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1.  Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase 
Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.
doi:10.1016/j.molbiopara.2014.01.003
PMCID: PMC4022148  PMID: 24548880
Entamoeba histolytica; low molecular weight protein tyrosine phosphatase (LMW-PTP); LMW-PTP crystal structure; substrate-trapping; LMW-PTP putative substrate identification
2.  Transcriptomic Evaluation of the Nicotinic Acetylcholine Receptor Pathway in Levamisole-resistant and -sensitive Oesophagostomum dentatum 
Nematode anthelminthic resistance is widespread for the 3 major drug classes commonly used in agriculture: benzamidazoles, macrocyclic lactones, and nicotinic agonists e.g. levamisole. In parasitic nematodes the genetics of resistance are unknown other than to the benzimidazoles which primarily involve a single gene. In previous work with a levamisole resistant Oesophagostomum dentatum isolate, the nicotinic acetylcholine receptor (nAChR) exhibited decreased levamisole sensitivity. Here, using a transcriptomic approach on the same isolate, we investigate whether that decreased nAChR sensitivity is achieved via a 1-gene mechanism involving 1 of 27 nAChR pathway genes. 3 nAChR receptor subunit genes exhibited ≥ 2-fold change in transcript abundance: acr-21 and acr-25 increased, and unc-63 decreased. 4 SNPs having a ≥ 2-fold change in frequency were also identified. These data suggest that resistance is likely polygenic, involving modulated abundance of multiple subunits comprising the heteropentameric nAChR, and is not due to a simple 1-gene mechanism.
doi:10.1016/j.molbiopara.2014.02.002
PMCID: PMC3992831  PMID: 24530453
resistance; levamisole; nAChR; nodular worm
3.  [No title available] 
PMCID: PMC4004664  PMID: 24389163
4.  Nitazoxanide: Nematicidal Mode of Action and Drug Combination Studies 
Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections, we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections, was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy.
doi:10.1016/j.molbiopara.2013.12.002
PMCID: PMC3972318  PMID: 24412397
5.  Chemical Biology Approaches for the study of Apicomplexan Parasites 
Chemical biology and the techniques the field encompasses provide scientists with the means to address biological questions in ever evolving and technically sophisticated ways. They facilitate the dissection of molecular mechanisms of cell phenomena on timescales not achievable by other means. Libraries of small molecules, bioorthogonal chemistries and technical advances in mass-spectrometry techniques enable the modern chemical biologist to tackle even the most difficult of biological questions. It is because of their broad applicability that these approaches are well suited to systems less tractable to more classical genetic methods. As such, the parasite community has embraced them with great success. Some of these successes and the continuing evolution of chemical biology applied to apicomplexans will be discussed.
doi:10.1016/j.molbiopara.2013.11.003
PMCID: PMC3945044  PMID: 24333788
Apicomplexan; chemical biology; small-molecule; bioorthogonal
6.  Use of Giardia, which appears to have a single nucleotide-sugar transporter for UDP-GlcNAc, to identify the UDP-Glc transporter of Entamoeba 
Nucleotide-sugar transporters (NSTs) transport activated sugars (e.g. UDP-GlcNAc) from the cytosol to the lumen of the endoplasmic reticulum or Golgi apparatus where they are used to make glycoproteins and glycolipids. UDP-Glc is an important component of the N-glycan-dependent quality control (QC) system for protein folding. Because Entamoeba has this QC system while Giardia does not, we hypothesized that transfected Giardia might be used to identify the UDP-Glc transporter of Entamoeba. Here we show Giardia membranes transport UDP-GlcNAc and have apyrases, which hydrolyze nucleoside-diphosphates to make the antiporter nucleoside-monophosphate. The only NST of Giardia (GlNst), which we could identify, transports UDP-GlcNAc in transfected Saccharomyces and is present in perinuclear and peripheral vesicles and increases in expression during encystation. Entamoeba membranes transport three nucleotide-sugars (UDP-Gal, UDP-GlcNAc, and UDP-Glc), and Entamoeba has three NSTs, one of which has been shown previously to transport UDP-Gal (EhNst1). Here we show recombinant EhNst2 transports UDP-Glc in transfected Giardia, while recombinant EhNst3 transports UDP-GlcNAc in transfected Saccharomyces. In summary, all three NSTs of Entamoeba and the single NST of Giardia have been molecularly characterized, and transfected Giardia provide a new system for testing heterologous UDP-Glc transporters.
doi:10.1016/j.molbiopara.2008.01.004
PMCID: PMC4258307  PMID: 18346800
Giardia; Entamoeba; Nucleotide sugar transporter; Apyrase; transfection; N-glycan-dependent quality control of protein folding
7.  Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas’ disease 
Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed.
doi:10.1016/j.molbiopara.2012.10.006
PMCID: PMC4197799  PMID: 23137716
Trypanosoma cruzi; Chagas's disease; phosphatase; drug design; crystal structure; inhibitors
8.  Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine 
Molecular and biochemical parasitology  2013;191(2):10.1016/j.molbiopara.2013.09.007.
Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.
doi:10.1016/j.molbiopara.2013.09.007
PMCID: PMC3851587  PMID: 24120444
adenosine; Crithidia fasciculata; equilibrative nucleoside transporter; ligand affinity; purine metabolism
9.  Kinetoplastid-specific histone variant functions are conserved in Leishmania major 
Molecular and biochemical parasitology  2013;191(2):10.1016/j.molbiopara.2013.09.005.
Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.
doi:10.1016/j.molbiopara.2013.09.005
PMCID: PMC3863619  PMID: 24080031
epigenetics; histone variants; transcriptional read-through; chromatin; trypanosomatid protozoa; genetics
10.  Plasmodium falciparum aldolase and the C-terminal cytoplasmic domain of certain apical organellar proteins promote actin polymerization 
The current model of Apicomplexan motility and host cell invasion is that both processes are driven by an actomyosin motor located beneath the plasma membrane, with the force transduced to the outside of the cell via coupling through aldolase and the cytoplasmic tail domains (CTDs) of certain type 1 membrane proteins. In Plasmodium falciparum (Pf), aldolase is thought to bind to the CTD of members of the thrombospondin-related anonymous protein (TRAP) family, which are micronemal proteins and represented by MTRAP in merozoites. Other type 1 membrane proteins including members of the erythrocyte binding antigen (EBA) and reticulocyte binding protein homologue (RH) protein families, which are also apical organellar proteins, have also been implicated in host cell binding in erythrocyte invasion. However, recent studies with Toxoplasma gondii have questioned the importance of aldolase in these processes. Using biolayer interferometry we show that Pf aldolase binds with high affinity to both rabbit and Pf actin, with a similar affinity for filamentous (F-) actin and globular (G-) actin. The interaction between Pf aldolase and merozoite actin was confirmed by co-sedimentation assays. Aldolase binding was shown to promote rabbit actin polymerization indicating that the interaction is more complicated than binding alone. The CTDs of some but not all type 1 membrane proteins also promoted actin polymerization in the absence of aldolase; MTRAP and RH1 CTDs promoted actin polymerization but EBA175 CTD did not. Direct actin polymerization mediated by membrane protein CTDs may contribute to actin recruitment, filament formation and stability during motor assembly, and actin-mediated movement, independent of aldolase.
doi:10.1016/j.molbiopara.2014.09.006
PMCID: PMC4251702  PMID: 25261592
Malaria; Merozoite; Motor; Actin; Aldolase; Invasion
11.  Cryptosporidium parvum has an active hypusine biosynthesis pathway 
The protozoan parasite Cryptosporidium parvum causes severe enteric infection and diarrheal disease with substantial morbidity and mortality in untreated AIDS patients and children in developing or resource-limited countries. No fully effective treatment is available. Hypusination of eIF5A is an important post-translational modification essential for cell proliferation. This modification occurs in a two step process catalyzed by deoxyhypusine synthase (DHS) followed by deoxyhypusine hydroxylase. An ORF of 1086 bp was identified in the C. parvum (Cp) genome which encodes for a putative polypeptide of 362 amino acids. The recombinant CpDHS protein was purified to homogeneity and used to probe the enzyme’s mechanism, structure, and inhibition profile in a series of kinetic experiments. Sequence analysis and structural modeling of CpDHS were performed to probe differences with respect to the DHS of other species. Unlike Leishmania, Trypanosomes and Entamoeba, Cryptosporidium contains only a single gene for DHS. Phylogenetic analysis shows that CpDHS is more closely related to apicomplexan DHS than kinetoplastid DHS. Important residues that are essential for the functioning of the enzyme including NAD+ binding residues, spermidine binding residues and the active site lysine are conserved between CpDHS and human DHS. N1-guanyl-1.7-diaminoheptane (GC7), a potent inhibitor of DHS caused an effective inhibition of infection and growth of C. parvum in HCT-8 cells.
doi:10.1016/j.molbiopara.2014.05.005
PMCID: PMC4176827  PMID: 24893338
Cryptosporidium parvum; Protozoan parasite; Deoxyhypsuine synthase; Hypusine pathway
12.  Characterization of the Phytochelatin Synthase from the Human Parasitic Nematode Ancylostoma ceylanicum 
Molecular and biochemical parasitology  2013;191(1):10.1016/j.molbiopara.2013.07.003.
Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.
doi:10.1016/j.molbiopara.2013.07.003
PMCID: PMC3823645  PMID: 23916800
Glutathione; Hookworm; Metal toxicity; Neglected tropical disease; Phytochelatin; Xenobiotic metabolism
13.  Strategies to construct null and conditional null Trypanosoma brucei mutants using Cre-recombinase and loxP 
Molecular and biochemical parasitology  2013;191(1):10.1016/j.molbiopara.2013.08.001.
We describe two gene-knockout (KO) strategies in Trypanosoma brucei using Cre recombinase and loxP sites. Due to the limited number of selection markers for T. brucei, it has been difficult to generate a mutant with two genes knocked out and impractical to simultaneously knockout more than two genes, deterring detailed studies of important cellular mechanisms. The first KO strategy described can overcome the marker problem by allowing continuous re-use of drug-resistance markers. The same KO vector can be used to make a conditional KO system, when a gene of interest is essential for cell viability. As a gene of interest is removed from its original chromosomal locus by the induction of Cre recombinase, deletion is complete and instantaneous. This makes it easier to identify primary effects rather than having secondary effects obscuring phenotypic assessment, as is often the case with RNAi silencing.
doi:10.1016/j.molbiopara.2013.08.001
PMCID: PMC3830529  PMID: 23954366
Trypanosoma brucei; Cre-recombinase; gene knockout; conditional gene knockout
14.  Identification of a family of four UDP-polypeptide N-acetylgalactosaminyl transferases in Cryptosporidium species 
Molecular and biochemical parasitology  2013;191(1):10.1016/j.molbiopara.2013.08.002.
Although mucin-type O-glycans are critical for Cryptosporidium infection, the enzymes catalyzing their synthesis have not been studied. Here, we report four UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl transferases (ppGalNAc-Ts) from the genomes of C. parvum, C. hominis and C. muris. All are Type II membrane proteins which include a cytoplasmic tail, a transmembrane domain, a stem region, a glycosyltransferase family 2 domain and a C-terminal ricin B lectin domain. All are expressed during C. parvum infection in vitro, with Cp-ppGalNAc-T1 and -T4 expressed at 24 h and Cp-ppGalNAc-T2 and -T3 at 48 and 72 h post-infection, suggesting that their expression may be developmentally regulated. C. parvum sporozoite lysates display ppGalNAc-T enzymatic activity against non-glycosylated and pre-glycosylated peptides suggesting that they contain enzymes capable of glycosylating both types of substrates. The importance of mucin-type O-glycans in Cryptosporidium–host cell interactions raises the possibility that Cp-ppGalNAc-Ts may serve as targets for intervention in cryptosporidiosis.
doi:10.1016/j.molbiopara.2013.08.002
PMCID: PMC3856541  PMID: 23954365
O-glycosylation; Mucin; Mucin-like glycoprotein; Cryptosporidium; UDP GalNAc:polypeptide; N-acetylgalactosaminyl transferase
15.  SLaP mapper: A webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes 
Graphical abstract
Highlights
•A web-server for identification of spliced-leader and polyadenylation addition sites.•Fully automated site quantification and gene assignment.•Multiple species within the Kinetoplastida.
The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ∼39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website.
doi:10.1016/j.molbiopara.2014.07.012
PMCID: PMC4222701  PMID: 25111964
RNA splicing; Trypanosoma; Leishmania; Polyadenylation; RNA-Seq; Kinetoplastida
16.  RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade 
The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Rather, their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3’-region were employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated ex vivo with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions.
doi:10.1016/j.molbiopara.2007.10.009
PMCID: PMC4130333  PMID: 18067980
schistosomes; cathepsin D; RNAi; schistosomules; hemoglobin proteolysis; RT-PCR
17.  The compact conformation of the Plasmodium knowlesi myosin tail interacting protein MTIP in complex with the C-terminal helix of myosin A 
The myosin motor of the malaria parasite’s invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a “compact” Plasmodium falciparum MTIP•MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an “extended” Plasmodium knowlesi MTIP•MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIP•MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously.
doi:10.1016/j.molbiopara.2013.06.004
PMCID: PMC3910325  PMID: 23831369
Malaria; Invasion; Glideosome; MTIP; X-ray structure
18.  Adenine and adenosine salvage in Leishmania donovani 
6-aminopurine metabolism in Leishmania is unique among trypanosomatid pathogens since this genus expresses two distinct routes for adenine salvage: adenine phosphoribosyltransferase (APRT) and adenine deaminase (AAH). To evaluate the relative contributions of APRT and AAH, adenine salvage was evaluated in Δaprt, Δaah, and Δaprt/Δaah null mutants of L. donovani. The data confirm that AAH plays the dominant role in adenine metabolism in L. donovani, although either enzyme alone is sufficient for salvage. Adenosine salvage was also evaluated in a cohort of null mutants. Adenosine is also primarily converted to hypoxanthine, either intracellularly or extracellularly, but can also be phosphorylated to the nucleotide level by adenosine kinase when the predominant pathways are genetically or pharmacologically blocked. These data provide genetic verification for the relative contributions of 6-aminopurine metabolizing pathways in L. donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation.
doi:10.1016/j.molbiopara.2013.06.005
PMCID: PMC3767402  PMID: 23845934
Leishmania donovani; purine salvage; adenine metabolism; adenosine metabolism; adenine phosphoribosyltransferase; adenine aminohydrolase
19.  A conserved domain targets exported PHISTb family proteins to the periphery of Plasmodium infected erythrocytes 
Graphical abstract
The extended PRESAN domain is a targeting domain used by multiple Plasmodium species to target PHISTb proteins to the cytoskeleton/plasma membrane of infected cells.
Highlights
•Multiple P. falciparum PHISTb proteins localise to the erythrocyte periphery.•Solubility profiling indicates that these proteins associate with the red cell cytoskeleton.•The PRESAN domain and a preceding N-terminal sequence is a novel targeting domain.•A protein targeted to the red cell periphery is essential for parasite survival.•P. knowlesi and P. vivax homologous domains also confer similar localisation.
During blood-stage infection, malaria parasites export numerous proteins to the host erythrocyte. The Poly-Helical Interspersed Sub-Telomeric (PHIST) proteins are an exported family that share a common ‘PRESAN’ domain, and include numerous members in Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi. In P. falciparum, PHIST proteins have been implicated in protein trafficking and intercellular communication. A number of PHIST proteins are essential for parasite survival. Here, we identify nine members of the PHISTb sub-class of PHIST proteins, including one protein known to be essential for parasite survival, that localise to the erythrocyte periphery. These proteins have solubility characteristics consistent with their association with the erythrocyte cytoskeleton. Together, an extended PRESAN domain, comprising the PRESAN domain and preceding sequence, form a novel targeting-domain that is sufficient to localise a protein to the erythrocyte periphery. We validate the role of this domain in RESA, thus identifying a cytoskeleton-binding domain in RESA that functions independently of its known spectrin-binding domain. Our data suggest that some PHISTb proteins may act as cross-linkers of the erythrocyte cytoskeleton. We also show for the first time that peripherally-localised PHISTb proteins are encoded in genomes of P. knowlesi and vivax indicating a conserved role for the extended PRESAN domain of these proteins in targeting to the erythrocyte periphery.
doi:10.1016/j.molbiopara.2014.07.011
PMCID: PMC4165601  PMID: 25106850
PHIST; PRESAN; Plasmodium; Cytoskeleton; Protein export; Malaria
20.  Antigenic variation in African trypanosomes 
Graphical abstract
Highlights
•Much of what we know about trypanosomatid biology has its origin in studies on VSGs.•Monotelomeric VSG expression and epigenetic switching are remarkable examples of allelic exclusion.•DNA repair processes allow a new VSG to be copied into the single transcribed locus.
Studies on Variant Surface Glycoproteins (VSGs) and antigenic variation in the African trypanosome, Trypanosoma brucei, have yielded a remarkable range of novel and important insights. The features first identified in T. brucei extend from unique to conserved-among-trypanosomatids to conserved-among-eukaryotes. Consequently, much of what we now know about trypanosomatid biology and much of the technology available has its origin in studies related to VSGs. T. brucei is now probably the most advanced early branched eukaryote in terms of experimental tractability and can be approached as a pathogen, as a model for studies on fundamental processes, as a model for studies on eukaryotic evolution or often all of the above. In terms of antigenic variation itself, substantial progress has been made in understanding the expression and switching of the VSG coat, while outstanding questions continue to stimulate innovative new approaches. There are large numbers of VSG genes in the genome but only one is expressed at a time, always immediately adjacent to a telomere. DNA repair processes allow a new VSG to be copied into the single transcribed locus. A coordinated transcriptional switch can also allow a new VSG gene to be activated without any detectable change in the DNA sequence, thereby maintaining singular expression, also known as allelic exclusion. I review the story behind VSGs; the genes, their expression and switching, their central role in T. brucei virulence, the discoveries that emerged along the way and the persistent questions relating to allelic exclusion in particular.
doi:10.1016/j.molbiopara.2014.05.001
PMCID: PMC4155160  PMID: 24859277
Immune evasion; In situ; Monoallelic; Polycistronic; Silencing; Trans-splicing
21.  Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii 
The intracellular parasite Toxoplasma gondii is a leading cause of congenital neurological defects. To cause disease, it must reiterate its lytic cycle through host cell invasion, replication,and parasite egress. This requires the parasite to sense changes in its environment and switch between the non-motile (for replication) and motile (for invasion and egress) states appropriately. Recently, we discovered a previously unknown mechanism of motility regulation in T. gondii, mediated by a lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). When AKMT is absent, activation of motility is inhibited, which compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Although the methyltransferase activity of AKMT has been established, the phylogenetic relationship of AKMT with other better studied lysine methyltransferases (KMTs) was not known. Also unknown was the functional relationships between different domains of AKMT. In this work we carried out phylogenetic analyses, which show that AKMT orthologs form a new subfamily of KMTs. We systematically generated truncation mutants of AKMT, and discovered that the predicted enzymatic domain alone is a very poor enzyme and cannot complement the function of AKMT in vivo. Interestingly, the N- and C-terminal domains of the AKMT have drastically different impacts on its enzyme activity, localization as well as in vivo function. Our results thus reveal that AKMT is an unusual, parasite-specific enzyme and identified regions and interactions within this novel lysine methyltransferase that can be used as drug targets.
doi:10.1016/j.molbiopara.2013.05.004
PMCID: PMC3740173  PMID: 23685344
lysine methyltransferase; AKMT; motility; Toxoplasma gondii; Plasmodium; KMT phylogeny
22.  Crystal structures of Plasmodium falciparum cytosolic tryptophanyl-tRNA synthetase and its potential as a target for structure-guided drug design 
Malaria, most commonly caused by the parasite Plasmodium falciparum, is a devastating disease that remains a large global health burden. Lack of vaccines and drug resistance necessitate the continual development of new drugs and exploration of new drug targets. Due to their essential role in protein synthesis, aminoacyl-tRNA synthetases are potential anti-malaria drug targets. Here we report the crystal structures of P. falciparum cytosolic tryptophanyl-tRNA synthetase (Pf-cTrpRS) in its ligand-free state and tryptophanyl-adenylate (WAMP)-bound state at 2.34 Å and 2.40 Å resolutions, respectively. Large conformational changes are observed when the ligand-free protein is bound to WAMP. Multiple residues, completely surrounding the active site pocket, collapsed onto WAMP. Comparison of the structures to those of human cytosolic TrpRS (Hs-cTrpRS) provides information about the possibility of targeting Pf-cTrpRS for inhibitor development. There is a high degree of similarity between Pf-cTrpRS and Hs-cTrpRS within the active site. However, the large motion that Pf-cTrpRS undergoes during transitions between different functional states avails an opportunity to arrive at compounds which selectively perturb the motion, and may provide a starting point for the development of new anti-malaria therapeutics.
doi:10.1016/j.molbiopara.2013.04.007
PMCID: PMC3680109  PMID: 23665145
Plasmodium falciparum; malaria; tryptophanyl-tRNA synthetase; crystal structure; conformational changes; drug design
23.  Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum 
In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.
PMCID: PMC4011076  PMID: 9497030
Plasmodium; Malaria; Salivary glands; Circumsporozoite protein; Mosquito; Sporozoite
24.  Transcripts analysis of the entomopathogenic nematode Steinernema carpocapsae induced in vitro with insect haemolymph☆ 
Steinernema carpocapsae is an insect parasitic nematode widely used in pest control programs. The efficacy of this nematode in controlling insects has been found to be related to the pathogenicity of the infective stage. In order to study the parasitic mechanisms exhibited by this parasite, a cDNA library of the induced S. carpocapsae parasitic phase was generated. A total of 2500 clones were sequenced and 2180 high-quality ESTs were obtained from this library. Cluster analysis generated a total of 1592 unique sequences including 1393 singletons. About 63% of the unique sequences had significant hits (e≤1e-05) to the non-redundant protein database. The remaining sequences most likely represent putative novel protein coding genes. Comparative analysis identified 377 homologs in C. elegans, 431 in C. briggsae and 75 in other nematodes. Classification of the predicted proteins revealed involvement in diverse cellular, metabolic and extracellular functions. One hundred and nineteen clusters were predicted to encode putative secreted proteins such as proteases, proteases inhibitors, lectins, saposin-like proteins, acetyl-cholinesterase, anti-oxidants, and heat-shock proteins, which can possibly have host interactions. This dataset provides a basis for genomic studies towards a better understanding of the events that occur in the parasitic process of this entomopathogenic nematode, including invasion of the insect haemocoelium, adaptations to insect innate immunity and stress responses, and production of virulence factors. The identification of key genes in the parasitic process provides useful tools for the improvement of S. carpocapsae as a biological agent.
doi:10.1016/j.molbiopara.2009.10.002
PMCID: PMC4010113  PMID: 19836423
Entomopathogenic nematode; Steinernema carpocapsae; EST; Nematode transcripts; Secreted proteins; Virulence factors
25.  Molecular and functional characterization of a putative PA28γ proteasome activator orthologue in Schistosoma mansoni 
PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753 bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28γ in larval growth/development of the S. mansoni.
doi:10.1016/j.molbiopara.2013.04.003
PMCID: PMC3712533  PMID: 23611749
Schistosoma mansoni; proteasome activator; PA28γ subunit; protease; stage-specific expression; molecular phylogeny; RNAi

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