Maria C. Rodriguez

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission‐blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock‐out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission‐blocking vaccines.

Characterization of two linear cationic antimalarial peptides in the scorpion Mesobuthus eupeus

Plasmodium falciparum is a pathogen of human malaria which causes millions of deaths per year due to the ever-increasing drug resistance by the parasite, and thus novel antimalarial agents are urgently needed. In this work, we report two cDNA clones from the scorpion Mesobuthus eupeus venom gland, which encode peptides inhibiting the development of Plasmodium berghei, killing intraerythrocytic P. falciparum, and toxic to the Drosophila S2 cell at micromolar concentrations. One peptide of 24 amino acids (named meucin-24) shares high sequence identity to the amino-terminus of a family of scorpion venom long-chain K(+) channel toxins (LcKTx) and two frog antimicrobial peptides (magainin1 and 2). Sequencing genomic DNA of meucin-24 identified this short peptide as a product of a putative guanine-to-adenine RNA editing from a M. eupeus LcKTx transcript. Another peptide, named meucin-25, contains 25 residues and does not share sequence similarity with any known peptides. Circular dichroism analysis of chemically synthesized peptides demonstrates that meucin-24 presents an essential random coil conformation in water, but its alpha-helical content largely increases in the presence of 50% trifluoroethanol, a membrane-mimicking environment. This finding was further verified by its NMR structure that showed an alpha-helical amphipathic architecture in the region of residues 4-20. CD results indicate that meucin-25 mainly adopts a beta-sheet structure in water but TFE promotes its alpha-helical formation, suggesting its conformational flexibility. Killing of intraerythrocytic P. falciparum without harming mammalian cells (erythrocytes and GC-2 cell) make them attractive candidates for antimalarial drug design.

SUPEROXIDE ANION IN ANOPHELES ALBIMANUS HEMOLYMPH AND MIDGUT IS TOXIC TO PLASMODIUM BERGHEI OOKINETES

The mechanisms of Plasmodium spp. elimination in resistant mosquitoes are not completely understood. Some resistant anopheline strains are able to melanize Plasmodium spp. ookinetes in their midguts. Because quinoid compounds are potent catalysts for free radical generation and because these radicals can be generated in association with melanogenesis, it is probable that they play an important role in the elimination of parasites. The production of the superoxide anion (O−2) in the hemolymph and midgut of Anopheles albimanus female mosquitoes and its cytotoxic effect on Plasmodium berghei ookinetes were analyzed. Ookinetes inoculated into the hemocoel of A. albimanus were covered with melanin and then encapsulated by hemocytes within 1 hr. The presence of O−2 in midguts and in hemolymph obtained by perfusion was verified by the reduction of 3-(4,5 dimethylthiazolil-2)-2,5-diphenyl tetrazolium bromide. O−2 was generated in the hemolymph obtained by perfusion and midguts only in the presence of dihydroxyphenylalanine (l-DOPA), and this reaction was inhibited by superoxide dismutase (SOD). Plasmodium berghei ookinetes exposed to hemolymph plus l-DOPA were killed in vitro, but addition of SOD prevented their killing. Prophenoloxidase transcripts were not observed in midgut epithelium, suggesting that toxic compounds may be imported from the hemolymph. These results suggest that A. albimanus hemolymph and midguts produce O−2 that may limit Plasmodium spp. parasite development.

Characterisation and expression of pbs25, a sexual and sporogonic stage specific protein of Plasmodium berghei.

Following gametogenesis and fertilisation in the bloodmeal within the mosquito midgut, the newly formed zygotes of the malaria parasite develop into motile invasive ookinetes. During this development, surface molecules are synthesised de novo including molecules of 21-28 kDa from the zygote-ookinete stages. An antiserum recognising a 26 kDa protein of Plasmodium berghei was used to clone the corresponding gene from a cDNA library, which was shown to be identical to the reported Pbs25 gene sequence. We show here that Pbs25 was detectable in preparations of gametes 30 min post-gametocyte activation, expression continued on zygotes, ookinetes and oocysts indicating there is a significant overlap of expression of the two immunogenic zygote-ookinete proteins belonging to the P25/28 protein family of sexual stage antigens. Biochemical analysis of Pbs25 demonstrates the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. Antibodies recognising Pbs25 impaired parasite development in the mosquito.

AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium activity

Antimicrobial defensins with the cysteine-stabilized alpha-helical and beta-sheet (CSalphabeta) motif are widely distributed in three eukaryotic kingdoms. However, recent work suggests that bacteria could possess defensin-like peptides (DLPs). Here, we report recombinant expression, in vitro folding, structural and functional characterization of a DLP from the myxobacterium Anaeromyxobacter dehalogenans (AdDLP). Circular dichroism analysis indicates that recombinant AdDLP adopts a typical structural feature of eukaryotic defensins, which is also consistent with an ab initio structure model predicted using I-TASSER algorithm. We found that AdDLP is an antimalarial peptide that led to more than 50% growth inhibition on sexual stages of Plasmodium berghei at micromolar concentrations and killed 100% intraerythrocytic Plasmodium falciparum at 10 microM in a time-dependent manner. These results provide functional evidence for myxobacterial origin of eukaryotic defensins. High-level production of the pure anti-Plasmodium peptide without harming mammalian red blood cells in Escherichia coli makes AdDLP an interesting candidate for antimalarial drug design.

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei.

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189 213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.

Cost of immune priming within generations: trade-off between infection and reproduction

Immune priming is a new paradigm in innate immunity. However, most studies have focused on the benefits of priming (enhanced survival and parasite clearance after a second challenge), while little attention has been paid to the costs. In this study, both factors were investigated in Anopheles albimanus primed against Plasmodium berghei. As previously observed in other invertebrates, compared to un-primed mosquitoes, those primed better controlled a challenge from the same parasite, and had a higher survival rate. Although there was no difference in the number of oviposited eggs between primed and control females, hatching rate was lower in primed than in control mosquitoes and it was more likely for control females to produce eggs than for primed females. Furthermore, a trade-off between parasite elimination and egg production was observed among primed mosquitoes, as primed females that successfully fought the infection were unable to produce eggs, but primed females that produced eggs were similarly infected as control un-primed ones. These results concord with recent mathematical models suggesting that reproduction affects immune priming outcomes, and may explain why in some species and under some conditions it seems that immune priming is not occurring.

A bioinformatics approach for integrated transcriptomic and proteomic comparative analyses of model and non-sequenced anopheline vectors of human malaria parasites

Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquitos luminal midgut brush border. Although the genome of the “model” African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax–An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.

Sequence variation of ookinete surface proteins Pvs25 and Pvs28 of Plasmodium vivax isolates from Southern Mexico and their association to local anophelines infectivity

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile-->Thr in Pvs25, while type B2 presented 87 Gln-->Lys/130 Ile-->Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn-->Asp, 110 Tyr-->Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivaxparasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p< or =0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p< or =0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.

Novel antimicrobial peptides isolated from skin secretions of the Mexican frog Hyla eximia.

In this work, we describe the original characterization of peptides and proteins present in the skin secretions of the Mexican amphibian Hyla eximia. To this purpose, a novel water/dark extraction method, as well as the classic electrical stimulation procedure, was applied in order to extract the skin secretion. Two novel antimicrobial peptides He-1 and He-2 were sequenced. In addition, a molecular mass fingerprint revealed more than one hundred different molecules. Eight peptides in homogeneous form were assayed against five species of bacteria. Thereafter, the peptide He-2 demonstrated high antiparasitic activity against ookinete forms of malaria parasites at low concentration.