angle X-ray scattering and spectral studies showed that PfIscU, a trimer, bound one [4Fe-4S]. In a deviation from reported complexes from other organisms, the P. falciparum desulfurase-scaffold complex assembled around a PfIscS tetramer instead of a dimer, resulting in a symmetric hetero-hexamer [2× (2PfIscS-2PfIsd11-2PfIscU)]. PfIscU directly transferred [4Fe-4S] to the apo-protein aconitase B thus abrogating the requirement of intermediary proteins for conversion of [2Fe-2S] to [4Fe-4S] before transfer to [4Fe-4S]-recipients. Among the putative cluster-carriers, PfIscA2 was more efficient than PfNifU-like protein; PfIscA1 primarily bound iron, suggesting its potential role as a Fe2+ carrier/donor. Our results identify the core P. falciparum ISC machinery and reveal unique features compared with those in bacteria or yeast and human mitochondria.

Plasmodium Ape1 is a multifunctional enzyme in mitochondrial base excision repair and is required for efficient transition from liver to blood stage infection.

The malaria parasite has a single mitochondrion which carries multiple tandem repeats of its 6 kb genome encoding three proteins of the electron transport chain. There is little information about DNA repair mechanisms for mitochondrial genome maintenance in Plasmodium spp. Of the two AP-endonucleases of the BER pathway encoded in the parasite nuclear genome, the EndoIV homolog PfApn1 has been identified as a mitochondrial protein with restricted functions. We explored the targeting and biochemical properties of the ExoIII homolog PfApe1. PfApe1 localized in the mitochondrion and exhibited AP-site cleavage, 3'-5' exonuclease, 3'-phosphatase, nucleotide incision repair (NIR) and RNA cleavage activities indicating a wider functional role than PfApn1. The parasite enzyme differed from human APE1 in possessing a large, disordered N-terminal extension. Molecular modelling revealed conservation of structural domains but variations in DNA-interacting residues and an insertion in the α-8 loop suggested differences with APE1. Unlike APE1, where AP-site cleavage and NIR activities could be mutually exclusive based on pH and Mg2+ ion concentration, PfApe1 was optimally active under similar conditions suggesting that it can function both as an AP-endonuclease in BER and directly cleave damaged bases in NIR under similar physiological conditions. To investigate the role of Ape1 in malaria life cycle, we disrupted the gene by double-cross-over homologous recombination. Ape1 knockout (KO) P. berghei parasites showed normal development of blood and mosquito stages. However, inoculation of mice with Ape1 KO salivary gland sporozoites revealed a reduced capacity to initiate blood stage infection. Ape1 KO parasites underwent normal liver stage development until merozoites egressed from hepatocytes. Our results indicated that the delay in pre-patent period was due to the inability of Ape1 KO merosomes to infect erythrocytes efficiently.

Plasmodium falciparum FIKK9.1 is a monomeric serine-threonine protein kinase with features to exploit as a drug target.

FIKK-9.1 is essential for parasite survival, but its structural and biochemical characterization will enable us to understand its role in the parasite life cycle. The recombinant FIKK9.1 kinase is monomeric with a native molecular weight of 60 ± 1.6 kDa. Structural characterization of FIKK9.1 kinase reveals that it consists of two domains: N-terminal FHA like domain and C-terminal kinase domain. The C-terminal domain has a well-defined pocket, but it displayed RMSD deviation of 1.38-3.2 Å from host kinases. ITC analysis indicates that ATP binds to the protein with a Kd of 45.6 ± 2.4 µM. Mutational studies confirm the role of Val-244, Met-245, Lys-320, 324, and Glu-366 for ATP binding. Co-localization studies revealed FIKK9.1 in the parasite cytosol with a component trafficked to the apicoplast and also to IRBC. FIKK9.1 has 23 pockets to serve as potential docking sites for substrates. Correlation analysis of peptides from the combinatorial library concluded that peptide P277 (MFDFHYTLGPMWGTL) was fitting nicely into the binding pocket. The peptide P277 picked up candidates from parasite and key players from RBC cytoskeleton. Interestingly, FIKK9.1 is phosphorylating spectrin, ankyrin, and band-3 from RBC cytoskeleton. Our study highlights the structural and biochemical features of FIKK9.1 to exploit it as a drug target.

PfKsgA1 functions as a transcription initiation factor and interacts with the N-terminal region of the mitochondrial RNA polymerase of Plasmodium falciparum.

The small mitochondrial genome (mtDNA) of the malaria parasite is known to transcribe its genes polycistonically, although promoter element(s) have not yet been identified. An unusually large Plasmodium falciparum candidate mitochondrial phage-like RNA polymerase (PfmtRNAP) with an extended N-terminal region is encoded by the parasite nuclear genome. Using specific antibodies against the enzyme, we established that PfmtRNAP was targeted exclusively to the mitochondrion and interacted with mtDNA. Phylogenetic analysis showed that it is part of a separate apicomplexan clade. A search for PfmtRNAP-associated transcription initiation factors using sequence homology and in silico protein-protein interaction network analysis identified PfKsgA1. PfKsgA1 is a dual cytosol- and mitochondrion-targeted protein that functions as a small subunit rRNA dimethyltransferase in ribosome biogenesis. Chromatin immunoprecipitation showed that PfKsgA1 interacts with mtDNA, and in vivo crosslinking and pull-down experiments confirmed PfmtRNAP-PfKsgA1 interaction. The ability of PfKsgA1 to serve as a transcription initiation factor was demonstrated by complementation of yeast mitochondrial transcription factor Mtf1 function in Rpo41-driven in vitro transcription. Pull-down experiments using PfKsgA1 and PfmtRNAP domains indicated that the N-terminal region of PfmtRNAP interacts primarily with the PfKsgA1 C-terminal domain with some contacts being made with the linker and N-terminal domain of PfKsgA1. In the absence of full-length recombinant PfmtRNAP, solution structures of yeast mitochondrial RNA polymerase Rpo41 complexes with Mtf1 or PfKsgA1 were determined by small-angle X-ray scattering. Protein interaction interfaces thus identified matched with those reported earlier for Rpo41-Mtf1 interaction and overlaid with the PfmtRNAP-interfacing region identified experimentally for PfKsgA1. Our results indicate that in addition to a role in mitochondrial ribosome biogenesis, PfKsgA1 has an independent function as a transcription initiation factor for PfmtRNAP.

YihA GTPases localize to the apicoplast and mitochondrion of the malaria parasite and interact with LSU of organellar ribosomes.

The YihA TRAFAC GTPases are critical for late-stage assembly of the ribosomal large subunit (LSU). In order to explore biogenesis of the reduced organellar ribosomes of the malaria parasite, we identified three nuclear-encoded homologs of YihA in Plasmodium falciparum. PfYihA1 targeted to the parasite apicoplast, PfYihA2 to the mitochondrion, and PfYihA3 was found in both the apicoplast and cytosol. The three PfYihA, expressed as recombinant proteins, were active GTPases and interacted with surrogate E. coli ribosomes in a nucleotide-independent manner. In vivo complexation of PfYihA with parasite organellar and/or cytosolic LSU was confirmed by co-immunoprecipitation using specific antibodies. Mitochondrial PfYihA2 carries a large C-ter extension with a strongly positively charged stretch. We hypothesise that this is important in compensating for the absence of helices of the central protuberance in the fragmented rRNA of Plasmodium mitoribosomes and may provide additional contact sites to aid in complex assembly. Combined with previous reports, our results indicate that P. falciparum mitochondria are likely to assemble ribosomes with the aid of PfEngA, PfObg1 and PfYihA2 GTPases while apicoplast ribosomes might use PfYihA1 and 3 in combination with other factors.

Plasmodium falciparum Apn1 homolog is a mitochondrial base excision repair protein with restricted enzymatic functions.

The malaria parasite carries two organelles, the apicoplast and mitochondrion, whose DNA genomes must be maintained for optimal function and parasite survival under genotoxic stress. DNA repair mechanism(s) operative within these organelles were explored by mining the Plasmodium falciparum nuclear genome for sequences encoding proteins of major DNA repair pathways with predicted targeting to either organelle. Of the panel of enzymes identified for base excision repair (BER), we characterized the apurinic/apyrimidinic (AP) endonuclease PfApn1-an EndoIV whose homolog is not known in humans. PfApn1 targeted to the mitochondrion and functioned as an AP endonuclease requiring both Zn2+ and Mn2+ ions for maximal activity. Mutation of the critical third metal-binding site residue H542 resulted in the loss of Mn2+ (but not Zn2+ ) binding indicating that Mn2+ bound PfApn1 at this site; this was further supported by molecular dynamic simulation. CD spectra analysis further showed requirement of both metal ions for the attainment of PfApn1 ß-strand-rich optimal conformation. PfApn1 also functioned as a 3'-phosphatase that would enable removal of 3'-blocks for DNA polymerase activity during BER. Interestingly, unlike Escherichia coli and yeast EndoIV homologs, PfApn1 lacked 3'-5' exonuclease activity and also did not cleave damaged bases by nucleotide incision repair (NIR). Uncoupling of endonuclease/phosphatase and exonuclease/NIR in PfApn1 suggests that amino acid residues distinct from those critical for endonuclease function are required for exonuclease activity and NIR. Characterization of a critical mitochondrion-targeted AP endonuclease provides evidence for a functional BER pathway in the parasite organelle.

Characterization of a Plasmodium falciparum rRNA methyltransferase.

The ribosomal RNA adenine dimethyltransferases (rAD) of KsgA/Dim1 family are universally conserved with eukaryotic rADs separated into distinct cytosolic Dim1 and organellar KsgA/TFB homologs. Among the two putative KsgA proteins encoded by the Plasmodium falciparum genome, we found that PfKsgA1 was dually localised in the cytoplasm and the mitochondrion. The protein interacted specifically with small ribosomal subunit as detected by ribosome pull-down using anti-PfKsgA1 antibodies. Recombinant PfKsgA1 exhibited methyltransferase activity which was further confirmed by complementation in an Escherichia coli KsgA knockout strain. Similar to the human mitochondrial KsgA homologs that can additionally function as transcription regulators, PfKsgA1 also interacted with DNA in a sequence non-specific manner suggesting more than one functional role of an important ribosome biogenesis protein in Plasmodium.

Characterization of mitochondrion-targeted GTPases in Plasmodium falciparum.

Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.

[Fe-S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite.

The relict plastid (apicoplast) of the malaria parasite is the site for important biochemical pathways and is essential for parasite survival. The sulfur mobilization (SUF) pathway of iron-sulfur [Fe-S] cluster assembly in the apicoplast of Plasmodium spp. is of interest due to its absence in the human host suggesting the possibility of antimalarial intervention through apicoplast [Fe-S] biogenesis. We report biochemical characterization of components of the Plasmodium falciparum apicoplast SUF pathway after the first step of SUF. In vitro interaction experiments and in vivo cross-linking showed that apicoplast-encoded PfSufB and apicoplast-targeted PfSufC and PfSufD formed a complex. The PfSufB-C2 -D complex could function as a scaffold to assemble [4Fe-4S] clusters in vitro and activity of the PfSufC ATPase was enhanced by PfSufD. Two carrier proteins, the NifU-like protein PfNfu and the A-type carrier PfSufA are homodimers, the former mediating transfer of [4Fe-4S] from the scaffold to a model [4Fe-4S] target protein with higher efficiency. Conditional knockout of SufS, the enzyme catalyzing the first step of SUF, by selective excision in the mosquito stages of Plasmodium berghei severely impaired development of sporozoites in oocysts establishing essentiality of the SUF machinery in the vector. Our results delineate steps of the complete apicoplast SUF pathway and demonstrate its critical role in the parasite life cycle.

Dehydroascorbate-derivatized chitosan particles for targeting antimalarial agents to infected erythrocytes.

The mammalian glucose transporter GLUT-1 and Plasmodium falciparum hexose transporter PfHT1 are overexpressed on human RBC infected with the parasite (iRBC), presumably for enhanced glucose uptake. Dehydroascorbic acid (DHA) competes out glucose in GLUT-1 binding. We prepared particles containing chloroquine phosphate using novel derivatives of chitosan (CSN). CSN was either pre-derivatized with DHA (PRE) or particles made of CSN were derivatized by surface-grafting DHA (POST). The optimized formulations were analyzed for size (170-200nm) drug content (about 40%) entrapment efficiency (50-57%), in vitro drug release (80% in 72h, Higuchi's model), hemolysis on exposure to whole blood or RBC at 5% hematocrit, cytotoxicity towards cultured HEK 293T (kidney) and HepG2 (hepatic) cells, targeting iRBC and in vitro efficacy against P. falciparum. PRE particles were superior to POST CSN particles in terms of uptake and extent of preferential targeting to iRBCs than RBCs. Unlike starch particles reported earlier, dextrose did not competitively inhibit uptake of DHA-derivatized CSN particles. Both formulations significantly induced parasite inhibition at 1nM while free drug showed comparable activity at 100nM. Both PRE and POST particles were superior to free drug in efficacy. Targeting with high efficiency promises dose reduction and possibility of overcoming efflux-based drug resistance.

Translation in Organelles of Apicomplexan Parasites.

The protein translation machineries of the apicoplast and mitochondrion-the two actively translating organelles of apicomplexan parasites-have potential sites for drug intervention against diseases caused by these organisms. Work in the past few years, particularly on Plasmodium falciparum and Toxoplasma gondii, has shown that a reduced machinery of enzymes and factors is sufficient for organellar translation, which is also supported by components shared with the cytosolic translation system. This interplay between eukaryotic and prokaryotic-like components for mRNA translation in organelles is reviewed here. We also discuss functional and structural aspects of factors mediating initiation, elongation, and termination of polypeptides, and recycling of the reduced ribosomes of the apicoplast and mitochondrion.

Polypeptide release factors and stop codon recognition in the apicoplast and mitochondrion of Plasmodium falciparum.

Correct termination of protein synthesis would be a critical step in translation of organellar open reading frames (ORFs) of the apicoplast and mitochondrion of the malaria parasite. We identify release factors (RFs) responsible for recognition of the UAA and UGA stop-codons of apicoplast ORFs and the sole UAA stop-codon that terminates translation from the three mitochondrial ORFs. A single nuclear-encoded canonical RF2, PfRF2Api , localizes to the apicoplast. It has a conserved tripeptide motif (SPF) for stop-codon recognition and is sufficient for peptidyl-tRNA hydrolysis (PTH) from both UAA and UGA. Two RF family proteins are targeted to the parasite mitochondrion; a canonical RF1, PfRF1Mit , with a variant codon-recognition motif (PxN instead of the conserved RF1 PxT) is the major peptidyl-hydrolase with specific recognition of the UAA codon relevant to mitochondrial ORFs. Mutation of the N residue of the PfRF1Mit PxN motif and two other conserved residues of the codon recognition domain lowers PTH activity from pre-termination ribosomes indicating their role in codon-recognition. The second RF imported by the mitochondrion is the non-canonical PfICT1 that functions as a dimer and mediates codon nonspecific peptide release. Our results help delineate a critical step in organellar translation in Plasmodium, which is an important target for anti-malarials.

Preferential targeting of human erythrocytes infected with the malaria parasite Plasmodium falciparumvia hexose transporter surface proteins.

Glucose uptake by Plasmodium-infected erythrocytes (RBC) is higher compared to uninfected RBC. Glucose is transported across the cell membrane by transporter proteins. Particles of median size 146.3±18.7 nm, containing anti-malarial agents in corn starch were prepared for investigating: (a) whether the glucose moiety in starch targets RBC via hexose transporter(s), (b) whether there are differences in the extent of targeting to uninfected RBC versus infected RBC (iRBC) in view of higher cell surface density of these proteins on iRBC and (c) whether targeting provides enhanced efficacy against P. falciparum in comparison to drugs in solution. Binding of these particles to RBC was target-specific, since it could be blocked by phloretin, an inhibitor of glucose transporters (GLUT), or competed out in a dose-dependent manner with d-glucose in a flow cytometry assay. Significant (P=0.048, t-test) differences in extent of targeting to iRBC versus RBC were observed in flow cytometry. CDRI 97/63 incorporated in particles was 63% more efficacious than its solution at 250 ng/ml, while quinine was 20% more efficacious at 6.25 ng/ml in a SYBR Green incorporation assay. Preferential targeting of iRBC using an inexpensive excipient promises advantages in terms of dose reduction and toxicity alleviation.