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Background: Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. Falciparum malaria is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat., Methods: The P. falciparum essential proteins were retrieved from the recently published studies. These proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analysed for druggability potential via in silico methods to possibly identify novel therapeutic targets. Finally, the PfAp4AH target was prioritized for pharmacophore modelling based virtual screening and molecular docking analyses to identify potent inhibitors from drug-like compounds databases., Results: The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not been catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein-protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. The pharmacophore features based virtual screening of Pharmit resource predicted a lead compound i.e. MolPort-045-917-542 as a promising inhibitor of PfAp4AH among prioritized targets., Conclusion: The prioritized protein targets may worthy to test in malarial drug discovery programme to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria., (© 2021. The Author(s).)

The development of the anti-malaria vaccine holds a promising future in malaria control. One of the anti-malaria vaccine strategies known as the transmission-blocking vaccine (TBV) is to inhibit the parasite transmission between humans and mosquitoes by targeting the parasite gametocyte. Previously, we found that P48/45 included in the 6-Cysteine protein family shared by Plasmodium sp. We also detected vaccine properties possessed by all human-infecting Plasmodium and could be used as a cross-species anti-malaria vaccine. In this study, we investigated the efficacy of P48/45 through the ancestral and consensus reconstruction approach. P48/45 phylogenetic and time tree analysis was done by RAXML and BEAST2. GRASP server and Ugene software were used to reconstruct ancestral and consensus sequences, respectively. The protein structural prediction was made by using a psipred and Rosetta program. Each protein characteristic of P48/45 was analyzed by assessing hydrophobicity and Post-Translational Modification sites. Meanwhile, the Epitope sequence for B-cell, T-cell, and HLA was determined using an immunoinformatics approach. Lastly, molecular docking simulation was done to determine native binding interactions of P48/45-P230. The result showed a distinct protein characteristic of ancestral and consensus sequences. The immunogenicity analysis revealed the number of epitopes in the ancestral sequence is greater than the consensus sequence. The study also found a conserved epitope located in the binding site and consists of specific Post-Translational Modification sites. Hence, our research provides detailed insight into ancestral and consensus P48/45 efficacy for the cross-species anti-malaria vaccine., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Upon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for designing antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Furthermore, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Furthermore, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Conceivably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Drug-resistant strains of malaria parasites have emerged for most of antimalarial medications. A new chemotherapeutic compound is needed for malarial therapy. Antimalarial activity against both drug-sensitive and drug-resistant P. falciparum has been reported for an isocryptolepine derivative, 8-bromo-2-fluoro-5-methyl-5H-indolo[3,2-c]quinoline (ICL-M), which also showed less toxicity to human cells. ICL-M has indoloquinoline as a core structure and its mode of action remains unclear. Here, we explored the mechanisms of ICL-M in P. falciparum by assessing the stage-specific activity, time-dependent effect, a proteomic analysis and morphology. Since human topo II activity inhibition has been reported as a function of isocryptolepine derivatives, malarial topo II activity inhibition of ICL-M was also examined in this study. The ICL-M exhibited antimalarial activity against both the ring and trophozoite stages of P. falciparum. Our proteomics analysis revealed that a total of 112 P. falciparum proteins were differentially expressed after ICL-M exposure; among these, 58 and 54 proteins were upregulated and downregulated, respectively. Proteins localized in the food vacuole, nucleus, and cytoplasm showed quantitative alterations after ICL-M treatment. A bioinformatic analysis revealed that pathways associated with ribosomes, proteasomes, metabolic pathways, amino acid biosynthesis, oxidative phosphorylation, and carbon metabolism were significantly different in P. falciparum treated with ICL-M. Moreover, a loss of ribosomes was clearly observed by transmission electron microscopy in the ICL-M-treated P. falciparum. This finding is in agreement with the proteomics data, which revealed downregulated levels of ribosomal proteins following ICL-M treatment. Our results provide important information about the mechanisms by which ICL-M affects the malaria parasite, which may facilitate the drug development of isocryptolepine derivatives., Competing Interests: The authors have declared that no competing interests exist.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed K m value of 26.3 ± 0.4 mM for l- glutamate and V max value of 2.15 ± 0.07 U mg -1 . Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Leishmaniasis is a parasitic disease caused by the protozoan of the Leishmania genus. While no human vaccine is available, drugs such as pentavalent antimonials, pentamidine and amphotericin B are used for treat the patients. However, the high toxicity of these pharmaceutics, the emergence of parasite resistance and/or their high cost have showed to the urgent need of identify new targets to be employed in the improvement of the treatment against leishmaniasis. In a recent immunoproteomics approach performed in the Leishmania infantum species, 104 antigenic proteins were recognized by antibodies in sera of visceral leishmaniasis (VL) dogs. Some of them were later showed to be effective diagnostic markers and/or vaccine candidates against the disease. Between these proteins, 24 considered as hypothetical were identified in the promastigote and amastigote-like extracts of the parasites. The present study aimed to use bioinformatics tools to select new drug targets between these hypothetical proteins. Their cellular localization was predicted to be seven membrane proteins, as well as eight cytoplasmic, three nuclear, one mitochondrial and five proteins remained unclassified. Their functions were predicted as being two transport proteins, as well as five with metabolic activity, three as cell signaling and fourteen proteins remained unclassified. Ten hypothetical proteins were well-annotated and compared to their homology regarding to human proteins. Two proteins, a calpain-like and clavaminate synthase-like proteins were selected by using Docking analysis as being possible drug targets. In this sense, the present study showed the employ of new strategies to select possible drug candidates, according their localization and biological function in Leishmania parasites, aiming to treat against VL., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.

Two hexaazatrinaphthylene derivatives, DGV-B and DGV-C previously known to induce an apoptotic-like process in Leishmania donovani parasites were used in this study. For this purpose, two different human protein commercial arrays were used to determine the proteomic profile of the treated parasites compared to non-treated ones. One of the commercial arrays is able to detect the relative expression of 35 human apoptosis-related proteins and the other one is able to identify 9 different human kinases. The obtained results showed that the two tested molecules were able to activate a programmed cell death process by different pathways in the promastigote stage of the parasite. The present study reports the potential application of two commercialised human apoptotic arrays to evaluate the action mechanism of active compounds at least against Leishmania donovani. The obtained data would be useful to establish the putative activated apoptosis pathways in the treated parasites and to further support the use of hexaazatrinaphthylene derivatives for the treatment of leishmaniasis in the near future. Nevertheless, further molecular studies should be developed in order to design and evaluate specific apoptotic arrays for Leishmania genus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable "next generation" target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are "druggable." One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background and Purpose: Identifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases., Experimental Approach: We used the simple, tractable biomedical model Dictyostelium to investigate the molecular mechanism of naringenin, a dietary flavonoid with antiproliferative and chemopreventive actions in vitro and in animal models of carcinogenesis. We then translated these results to a mammalian kidney model, Madin-Darby canine kidney (MDCK) tubule cells, grown in culture and as cysts in a collagen matrix., Key Results: Naringenin inhibited Dictyostelium growth, but not development. Screening of a library of random gene knockout mutants identified a mutant lacking TRPP2 (polycystin-2) that was resistant to the effect of naringenin on growth and random cell movement. TRPP2 is a divalent transient receptor potential cation channel, where mutations in the protein give rise to type 2 autosomal dominant polycystic kidney disease (ADPKD). Naringenin inhibited MDCK cell growth and inhibited cyst growth. Knockdown of TRPP2 levels by siRNA in this model conferred partial resistance to naringenin such that cysts treated with 3 and 10 μM naringenin were larger following TRPP2 knockdown compared with controls. Naringenin did not affect chloride secretion., Conclusions and Implications: The action of naringenin on cell growth in the phylogenetically diverse systems of Dictyostelium and mammalian kidney cells, suggests a conserved effect mediated by TRPP2 (polycystin-2). Further studies will investigate naringenin as a potential new therapeutic agent in ADPKD., (© 2013 The British Pharmacological Society.)

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin-treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy for the development of vaccines against blood stage malaria parasites., (© 2013 John Wiley & Sons Ltd.)

Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The rapid development by malaria parasites of resistance to almost all the chemotherapeutic agents so far used for their control means that constant efforts to develop new drugs are necessary. In this review, we propose that the exploration of protein-protein interactions as a new strategy to identify antimalarial drug targets is an attractive and a promising area of research. Nevertheless, one of the most important criteria is that the targeted gene should encode an essential protein within a complex that is able to affect parasite survival. Recently, our research on the biology of Plasmodium falciparum allowed us to identify the interaction of Protein Phosphatase type 1 and actin with two essential partners, PfLRR1 and PfLRR7 respectively, both of which belong to the Leucine Rich Repeat (LRR) protein family. LRR-containing proteins are composed of several consensus LRR motifs LXLXXNXL (where X is any amino acid) that provide sites for the assembly of protein interactions. The LRR combines structural versatility, adaptability and more importantly a high degree of interaction specificity. In addition, it has been shown that a single mutation in a particular LRR motif abolishes the protein-protein interaction and contributes to the expression of severe pathology in humans. This clearly infers that blocking the interaction related to 'hot spots' of LRR motifs can be considered as good targets to block parasite growth and development. Thus, the inhibition of protein-protein interactions by peptides, peptidomimetics or small-molecule inhibitors that interfere with binding domains can contribute to defining new potential drug targets.

Tanzania implemented artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria in November of 2006 because of resistance to sulfadoxine-pyrimethamine. AL remains highly efficacious, but widespread use may soon facilitate emergence of artemisinin tolerance/resistance, which initially may be detected at the molecular level as temporal changes in the frequency of single nucleotide polymorphisms (SNPs) in the Pfmdr-1 gene associated with AL resistance. In Tanzania, 830 Plasmodium falciparum-positive samples collected between 2003 and 2010 were examined for SNPs of Pfmdr-1 at codons 86, 184, and 1246. Both the N86 and 184F increased from 2006 to 2010 (logistic regression; N86: odds ratio [95% confidence interval] = 1.35 [1.07-1.71], P = 0.01; 184F: odds ratio = 1.42 [1.07-1.88], P = 0.02), and no change was found for D1246 (odds ratio = 1.01 [0.80-1.28], P = 0.9). The observed changes may be because of introduction of AL, and if so, this finding gives cause for concern and argues for continued surveillance of these molecular markers.

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the β-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30-35% of the total predicted proteome of Leishmania spp. have been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches on how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth., (2010 Wiley-Liss, Inc.)

Background: The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level., Methods: A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ)., Results: After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis., Conclusions: In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.

Plasmodium vivax is the second leading cause of malaria worldwide. Invasion of human erythrocytes by P. vivax merozoites is dependent upon the interaction between the parasite Duffy binding protein (PvDBP) and the erythrocyte Duffy antigen receptor. Therefore, disruption of this vital interaction is an attractive target for therapeutic intervention. Although Aotus nancymaae is a commonly used primate model for human P. vivax infections, it has not been confirmed that the interaction between Ao. nancymaae erythrocytes and P. vivax is Duffy antigen dependent. Our results indicate that normal Ao. nancymaae erythrocytes readily bind to PvDBPII and that this binding is completely abolished with chymotrypsin treatment of the erythrocytes. Furthermore, the results of our inhibition assays show a dose-dependent decrease in binding with increasing amounts of anti-PvDBPII polyclonal rabbit sera or anti-Fy6 monoclonal antibody. These data indicate that the interaction between Ao. nancymaae erythrocytes and P. vivax DBPII is Duffy antigen dependent, validating this model system for in vivo studies of anti-PvDBP inhibition.

Purpose of Review: This review summarizes recent advances in our understanding of the interaction between malaria parasites and blood group antigens and discusses how the knowledge gleaned can be used to target the development of new antimalarial treatments and vaccines., Recent Findings: Studies of the interaction between Plasmodium vivax and the Duffy antigen provide the clearest example of the potential for basic research on blood groups and malaria to be translated into a vaccine that could have a major impact on global health. Progress is also being made in understanding the effects of other blood group antigens on malaria. After years of controversy, the effect of ABO blood groups on falciparum malaria has been clarified, with the non-O blood groups emerging as significant risk factors for life-threatening malaria, through the mechanism of enhanced rosette formation. The Knops blood group system may also influence malaria susceptibility, although conflicting results from different countries mean that further research is required. Unanswered questions remain about the interactions between malaria parasites and other blood group antigens, including the Gerbich, MNS and Rhesus systems., Summary: The interplay between malaria parasites and blood group antigens remains a fascinating subject with potential to contribute to the development of new interventions to reduce the global burden of malaria.

In this study, we investigated the immunological effects of bovine heat shock protein 70 (HSP70) on the major Theileria sergenti surface protein (p33). The gene encoding p33 was expressed as a fusion protein with bovine HSP70 from a plasmid vector. The adjuvant function of HSP70 on p33 was evaluated with regard to antibody response, cytokine production, and a challenge experiment in mice or cattle. HSP-p33 fusion protein provoked higher humoral and cellular immunity than either Escherichia coli-expressed p33 or piroplasm soluble protein. The HSP adjuvant activity toward p33 was also possible to detect in the inoculated cattle. The overall growth of parasites in cattle was significantly restrained in the HSP-p33-inoculated group, up to 50-52 days longer than in the controls. The present results indicate that HSP-p33 fusion protein is a promising candidate vaccine for clinical theileriosis in the field.

Objective: In China, Chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) were abandoned for the treatment of falciparum malaria 20 years ago due to resistance. Subsequent field studies showed a trend of declining CQ and SP resistance in the country. The main purpose of this study was to analyse the molecular markers of antimalarial resistance and thereby to assess the possibility of reintroduction of CQ or SP for falciparum malaria treatment., Methods: Plasmodium falciparum field isolates were collected in 2006-2007 from Hainan and Yunnan provinces, China. Nested PCR-sequencing assays were applied to analyse the SNPs in four genes: P. falciparum chloroquine resistance transporter (pfcrt) gene, multi-drug resistance 1 (pfmdr1) gene, dihydrofolate reductase (dhfr) gene and dihydropteroate synthetase (dhps) gene., Results: We found the widespread presence of point mutations in the dhfr and dhps genes which are associated with SP treatment failure. The molecular analyses also showed the fairly high prevalence of point mutation in the pfcrt gene which is linked to CQ resistance., Conclusion: The results of the present study indicate that CQ and SP should not be reintroduced for falciparum malaria treatment in the near future in China.

Cryptosporidium parvum is a member of the Apicomplexa that lacks a plastid and associated nuclear-encoded genes, which has hampered its use in evolutionary comparisons with algae and eliminated a pool of potentially useful drug targets. Here we show that apicomplexan parasites possess an unusual family of class II histone deacetylase (HDAC) proteins with orthologues that are present in other chromalveolates and primitive algae. A striking feature of these HDAC proteins is the presence of ankyrin repeats in the amino-terminus that appear to be required for enzyme activity. In vitro and in vivo analyses of the C. parvum orthologue indicate that this subclass of chromatin-remodelling proteins is targeted by the anti-cancer drug suberoylanilide hydroxamic acid and that these proteins are most likely involved in the essential process of H4 histone deacetylation that coincides with DNA replication. We propose that members of this novel class of histone deacetylase can serve as promising new targets for treatments against debilitating diseases such as cryptosporidosis, toxoplasmosis and malaria.

Background: Artemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 x 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required., Methods: One hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers msp-1 and msp-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in pfmdr1 was also carried out in the samples obtained from patients before and after the treatment., Results: The PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of pfmdr1 gene., Conclusion: AL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in pfmdr1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.

Background: Malaria is caused by the intracellular parasite Plasmodium falciparum. The constant need for novel malaria therapies is due to the development of resistance against existing drugs., Objective: To summarise attempts to investigate parasitic aquaporins as drug targets in malaria., Methods: Starting with a summary of the history of malaria we present aquaporin structure and function relationships. Potential interactions of inhibitors with plasmodial AQP (PfAQP) are discussed. PfAQP blockage is examined in the light of recent work on knock-out parasites. Since PfAQP is able to transport other small solutes the parasites are sensitive to other compounds which are harmless to the human host., Results/conclusions: Total blockage of PfAQP may not lead to the death of the parasite but application of PfAQP as a vehicle for toxic substances may be a further pathway for research.

The understanding of the biology and the biochemistry of malaria parasites has considerably increased over the past two decades with the discovery of many potential targets for new antimalarial drugs. The decrypted genomes of several Plasmodium species and the new post-genomic tools further enriched our "reservoir" of targets and increased our ability to validate potential drug targets or to study the entire parasite metabolism. This review discusses targets involved in calcium metabolism, protein prenylation and apicoplast functions that have emerged by different approaches.

Carbohydrate-based therapy, known as glycotherapeutics, is a new and emerging field that promises to be the future hope for combating kinetoplastid infections more efficiently and effectively. Targeting novel glycoproteins/lipids, which are important disease determinants of kinetoplastid diseases, have helped in the development of this field. Better and refined understanding of all the available data would possibly help us in providing a future direction for rational drug design and better disease management. This review intends to focus on such lines, which will give us an insight into the future hope for development of novel therapeutic strategies through glycobiological platform for combating kinetoplastid infections.

Background: Observations in natural Plasmodium falciparum populations after removal of failing drugs suggest that there is a fitness cost of drug resistance., Methods: To examine the effect of transient removal of drug pressure, we analyzed seasonal changes in the prevalence of chloroquine (CQ)-resistant parasite genotypes in The Gambia. Parasite isolates from 441 children presenting with uncomplicated falciparum malaria over 5 seasons (1998-2002) were linked to weekly rainfall data., Results: The prevalence of CQ-resistant parasites increased slightly over 5 years, with the 76T allele of pfcrt (odds ratio [OR] per year, 1.16; P=.03) and the 86Y allele of pfmdr1 (OR per year, 1.18; P=.02) becoming significantly more common. However, intraseasonal analysis showed that these alleles decreased in prevalence each dry season. Wild-type parasites with respect to both loci predominated as transmission began each year, with resistant parasites becoming more common as drug use increased. This pattern was seen for both pfcrt-76T (OR per week, 1.09; P=.001) and pfmdr1-86Y (OR per week, 1.07; P=.001) and could not be explained by seasonal changes in the clonal complexity of infections., Conclusions: The fitness cost of CQ resistance works against the persistence of resistant parasites through the dry season.

Alkyl-lysophospholipids (ALPs), developed initially to be antitumor agents, have proved highly effective in the treatment of visceral leishmaniasis, a disease caused by the species making up the protozoan complex Leishmania donovani. Although their effectiveness is known, the mode of action against this parasite is not completely understood. In the present work, we have studied the effect of 3 derivatives, edelfosine, miltefosine, and ilmofosine. Using nuclear magnetic resonance spectroscopy ('H-NMR), we have examined the excreted catabolites from glucose metabolism in the promastigote forms treated with these compounds. The ALPs at concentrations of 19 and 38 microM inhibit the excretion of acetate, succinate, and pyruvate. The effect of edelfosine, miltefosine, and ilmofosine on the activity of the enzymes hexokinase, glycerolkinase 3-PD, phosphoglucose isomerase, superoxide dismutase, and phospholipase C were also examined. Glycerolkinase 3-PD and phosphoglucose isomerase are generally insensitive to the compounds, whereas hexokinase and superoxide dismutase are inhibited by miltefosine and ilmofosine. The ALPs exhibited an activated effect against the phospholipase C activity. Alkyl-lysophospholipids were shown to have a significant effect on several enzymes in important biochemical pathways indispensable for the survival of L. donovani promasigotes.

Cyclophilins (CyPs) are enzymes involved in protein folding, catalyzing the isomerisation of peptidyl prolyl bonds in proteins and peptides between the cis- and trans-conformations. They are also the major cellular target for the immunosuppressive drug Cyclosporin A (CsA). In Trypanosoma cruzi, the most abundantly expressed CyP is an isoform of 19 kDa, TcCyP19, in which the enzymatic activity is inhibited by CsA. Among a reported set of CsA analogues, two non-immunosuppressive compounds, H-7-94 and F-7-62, proved to be the best inhibitors of TcCyP19 enzymatic activity as well as the most efficient trypanocidal drugs. With the objective of analysing, at the molecular level, how the structural differences between the three above-mentioned inhibitors justify their different inhibitory activity on TcCyP19, three-dimensional molecular modelling structures were generated to computationally simulate behaviours and interactions. An energy-minimized model of each binary complex in water with ions was obtained. These models were then used as starting point for molecular dynamic simulations, performed with GROMOS96 program. With the resulting set of co-ordinates and energies, a comparison of the interaction between CsA and both CsA analogues in T. cruzi and human cyclophilins were performed. Within the different magnitudes analysed, the total potential complex energy exhibited the best correlation with the experimental data. The results obtained in this study support the use of this methodology when designing new lead inhibitor compounds.

Background: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals., Methodology/principal Findings: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported., Conclusion/significance: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.

We previously hypothesized that the key site of chloroquine (CQ) antimalarial action and resistance development is in nucleus of malaria parasite, and that lysosomal (food vacuolar) accumulation of CQ may benefit parasite's survival through reducing the drug pressure on the key site. To further support our hypothesis we propose that Plasmodium falciparum chloroquine resistance marker protein (Pfcrmp) may be a CQ target protein in nucleus and that CQ resistance in malaria parasites may be caused by genetic alterations in Pfcrmp gene.

Malaria is undoubtedly the world's most devastating parasitic disease, affecting 300 to 500 million people every year. Some cases of Plasmodium falciparum infection progress to the deadly forms of the disease responsible for 1 to 3 million deaths annually. P. falciparum-infected erythrocytes adhere to host receptors in the deep microvasculature of several organs. The cytoadhesion of infected erythrocytes to placental syncytiotrophoblast receptors leads to pregnancy-associated malaria (PAM). This specific maternal-fetal syndrome causes maternal anemia, low birth weight and the death of 62,000 to 363,000 infants per year in sub-Saharan Africa, and thus has a poor outcome for both mother and fetus. However, PAM and non-PAM parasites have been shown to differ antigenically and genetically. After multiple pregnancies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia and clinical symptoms of PAM. The recent description of a new parasite ligand encoded by the var2CSA gene as the only gene up-regulated in PAM parasites renders the development of an anti-PAM vaccine more feasible. The search for a vaccine to prevent P. falciparum sequestration in the placenta by eliciting adhesion-blocking antibodies and a cellular immune response, and the development of new methods for evaluating such antibodies should be key priorities in mother-child health programs in areas of endemic malaria. This review summarizes the main molecular, immunological and physiopathological aspects of PAM, including findings related to new targets in the P. falciparum var gene family. Finally, we focus on a new methodology for mimicking cytoadhesion under blood flow conditions in human placental tissue.

A previous study showed that the 1,10-phenanthroline skeleton was active in vitro against chloroquine-resistant and sensitive strains of Plasmodium falciparum. Based on this skeleton, 8 derivatives of N-alkyl and N-benzyl-1,10-phenanthrolines have been synthesized. This study was conducted to evaluate the in vitro antiplasmodial activity and cytotoxicity of these compounds. The in vitro antiplasmodial activity was tested on two strains of P. falciparum, FCR-3 chloroquine-resistant and D10 chloroquine-sensitive strains, while their cytotoxicity was tested on the Vero cell line. The parasite and cell growth were estimated by hypoxantine-[2,8-3H] uptake after 24- and 72-hour incubation with each compound tested. The control parasite or cell free from any compounds was referred to as having 100% growth. For this radioactive method, the IC50 value showing concentration inhibiting 50% of the parasite growth was determined by probit analysis. The results showed that the highest antiplasmodial activity was observed with (1)-N-benzyl-1,10-phenanthrolinium iodide with the IC50 0.18-0.45 microM, and the IC50 of the compound on Vero cells ranged from 2,582.30 to 7,057.71 microM. The cytotoxic/ antiplasmodial ratio indicates that this compound has high selectivity (10,993 +/- 330.79-38,965 +/- 6,888.27).

New effective antimalarial agents are urgently needed due to increasing drug resistance of Plasmodium falciparum. Phosphorothioate antisense oligodeoxynucleotides (ODNs) silencing of malarial topoisomerase II gene have shown to possess promising features as anti malarial agents. In order to improve stability and to increase intracellular penetration, ODNs were complexed with the biodegradable polymer chitosan to form solid nanoparticles with an initial diameter of approximately 55 nm. The particle zetapotential depended on the chitosan/ODN mass ratio. Nanoparticles with mass ratio of 2:1 displayed a positive surface charge (+15 mV) whereas particles with 1:1 mass ratio were negatively charged (-20 mV). Additionally nanoparticles were found to protect ODNs from nuclease degradation. P. falciparum K1 strain was exposed to the chitosan/ODN-nanoparticles for 48 h in order to examine the effects of chitosan/antisense (AS) and chitosan/sense (S) oligodeoxynucleotide nanoparticles on malaria parasite growth. Both negatively and positively charged antisense nanoparticles as well as free antisense ODNs (in a final concentration of 0.5 microM) showed sequence specific inhibition compared with sense sequence controls. However, nanoparticles were much more sequence specific in their antisense effect than free ODNs. Nanoparticles with negative surface charge exhibited a significantly stronger inhibitory effect ( approximately 87% inhibition) on the parasite growth in comparison to the positive ones ( approximately 74% inhibition) or free ODNs ( approximately 68% inhibition). This is the first study demonstrating the susceptibility of P. falciparum to antisense nanoparticles.

Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death. In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.

Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction.

The malaria parasite, Plasmodium falciparum, infects and replicates in human erythrocytes. Through the use of substrate-specific transport proteins, P. falciparum takes up nutrients from the erythrocyte's cytoplasm. The sequencing and publishing of the P. falciparum genome have made it possible to identify, clone and characterise a number of these transport proteins from the parasite. Since the P. falciparum transport proteins differ from their human homologues, they may provide potential drug targets in the treatment of malaria. An example of a P. falciparum transport protein which seems promising as a drug target is the parasite's hexose transporter. Furthermore, the antimalarial drug artemisinin has been shown to interact specifically with the parasite's Ca2+ pump. A number of other transport proteins are also discussed as possible drug targets.

Background: Over the last decades molecular biologic techniques have been developed to alter the genome and proteome of Tetrahymena thermophila thereby providing the basis for recombinant protein expression including functional human enzymes. The biotechnological potential of Tetrahymena has been proved in numerous publications, demonstrating fast growth, high biomass, fermentation in ordinary bacterial/yeast equipment, up-scalability, existence of cheap and chemical defined media. For these reasons Tetrahymena offers promising opportunities for the development of a high expression system. Yet optimised high yield strains with protease deficiency such as commonly used in yeast and bacterial systems are not available., Results: This work presents the molecular identification of predominant proteases secreted into the medium by Tetrahymena thermophila. A one-step purification of the proteolytic enzymes is described., Conclusion: The information provided will allow silencing of protease activity by either knock out methods or by Tetrahymena specific antisense-ribosome-techniques. This will facilitate the next step in the advancement of this exciting organism for recombinant protein production.

Plasmodium vivax uses a single member of the Duffy binding-like (DBL) receptor family to invade erythrocytes and is not found in West Africa where its erythrocyte ligand, the Duffy blood group antigen, is missing. In contrast, Plasmodium falciparum expresses four members of the DBL family, and remarkably, single-point mutations of two of these receptors (BAEBL and JESEBL) bind to entirely different erythrocyte ligands, greatly expanding the range of erythrocytes that P. falciparum can invade. In this article, we describe the molecular basis of the binding specificity for one BAEBL variant (VSTK) that binds to glycophorin C. We demonstrate that soluble glycophorin C completely blocks the binding of BAEBL (VSTK) to human erythrocytes, requiring 0.7 microM for 50% inhibition, a concentration similar to that required by glycophorin A to block the binding of erythrocyte-binding antigen 175 to erythrocytes. BAEBL (VSTK) does not bind to Gerbich-negative erythrocytes that express a truncated form of glycophorin C because it lacks exon 3. The N-linked oligosaccharide of Gerbich-negative glycophorin C has a markedly different composition than the wild-type glycophorin C. Moreover, removal of the N-linked oligosaccharide from the wild-type glycophorin C eliminates its ability to inhibit binding of BAEBL (VSTK) to erythrocytes. These findings are consistent with the ligand for BAEBL (VSTK) being, in part, the N-linked oligosaccharide and suggest that single-point mutations in BAEBL allow P. falciparum to recognize oligosaccharides on different erythrocyte surface glycoproteins or glycolipids, greatly increasing its invasion range.

Cruzain is an essential cysteine protease of Trypanosoma cruzi and a therapeutic target for Chagas' disease. Eight dogs were infected with T. cruzi; three were treated with an inhibitor of cruzain, K777, for 14 days. Treatment with K777 abrogated myocardial damage by T. cruzi, as documented by histopathological lesion scores and serum troponin I levels.

A Plasmodium falciparum gene closely linked to the chloroquine resistance locus encodes PfCG2, a predicted 320-330kDa protein. In the parasitized erythrocyte, PfCG2 expression rises sharply in the trophozoite stage and is detected in electron-dense patches along the parasitophorous vacuolar membrane (PVM), in the cytoplasm and in the digestive vacuole (DV). Results of extraction and partitioning experiments show that PfCG2 is a peripheral membrane protein. Exposure of trophozoite-infected erythrocytes to trypsin-containing buffer after streptolysin O permeabilization indicates that PfCG2 is exposed to the erythrocyte cytosol at the outer face of the PVM. PfCG2 is highly susceptible to hydrolysis by aspartic and cysteine proteases and shows dose-dependent accumulation in the presence of protease inhibitors. These results suggest that PfCG2 is delivered from the outside face of the PVM to the DV, where it is broken down by parasite proteases. PfCG2 interacts with erythrocyte cytoplasm and may be associated with processes of hemoglobin uptake and digestion by erythrocytic-stage parasites.

Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex.

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

Background: The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM)., Methods: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied., Results: P. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described., Conclusion: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved.

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.

Members of heat shock proteins (Hsp70) family have been considered to respond to a large variety of stressful conditions. But it was suggested that, in pulmonary cells, Hsp response depends more closely on the type of stimulus. The lungs are critical organs potentially subjected to air pollution affecting respiratory function and, therefore, these organs are of particular interest with regard to the stress response. To investigate the stress dependence of Hsp70 response in lungs, we created transgenic mice where the firefly luciferase reporter gene is under the control of the murine hsp70-1 promoter and exposed them to different sublethal toxic conditions. For each condition, the level of transgene induction and pulmonary toxicity were assessed. We found that hsp70-1 promoter was stimulated by heat shock and cadmium but not by ozone, paraquat, and parathion, even if these chemicals induced respiratory distress and lung inflammation. Similar observations were made when expression of the endogenous hsp70-1 gene was analyzed, indicating that our transgenic model was accurately detecting hsp70-1 induction. Thereby, it appeared that hsp70-1 response is selective and depends on signaling pathways triggered by the toxicants rather than by their pathologic toxicity per se. Furthermore, because all the chemicals used in our study have been previously described to increase the level of oxidative stress, it indicates that there is no direct and simple correlation between hsp70-1 response and the level of oxidative stress, but more specific oxidative patterns should be involved in Hsp regulation.