The Armadillo Repeat Protein PF16 Is Essential for Flagellar Structure and Function in Plasmodium Male Gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world\u27s population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite\u27s flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent

Transmission of Artemisinin-Resistant Malaria Parasites to Mosquitoes under Antimalarial Drug Pressure.

Resistance to artemisinin-based combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested. Here, we analyzed P. falciparum clinical isolates from the Greater Mekong Subregion, each demonstrating delayed clinical clearance and known resistance-associated polymorphisms in the Kelch13 (PfK13var) gene. As well as demonstrating reduced asexual sensitivity to drug, certain PfK13var isolates demonstrated a marked reduction in sensitivity to artemisinin in an in vitro male gamete formation assay. Importantly, this same reduction in sensitivity was observed when the most resistant isolate was tested directly in mosquito feeds. These results indicate that, under artemisinin drug pressure, while sensitive parasites are blocked, resistant parasites continue transmission. This selective advantage for resistance transmission could favor acquisition of additional host-specificity or polymorphisms affecting partner drug sensitivity in mixed infections. Favored resistance transmission under ACT coverage could have profound implications for the spread of multidrug-resistant malaria beyond Southeast Asia

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage; Plasmodium falciparum; and; Cryptosporidium parvum; in cell-culture studies. Target deconvolution in; P. falciparum; has shown that cladosporin inhibits lysyl-tRNA synthetase (; Pf; KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both; Pf; KRS1 and; C. parvum; KRS (; Cp; KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED; 90; = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between; Pf; KRS1 and; Cp; KRS. This series of compounds inhibit; Cp; KRS and; C. parvum; and; Cryptosporidium hominis; in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for; Pf; KRS1 and; Cp; KRS vs. (human); Hs; KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis

MOESM1 of An inexpensive open source 3D-printed membrane feeder for human malaria transmission studies

3D CAD file of the bottom part of the membrane feeder

Characterization of Two Autographa californica Nucleopolyhedrovirus Proteins, Ac145 and Ac150, Which Affect Oral Infectivity in a Host-Dependent Manner

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent