Fig 2: The PDI inhibitor bacitracin inhibits transmission in Plasmodium berghei and Plasmodium falciparum. (A–C) Triplicate P. berghei standard membrane feeding assays with Bacitracin compared to control at concentrations of 0.3, 1 and 3 mM. Individual data points represent the number of oocysts found in individual mosquitoes 12-days post feed. Horizontal bars indicate mean intensity of infection, whilst error bars indicate S.E.M within individual samples. Asterisks indicate P value < 0.05 Mann-Whitney U test, ns indicate P value not significant. (D–F) Triplicate P. falciparum standard membrane feeding assays with Bacitracin compared to control at a concentration of 3 mM. Individual data points represent the number of oocysts found in individual mosquitoes 8-days post feed. Horizontal bars indicate mean intensity of infection, whilst error bars indicate S.E.M within individual samples. Asterisks indicate P value < 0.05 Mann-Whitney U test.

Fig 3: The PDI inhibitor bacitracin reversibly inhibits Plasmodium berghei fertilization. (A) Exflagellation centers in the presence of Bacitracin at 0, 0.03, 0.3, 1 mM. Asterisks indicate P value < 0.05 Paired t test. (B) In vitro ookinete development assay supplemented with Bacitracin at 0, 0.03, 0.3, 1 and 3 mM. Results are shown as percent inhibition in ookinete conversion. Asterisks indicate P value < 0.05 Paired t test, ns indicate P value not significant. (C) Exflagellation centers after 30 minutes in the presence of Bacitracin at 0, 0.03, 0.3, 1. P value < 0.05 Paired t test indicates P value not significant at any concentration. (D) In vitro ookinete development assay supplemented with Bacitracin at 0, 0.03, 0.3, 1 and 3 mM for 30 min prior to removal of Bacitracin. Results are shown as percent inhibition in ookinete conversion. P value < 0.05 Paired t test indicates P value not significant at any concentration. (E) Triplicate counts of free floating male gametes and male gametes in exflagellation centers. Represented as a percentage of total events observed in the presence of 0, 0.3, 1 and 3 mM Bacitracin.

Fig 4: Deletion of PDI-Trans strongly inhibits transmission and is male specific. (A) Diagnostic PCR with genomic DNA templates and primers 69 and 70 to test for the presence of PDI-Trans, and primers 72 and 9 to detect a unique 930 bp product across the integrations site. (B) The bar chart shows ookinete conversion rates for wild type and both ΔPDI-Trans clones. Conversion rate is expressed as a percentage of P28-positive parasites that had progressed to the ookinete stage (error bar indicates SEM; n = 3). Asterisks indicate P value < 0.05 Paired t test (C). In vitro ookinete conversion analysis demonstrates that PDI-Trans mutant shows production cross-fertilisation with the Δnek4 sterility mutant, which produces functional males only, and not with Δmap2 mutant, which produces functional females only. (error bar indicates SEM; n = 3). (D) PDI activity of purified whole parasite active and non-activated gametocytes from wild type, ΔPDI-Trans and ΔPDI-Trans Comp parasite lines. PDI activity is expressed as a percent relative to the positive control (human recombinant PDI). Bacitracin at 1 mM was used on human recombinant PDI as a control for a 50% reduction of activity as outlined by kit protocol. Experiments were performed in triplicate (error bar indicates SEM; n = 3). Asterisks indicate P value < 0.05 Paired t test. (E) Bar chart of ookinete conversion rates for wild type ΔPDI-Trans and ΔPDI-Trans Comp parasite lines. Conversion rate is expressed as a percentage of P28-positive parasites that had progressed to the ookinete stage (error bar indicates SEM; n = 3). Asterisks indicate P value < 0.05 Paired t test.

Fig 5: Constitutive expression of Plasmodium berghei PDI-Trans, and localization on the surface of gametocytes and ookinetes. (A) RT-PCR analysis of PDI-Trans in asexual blood stages using the non-gametocyte producing strain 2.33; non-activated (Gc-) and activated (Gc+) gametocytes; purified in vitro ookinetes and day 21 salivary gland dissected sporozoites. The analysis was complemented with alpha-tubulin loading controls (B). PCR confirmation of integration of egfp into the PDI-Trans locus. Oligonucleotides 35 and 14 were used to detect integration. Oligonucleotide 91 and 92 were used to detect DHFR presence, pbs25 oligonucleotides were used as positive controls. P. berghei WT 2.34 gDNA was used as a negative control for integration. (C) IFA of fixed, non-permeablised PDI-Trans-GFP parasites probed with anti-GFP; exflagellating male gametocytes (top) and ookinetes (bottom). Each panel shows an overlay of GFP fluorescence (green) and DNA labelled with DAPI (blue). White scale bar = 5 μm.