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The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei. [ABSTRACT FROM AUTHOR]

Multigene families optimise fitness by providing a set of related genes with possibly different temporal and/or topological expression patterns. We analyse here the structural organisation and sequence diversity of the rDNA, sera and var C Plasmodium falciparum families, and discuss their consequences for parasite biology. The low rDNA copy number, which reduces reshuffling, is probably the corollary of the need for functionally distinct rRNAs in the insect and in the vertebrate host. The unusual intra-genome and population rDNA sequence diversity results in cells equipped with mosaic ribosome sets. The functional constraints are such that ribosome compatibility could influence parasite fitness and contribute to population structuring. Unlike the dispersed rDNA units, the sera family is arranged as a tandem gene cluster, with seven contiguous similar genes, and one more distantly related paralog. We address the question of the inclusion criteria in family definition. We discuss the results concerning the SERA proteins expression and function in the context of the long overlooked multigene family. The var C module is shared by var genes, ‘orphan’ var C and var C pseudogenes. Analysis of 125 var C deduced protein sequences highlights a well-conserved framework, including putative phosphorylation sites, consistent with the proposed function of mediating interaction with cytoskeletal proteins. The 5′ and 3′ flanking sequences of the var C pseudogenes are heterogeneous. In contrast, the flanking sequences of the uninterrupted var C modules show remarkable conservation. This is interesting in view of the silencing activity of the var intronic sequence on var expression. The 5′ flanking sequence dichotomy reported for internal and sub-telomeric var genes extends to the 3′ flanking sequences. This has profound implications for transcription regulation and generation of diversity. The var C family suggests a role for pseudogenes as a diversity reservoir and in genome dynamics by promoting ectopic recombination. [Copyright &y& Elsevier]

The constant selection and propagation of multi‐resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as‐yet untargeted metabolic pathways. Subtilisin‐like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro‐region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full‐length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro‐region. High‐resolution 3D structures of PvS1Cat, alone and in complex with an α‐ketoamide substrate‐derived inhibitor (MAM‐117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α‐keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate‐derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1‐specific inhibitors that may define a novel class of antimalarial candidates. [ABSTRACT FROM AUTHOR]

Background In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. Objectives To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. Methods For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. Results Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. Conclusions The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs. [ABSTRACT FROM AUTHOR]

Abstract: As the malarial parasite Plasmodium falciparum develops inside the erythrocyte, parasite-derived membrane structures, referred to as Maurer''s clefts, play an important role in parasite development by delivering parasite proteins to the host cell surface, and participating in the assembly of the cytoadherence complex, essential for the pathogenesis of cerebral malaria. PfSBP1 is an integral membrane protein of the clefts, interacting with an erythrocyte cytosolic protein, identified here as the human Lantibiotic synthetase component C-like protein LANCL1. LANCL1 is specifically recruited to the surface of Maurer''s clefts in P. falciparum mature blood stages. We propose that the interaction between PfSBP1 and LANCL1 is central for late steps of the parasite development to prevent premature rupture of the red blood cell membrane. [Copyright &y& Elsevier]

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4–P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH 2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC 50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 μM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC 50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC 50 value of 23 μM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8 , were solved at high resolution to decipher the binding mode of these compounds. [Display omitted] • 41 α-ketoamide pseudopeptidic SUB1 inhibitors were designed and synthesized on the solid phase. • Several inhibited SUB1 from P. falciparum and P. vivax with IC 50 values in the 10 nM range. • The best compounds inhibited the parasite growth in culture with IC 50 close to 20 μM. • The crystal structures of three complexes of PvSUB1 with inhibitors were resolved. • The plasmodial SUB-1 peptidase is further supported as a promising antimalaria target. [ABSTRACT FROM AUTHOR]

Background: Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments.Objectives: To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity.Methods: In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine.Results: Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7-18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1-11.8) for Pfk13 WT parasites and was 12.9 nM (9.5-20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5-20.5) versus 9.1 nM (7.9-10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene.Conclusions: Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: A flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [3H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion. Methods: The method is based on the staining of parasites with hydroethidine (HE) and thiazole orange (TO) which allow differential identification of early, trophozoite and late stage of the parasite by flow cytometry. Late stages of the parasites are obtained by incubation in culture for 24 hours. Reinvasion is followed by culturing parasitized red blood cells for 24 h more. Results: Compared to the standard [3H]-hypoxanthine incorporation assay, it gave similar results as expressed by 50% inhibitory concentrations for chloroquine of laboratory strains and “field” isolates. The effect of quinine on the schizont-ring transition was also explored using this method. First data on the inhibition of re-invasion induced by quinine are presented for both P. falciparum-cultured strains and field isolates. Discussion: This method is simple to use event for field isolate study. It is suitable to analyse effect of drugs on steps of the parasite life cycle different for the maturation one. Using this method quinine was found to have a inhibitory effect on re-invasion of red cells by Plasmodium. [ABSTRACT FROM AUTHOR]

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries. [ABSTRACT FROM AUTHOR]

In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies. [ABSTRACT FROM AUTHOR]

Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. Wecharacterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50μM for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1(compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial. [ABSTRACT FROM AUTHOR]

Background: Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC50 in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity. Findings: Molecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged 25.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P.falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1. Conclusions: Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering. [ABSTRACT FROM AUTHOR]

In the course of our work on infectious diseases, we were led to prepare 6-bromo-2-chloro-4-methylquinoline as a starting material. Since surprisingly little has been reported in the literature, the two synthetic steps to this compound were investigated. The synthesis involves a condensation between b-keto esters and 4-bromoaniline and the cyclization of the resulting anilides into 6-bromoquinolin- 2(1H)-one, otherwise known as the Knorr reaction. The 1H NMR monitoring of the first step allowed us to optimize the conditions leading specifically to the anilide without the occurrence of the alternative crotonate. To illustrate the scope of our finding, few additional anilides featuring electron-attracting groups were prepared. The study of their cyclization revealed some unsuspected steric effect governing this second step. Aside from rectifying a few claims in this chemistry, this study led to a three-step preparation of 6-bromo-2-chloro-4-methylquinoline in 48% overall yield from 4- bromoaniline. [ABSTRACT FROM AUTHOR]

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas’ disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation. [ABSTRACT FROM AUTHOR]

Background: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. Results: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retrotranscription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-?, MIF, TGF-β1 and TNF-a mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. Conclusion: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows fora genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications. [ABSTRACT FROM AUTHOR]