Distinct effects of HIV protease inhibitors and ERAD inhibitors on zygote to ookinete transition of the malaria parasite

In an effort to eradicate malaria, new interventions are proposed to include compound/vaccine development against pre-erythrocytic, erythrocytic and mosquito stages of Plasmodium. Drug repurposing might be an alternative approach to new antimalarials reducing the cost and the time required for drug development. Previous in vitro studies have examined the effects of protease inhibitors on different stages of the Plasmodium parasite, although the clinical relevance of this remains unclear. In this study we tested the putative effect of three HIV protease inhibitors, two general aspartyl protease inhibitors and three AAA-p97 ATPase inhibitors on the zygote to ookinete transition of the Plasmodium parasite. Apart from the two general aspartyl inhibitors, all other compounds had a profound effect on the development of the parasites. HIVPIs inhibited zygote to ookinete conversion by 75%–90%, while the three AAA-p97 ATPase inhibitors blocked conversion by 50%–90% at similar concentrations, while electron microscopy highlighted nuclear and structural abnormalities. Our results highlight a potential of HIV protease inhibitors and p97 inhibitors as transmission blocking agents for the eradication of malaria

Serpin overexpression in Plasmodium-infected midgut cells

Summary The design of effective, vector-based malaria transmission blocking strategies relies on a thorough understanding of the molecular and cellular interactions that occur during the parasite sporogonic cycle in the mosquito. During Plasmodium berghei invasion, transcription from the SRPN10 locus, encoding four serine protease inhibitors of the ovalbumin family, is strongly induced in the mosquito midgut. Herein we demonstrate that intense induction as well as redistribution of SRPN10 occurs specifically in the parasite-invaded midgut epithelial cells. Quantitative analysis establishes that in response to epithelial invasion, SRPN10 translocates from the nucleus to the cytoplasm and this is followed by strong SRPN10 overexpression. The invaded cells exhibit signs of apoptosis, suggesting a link between this type of intracellular serpin and epithelial damage. The SRPN10 gene products constitute a novel, robust and cell-autonomous marker of midgut invasion by ookinetes. The SRPN10 dynamics at the subcellular level confirm and further elaborate the 'time bomb' model of P. berghei invasion in both Anopheles stephensi and Anopheles gambiae. In contrast, this syndrome of responses is not elicited by mutant P. berghei ookinetes lacking the major ookinete surface proteins, P28 and P25. Molecular markers with defined expression patterns, in combination with mutant parasite strains, will facilitate dissection of the molecular mechanisms underlying vector competence and development of effective transmission blocking strategies

Toward Anopheles transformation: Minos element activity in anopheline cells and embryos

The ability of the Minos transposable element to function as a transformation vector in anopheline mosquitoes was assessed. Two recently established Anopheles gambiae cell lines were stably transformed by using marked Minos transposons in the presence of a helper plasmid expressing transposase. The markers were either the green fluorescent protein or the hygromycin B phosphotransferase gene driven by the Drosophila Hsp70 promoter. Cloning and sequencing of the integration sites demonstrated that insertions in the cell genome occurred through the action of Minos transposase. Furthermore, an interplasmid transposition assay established that Minos transposase is active in the cytoplasmic environment of Anopheles stephensi embryos: interplasmid transposition events isolated from injected preblastoderm embryos were identified as Minos transposase-mediated integrations, and no events were recorded in the absence of an active transposase. These results demonstrate that Minos vectors are suitable candidates for germ-line transformation of anopheline mosquitoes

<i>Plasmodium</i> M50 proteases.

<p>(A) Conserved catalytic motifs (HExxH and NxxPxxxxDG- highlighted red in grey boxes) from a multiple sequence alignment of S2P orthologues from <i>Plasmodium</i> species and related apicomplexan parasites. (B) 3D homology model of <i>Pb</i>S2P (right panel—PbANKA_1404100) using the open conformation of <i>Methanocaldococcus jannaschii</i> S2P (left panel—PDB id: 3B4R) as a template and Phyre2 as program. The first transmembrane domain is labelled in orange, the second to fourth in blue, and the fifth and sixth in lime green, respectively. The catalytic zinc atom is depicted in red and the catalytic residues are shown surrounding the zinc atom as blue sticks. The orientation within the lipid membrane is also indicated. (C) Magnification of the active site in the <i>Pb</i>S2P homology model, illustrating the structural conservation of the catalytic residues. Strictly conserved residues are shown as sticks and are labelled in black for <i>Pb</i>S2P and blue for the respective homologous amino acid residues in <i>M</i>. <i>jannaschii</i> S2P.</p

<i>s2p(-)</i> parasites show no defects in sexual development nor sporogony.

<p>(A) Exflagellation assay showing formation of exflagellation centres. Mean values (±SD) from three independent experiments are shown. Differences were non-significant (Mann-Whitney test). (B) Ookinete conversion rates of WT and <i>s2p(-)</i> parasites after staining with an antibody against the surface antigen P28 and enumeration of ookinetes, zygotes and macrogametes. Shown are mean values (±SD) from three independent experiments. Differences were non-significant (2way-ANOVA). (C) Immunofluorescence analysis of <i>Anopheles gambiae</i> epithelia sheets infected with WT or <i>s2p(-)</i> parasites. Both strains induce an epithelial response as shown by the SRPN6 antibody (red). Ookinetes are stained with an antibody against surface protein P28 (green) and nuclei are stained with TO-PRO 3 (blue). Scale bar 10 μM. (D) Oocyst numbers of <i>s2p(-)</i> strain compared to the parental WT line after standard membrane feeding assay of <i>An</i>. <i>gambiae</i> mosquitoes from two independent experiments. Black bars show mean values (±SEM). Differences were non-significant (Mann-Whitney test).</p

Formalization of the SPECTRUM methodology in DEVA Signature and logical calculus

The signature and logical calculus of the algebraic specification language SPECTRUM are formalized in the generic language DEVA. This language is designed to express formal methods as well as proofs of propositions about the objects of such methods and the relations between them. This work is understood as a first step towards a formalization of the software development methodology induced by specifying software in SPECTRUMSIGLEAvailable from TIB Hannover: RN 2856(93-04) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman

<i>Pb</i>S2P shows partial co-localisation with the cis-Golgi marker ERD2.

<p>(A) Double labelling IFA of <i>P</i>. <i>berghei</i> schizont cultures using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and α-ERD2 as a Golgi marker (green) showing partial, or in some cases complete, co-localisation. Nuclei are stained with Hoechst (blue). Scale bar 5 μM.</p

Expression and localisation of <i>Pb</i>S2P.

<p>(A) Relative expression levels of <i>PbS2P</i> as determined by qRT-PCR from cDNAs of schizonts (schz), sporozoites (spz), 24h liver stages (LS24) and 48h liver stages (LS48). Transcript levels were normalised to <i>PbHSP70</i> and <i>GFP</i>. (B) Western blot analysis of <i>Pb</i>S2P-HA whole protein extract from purified schizonts of transgenic <i>PbS2P-HA</i> parasites using an α-HA antibody. <i>Pb</i>S2P-HA migrates at 35kDa. (C) Immunofluorescence analysis (IFA) of <i>Pb</i>S2P-HA merozoites, ookinete, oocyst, and salivary gland sporozoites using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and Hoechst stain for the nucleus (blue). For delineation of parasites the following antibodies (green) were used: α-HSP70, schizonts/merozoites; α-MTIP, ookinete and sporozoite; α-PbCap380, oocyst. Prominent localisation of <i>Pb</i>S2P in proximity to the nucleus is present in all invasive stages. Star, apical end of ookinete. Scale bar 5 μM.</p