Summary
Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. The lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of the PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans peptide antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first primary evidence that protein disulphide isomerase function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking compounds or vaccines.
Introduction
Malaria remains a major global health challenge with an estimated 216 million new cases and 445,000 deaths in 2016 [1]. Current tools have substantially reduced the global burden of disease, but recent progress has stalled [1], and it is widely accepted that a range of new tools will be needed to achieve malaria elimination [2]. The causative agent of malaria, the protozoan parasite Plasmodium, is transmitted almost exclusively by mosquitoes of the genus Anopheles. Transmission of Plasmodium from humans to mosquitoes is entirely dependent on the presence of sexually committed gametocytes in circulating blood, which rapidly undergo the process of activation and differentiate into male (micro) and female (macro) gametes upon uptake by the mosquito within a blood meal. The essential process of fertilization is then initiated by gamete adhesion, followed by membrane fusion [3,4]. A small number of proteins have been previously been implicated in plasmodial fertilization; the 6-Cys protein family members P48/45, P47 and P230 have been shown to have a demonstrable role in the mutual recognition and adhesion of micro- and macro-gametes [5-7], whereas the conserved male-specific Class II fusion protein HAP2/GCS1 has been shown to be the key driver of membrane fusion by mediating merger of lipid bilayers [3-4]. Following successful fertilization, resulting zygotes develop into motile ookinetes, establishing infection in the insect host by migration and invasion of the mosquito midgut, allowing for the progression of the parasitic lifecycle. Despite the obvious biological importance of parasitic transmission and its proven previous targeting as a potential point to disrupt the parasitic lifecycle with multiple therapeutics [8], our knowledge of the cellular and molecular mechanisms underlying fertilization and subsequent zygote formation in Plasmodium are surprisingly sparse.
It is widely recognized that to achieve malarial eradication, it will be necessary to use interventions that inhibit the transmission of parasites from humans to mosquitoes [2]. A potential manner of achieving this is by targeting Plasmodium using transmission-blocking interventions (TBIs); i.e. transmission blocking vaccines (TBVs), or transmission blocking drugs (TBDs) against parasitic sexual stages [9]. Antibodies targeted to three of the five currently proven, potent TBV targets have confirmed localization to proteins found on the surface of the plasma membrane of the gametes [10-20], clearly indicating the potential value of targeting this stage of the parasite lifecycle. Additionally, multiple anti-malarial compounds have been demonstrated to have activity against this parasitic stage [21-25]. In summary, the comparatively short life span, increased fragility and availability of proven surface-localized proteins on the male gamete of Plasmodium make targeting this gamete stage of the lifecycle a potential method of inhibiting transmission [26,27]. Similarly, potent TBIs targeting the parasitic ookinete post-fertilization are well characterized in multiple vaccine and drug studies [16,17,25, 28-30].
Protein Disulphide Isomerase (PDI) (EC: 5.3.4.1) is a multifunctional member of the thioredoxin superfamily of redox proteins, characterized by the presence of the βαβαβαββα fold [31]. PDIs typically have three catalytic activities; disulphide isomerase, thiol-disuphide oxidoredctase, and redox-dependent chaperone. PDI homologues have been identified in multiple species, where they are “classically” located in the endoplasmic reticulum (ER) and facilitate the folding and assembly of secretory and membrane proteins within the lumen [32]. In Plasmodium, a small number of proteins have been putatively identified (by sequence homology) as PDI-like molecules in Plasmodium falciparum, vivax, knowlesi, berghei and yoelii [33,34]. Conclusive demonstration of PDI activity has currently only been demonstrated with PF3D7_0827900/PDI-8 [33], with transcription and translation demonstrated in asexual blood schizonts, gametocytes and sporozoites. Knowledge regarding the process of disulphide bond-dependent protein folding in Plasmodium is scarce. Similarly, to target the sexual stages of the malaria parasite further, a deeper understanding of transmission and specifically, the mechanism of fertilization within Plasmodium is vital, and offers the potential for the development of new, effective anti-malarial TBIs. Here, we describe the identification, characterization and role of a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito host in P. berghei. We demonstrate that PDI-Trans is transcribed and translated across the entire parasitic lifecycle, but exhibits activity at the sexual stages of the lifecycle, when fertilization of gametes occurs. We show that PDI-Trans is male specific, essential for successful fertilization/transmission, and exhibits disulphide isomerase function which is up-regulated post-gamete activation. Furthermore, we show that PDI-Trans is a viable anti-malarial drug and vaccine target, expressed on the surface of the sexual stages of Plasmodium, by blocking malarial transmission with the use of repurposed compounds that target PDI activity, and anti-PDI-Trans peptide antibodies. These results demonstrate that protein disulphide isomerase function is essential for malarial transmission, emphasize the potential of anti-PDI agents to act as anti-malarials, and demonstrate the potential utility of rationally selected targets to facilitate the development of novel anti-malarial transmission-blocking interventions.
Results
PDI-Trans is located on the surface on the transmission stages of P. berghei
Previous proteomic analysis of a P. berghei male gamete proteome generated in [35] followed by advanced bioinformatics analysis encompassing a suite of functional and localization-based algorithms [36] identified the expression of PDI-Trans (PBANKA_0820300) in the male gamete, and suggested that the resulting transmembrane protein was potentially located on the surface of the plasma membrane of male gametes. A brief analysis of PDI-Trans is described within [37], where following a BarSeq Screen for asexual growth on an extensive library of non-clonal P. berghei KO parasites, it was posited that the gene is dispensable for the progression of blood-stage parasitemia. Our subsequent analysis of transcription levels by RT-PCR support this, demonstrating that PDI-Trans transcripts were present in wild-type asexual erythrocytic stages of gametocyte deficient strain 2.33, in addition to inactive (Gc-) and activated (Gc+) gametocytes, ookinetes and sporozoites (Figure 1A). To investigate the cellular localization of PDI-Trans across the parasitic lifecycle targeted-single homologous recombination was utilized to generate a transgenic P. berghei parasite expressing the endogenous PDI-Trans protein with a C-terminal EGFP fusion tag. Successful integration following drug selection was confirmed by PCR (Figure 1B). The presence of the EGFP tag caused no observable impact on blood or sexual stages, and did not impact transmission through An. stephensi mosquitoes. Immunofluorescence microscopy on non-permeablized parasites confirmed PDI-Trans-GFP expression on the surface of activated male gametes and ookinetes (Figure 1C). Live microscopy of mixed blood stages and fixed immunofluorescence of sporozoites demonstrated that PDI-Trans-GFP is expressed across the entire parasitic lifecycle (Figure S1).
PDI-Trans is essential for parasite transmission, is male specific and demonstrates classical PDI activity
To investigate the function of PDI-Trans targeted gene disruption was used to replace the entire PDI-Trans coding sequence. This was performed by double homologous recombination as described in [38.39], with constructs designed and manufactured by PlasmoGem (Sanger Institute, UK). Following dilution cloning of drug-resistant parasites, genotyping by PCR of two independently produced clones (Figure 2A) indicates that the replacement construct had integrated at the targeted site, disrupting the endogenous locus. Consistent with previous predictions [37], examination of mice infected with ΔPDI-Trans clones showed that the parasites underwent normal asexual development in erythrocytes (Figure S2). Rates of gametocytogenesis and sex ratio were unaffected, and gametocytes were able to emerge from their host cells and differentiate into gametes when exposed to standard gamete activation conditions (i.e. drop in pH or temperature, presence of xanthaurenic acid). To examine for a specific role during fertilization we specifically examined in vitro ookinete formation in blood collected from mice infected with ΔPDI-Trans parasites. Blood cultures from mice infected with ΔPDI-Trans parasites failed to produce ookinetes, a finding confirmed by triplicate experiments on two independent ΔPDI-Trans clones (Figure 2B). To further explore this phenotype in vivo, An. stephensi mosquitoes were fed on mice infected with ΔPDI-Trans parasites in triplicate, and 12 days later microscopy was used to examine the presence of oocysts (Figure S3). Triplicate experiments of each clone showed a mean reduction of 94.38% inhibition in intensity and 63.68% inhibition in prevalence with in ΔPDI-Trans clone 1, and a 96.43%/65.62% inhibition in intensity/prevalence with ΔPDI-Trans clone 2 when compared to wild type P. berghei (Table 1). These results suggest that Trans-PDI plays a key role in the successful transmission of Plasmodium.
Cross-fertilization experiments, with known gender-specific sexual mutants, such as the male-deficient map2 or the female-defective nek4 mutant [40,41], make it possible to detect gender-specific sterility phenotypes in P. berghei. As shown in Figure 2C, neither Δmap2 nor Δnek4 strains produce ookinetes when cultured in isolation, but when cultures containing both KO lines were mixed, Δnek4 male gametes are able to fertilize map2 female gametes, restoring the capacity to form ookinetes (Figure 2C). Reduced conversion rates (compared with wild type parasites) are expected [3, 40, 41], due to the persistence of Δnek4 female and Δmap2 gametes which are unable to fertilize. In ΔPDI-Trans /Δnek4 crosses the PDI-Trans female gametes were fertilized by Δnek4 male gametes, but Δmap2 females remained unable to differentiate into ookinetes in ΔPDI-Trans/Δmap2 crosses (Figure 2C), indicating that ΔPDI-Trans males are sterile. Thus, these results demonstrate that during plasmodial fertilization, PDI-Trans is essential for microgamete (male) fertility.
PDI enzymes typically catalyze the rearrangement of disulphide bonds between cysteine residues within proteins. To determine whether PDI-Trans exhibits classical PDI activity we utilized a fluorescent PDI insulin-reduction assay to determine reductase activity. Recombinant human PDI was used as a positive control for PDI activity, and the well-characterized PDI inhibitor bacitracin was used as a negative control. Gametocytes from ΔPDI-Trans, ΔPDI-Trans Comp and wild type lines were purified on a density gradient and used within the assay in either an inactive, or activated form. PDI activity was expressed as a percentage relative to the positive control (Figure 2D). In wild type parasites, PDI activity is increased post-activation, implicating broad PDI activity throughout gamete activation/fertilization. The activated gametes of the ΔPDI-Trans line had significantly reduced PDI activity with respect to wild type gametes, specifically indicating that PDI-Trans exhibits true PDI reductase function during fertilization. PDI activity was significantly increased when ΔPDI-Trans was complemented with the endogenous gene (ΔPDI-Trans Comp). To investigate whether complementation restored not only PDI activity, but the ability of these parasites to successfully fertilize we performed ookinete conversion assays. Wild type parasites had a mean conversion rate of 77.98%. Ookinete conversion was not observed in the ΔPDI-Trans parasite line. Conversely, ΔPDI-Trans Comp parasites exhibited a mean ookinete conversion rate of 72.25% indicating that complementation of the PDI-Trans restored the ability to of male gametes to fertilize. Assays were performed in triplicate (Figure 2E).
Malarial transmission is inhibited reversibly by the PDI inhibitor Bacitracin in P. berghei and P. falciparum
To further explore PDI-Trans activity, and to examine the ability of specific PDI inhibitors to block malarial transmission, we utilized the classical PDI inhibitor bacitracin [42]. The addition of bacitracin during fertilization in vitro resulted in a dose-dependent reduction of observable exflagellation centres (motile male and female gametes bound to each other), with a complete inhibition in formation of exflagellation centers at 3 mM (Figure 3A). This subsequently inhibited the ability of mature gametocytes to form ookinetes, with complete inhibition of ookinete conversion at 3 mM bacitracin (Figure 3B). In order to test whether bacitracin has a broad and non-specific toxic effect on parasites, potentially unrelated to PDI-Trans function, gametocytes were pre-incubated in bacitracin at a range of concentrations for 30 minutes, washed to remove the PDI inhibitor, then assayed for formation of exflagellation centers/ookinetes respectively (post-wash). Results show that parasites pre-incubated with bacitracin, then washed, resulted in no significant difference (Paired t test) in the number of exflagellation centres compared to the untreated control across all concentrations examined (Figure 3C-D).
In an attempt to further examine the activity of PDI-Trans and the mechanism of PDI-inhibitor based blockade of transmission, following bacitracin treatment we examined the number of visible free floating male gametes present, compared with the number of exflagellation centers present (defined as three or more male gametes adhered to a female gamete). As bacitracin concentrations increased. the number of visible exflagellation centres decreased, conversely, the number of free floating observed microgametes was unaffected (Figure 3E), suggesting that PDI activity is essential for the association of male and female gamete to form exflagellation centers, but not for the ability of gametogenesis/activation to form microgametes.
To test the ability of bacitracin to block the transmission of P. berghei ex vivo we performed standard membrane feeding assays (SMFA) in triplicate with 0.3, 1 and 3 mM doses of bacitracin (Figure 3 F-H). Bacitracin inhibited transmission at all concentrations in a dose-dependent manner with a maximal inhibition in oocyst intensity of 98.21% and infection prevalence of 92.48% with 3 mM bacitracin (Table 2).
To investigate if PDI function is implicated in fertilization in additional Plasmodium species, and to extend our observations in P. berghei to human malaria parasites, we performed SMFAs with P. falciparum gametocyte cultures in the presence of bacitracin. Given the results observed with P. berghei we chose to perform the P. falciparum feeds at the highest concentration of bacitracin (3 mM) in triplicate to detect maximal effect (Figure 3 I-K). The addition of bacitracin significantly inhibited transmission, with a mean inhibition in oocyst intensity of 95.05% and an inhibition of oocyst prevalence of 81.71% observed (Table 3).
PDI-Trans can be targeted specifically with antibodies to block transmission in vitro and ex vivo
Considering the surface localization of PDI-Trans on the surface of the microgamete and ookinete, the ability of anti-PDI-Trans antibodies to initiate a specific anti-parasitic transmission blocking response was additionally examined. A polyclonal peptide antibody raised against residues bioinformatically predicted to be within the extracellular domain of PDI-Trans (amino acids 30 - 43). Antibodies were raised in rabbits, IgG purified, and examined for their ability to inhibit transmission in vitro and ex vivo. Anti-PDI-Trans IgG recognized both non-permablized gametocyte and ookinetes by IFA (Figure 4A). Staining was absent in secondary only controls, indicating the ability of these antibodies to specifically recognize natively folded PDI-Trans on the gamete and ookinete surface (Figure 4B).
To examine the ability of PDI-Trans to act a transmission blocking antigen, we performed in vitro ookinete conversion assays in the presence of anti-PDI-Trans. Anti-PDI-Trans inhibited ookinete conversion in a dose-dependent manner, further suggesting specificity. At antibody concentrations of 50, 100, 250 and 500 μg/ml, ookinete formation was inhibited by 14.3%, 33.2%, 38.7% and 75.4% respectively. In contrast, as previously demonstrated [4], the presence of UPC10 (negative control) had no effect on ookinete conversion (Figure 4C).
The transmission blocking activity of anti-PDI-Trans antibodies were additionally assessed by triplicate SMFA (Figure 4D-F). Given the in vitro results observed previously (Figure 4D), we assessed the in vivo transmission blocking ability of these antibodies only at the highest concentration where an effect in the in vitro ookinete assay was demonstrated. Anti-PDI-Trans antibodies significantly inhibited P. berghei transmission in all experiments. At a concentration of 500 μg/ml anti-PDI-Trans antibodies inhibited oocyst intensity by a mean of 66.22% and reduced prevalence of infection by 33.16% (Table 4).
Discussion
Fertilization is a key process in the Plasmodium lifecycle, encompassing the active fusion of activated male (micro) and female (macro) gametes to form a zygote within the mosquito bloodmeal. The resulting diploid zygote develops into a motile ookinete, establishing infection in the mosquito midgut and further progression throughout the Anopheline host. Despite its essential nature to the success of the parasitic lifecycle, the cellular and molecular mechanisms that underlie gamete fertilization of male and female gametes remain largely opaque in Plasmodium (and for the majority of Apicomplexa). Gamete interaction is a two-phase process; in the first phase, cell adhesion molecules displayed on the surfaces of the male and female gametes are responsible for gamete-gamete recognition. This initial recognition/adhesion step initiates a signal transduction cascade that activates the sperm and exposes new, fusogenic regions of the sperm plasma membrane. In Plasmodium, only three proteins have been discovered that have a demonstrable role in the mutual recognition of gametes; the 6-Cys family members, P48/45, P47 and P230 [5]. The specific mechanism of action of these proteins are currently unknown. In the absence of many of these surface proteins, there is a significant reduction in the formation of zygotes, however, low levels of fertilization still occur, particularly in vitro [3,6,7], indicating that gametes potentially use alternative, currently unknown, molecules to recognize each other. In the second phase of fertilization, the plasma membranes of the two gametes come into intimate contact and then fuse, bringing about cytoplasmic continuity. The conserved class II fusion transmembrane protein HAP2 is essential for gamete fusion during fertilization, and initiates merger of lipid bilayers post gamete adhesion. Following a (currently uncharacterized) trigger, it is hypothesized that a short-conserved region within the Plasmodium HAP2 ectodomain becomes exposed on the microgamete membrane surface, leading to the alignment of protein subunits parallel to each other, favoring trimerization [4,43,44]. Polar residues on the fusion loop are subsequently inserted into the target (macrogamete) membrane, followed by a conformational change in HAP2 domain III which distorts the target membrane, leading to hemifusion and then fusion/cytoplasmic continuity. Further related upstream and downstream effector molecules that specifically mediate the process of fertilization are at present unclear.
Here, we demonstrate that protein disulphide isomerase function, specifically encoded by a single plasmodial gene (PDI-Trans/PBANKA_0820300) is essential for malarial transmission. We demonstrate that PDI-Trans is constitutively expressed throughout the parasitic lifecycle, in both the blood and mosquito stages, but is only essential in the male gamete, where it is surface expressed. Absence of PDI-Trans only confers a detectable effect post-gamete activation, prior to gamete adhesion, and is null for male fertility, and consequently, zygote/ookinete formation, and transmission to the mosquito host. Complementation of the disrupted locus restores fertility. Furthermore, we conclusively demonstrate specific reductase activity, indicative of “classical” PDI activity, Inhibition of PDI-Trans using the widely available (topical) antibiotic and PDI inhibitor, bacitracin, reversibly blocks plasmodial transmission in vivo and ex vivo. Specifically, the process of gamete activation remains unaffected by bacitracin, whereas the ability of treated gametes to adhere to other cells (i.e. form exflagellation centers) appears to be compromised. Bacitracin-derived transmission blockade is observed in both P. berghei and P. falciparum. Finally, we show that antibodies specifically raised against the extracellular region of PDI-Trans can recognize the surface of the sexual stages of the parasite by immunofluorescence, and can initiate transmission-blocking activity both in vitro and ex vivo.
In all living cells, the appropriate formation and cleavage of disulphide bonds between cysteine residues in secreted and membrane-anchored proteins is essential for native conformation, and therefore, function. PDIs are traditionally known to be versatile enzymes with key roles in the mediation of disulfide bond formation, isomeration and reduction in the endoplasmic reticulum [32]. PDI function is also associated with varied chaperone activity [31]. Little is known regarding the expression and function of PDI-like proteins in Plasmodium. A previous study [33] has bioinformatically identified nine PDI-like molecules across five species of malaria parasites (four in falciparum, one in vivax, berghei, knowlesi and yoelii), indicated by the presence of classical thioredoxin domains. A more detailed analysis of one of these PDI candidates in P. falciparum; PfPDI-8 (PF3D7_0827900), demonstrated expression within the endoplasmic reticulum of asexual blood schizonts, gametocytes and sporozoites, with biochemical analysis indicating a function in the disulfide-dependent conformational folding of a recombinant form of the erythrocyte-binding protein (and putative bloodstage vaccine target) EBA-175. As further evidence of the chaperone function of PDI enzymes, studies utilizing the overexpression of PfPDI-8 resulted in the enhanced expression and folding of the transmission-locking vaccine candidate, Pf25, in a Picha pastoris expression system [59]. Broadly, PDI function is bioinformatically predicted to be conferred by multiple ORF throughout the parasitic genome. Expression, localization and function of these proteins are still largely undefined. Future study to further dissect the function of PDIs (and associated mechanisms of protein folding) in Plasmodium may be advantageous.
Although classically considered to be key mediators of protein folding in the endoplasmic reticulum, key evidence showing localization and function of PDIs in other cell compartments does exist. In some organisms, PDIs have been demonstrated to on occasion escape the ER, and exhibit cytoplasmic and cell surface localization, where their predominant function appears to be the reduction of disulphide bonds [32,45]. In terms of fertilization, PDI activity on the sperm head has previously proved to be essential for sperm-egg cell fusion in multiple vertebrates [31,46-48], and implicated in male fertility in mice [49.50]. PDI function has previously been implicated in the progression of multiple infectious diseases, with a specific role in mediating pathogen entry. In viruses, overexpression of PDI enhances the fusion of vial membranes, leading to increased internalization of HIV-1 [51]. Cell surface PDI has been shown to facilitate the infection of HeLa cells by mouse polyoma virus [52], and in endothelial cells a surface localized (lipid-raft associated) PDI reduces β1 and β2 integrins, allowing for the entry of dengue virus [53,54]. Its function is also essential for release of cholera toxin active chain A from the ER to the cytosol of the infected cell [54,55]. In protozoan pathogens, previous experimentation has demonstrated that increased levels of PDI increase the phagocytosis of the L. chagasi promastigote (but not the amastigote) [56]. It has previously been hypothesized that T. gondii and L. donovani PDI could be putative targets for vaccine development [57.58]. The specific function of PDI-Trans within the parasite is still unknown, however, it is clear that the process of successful fertilization in Plasmodium requires the presence and function of a range of proteins with conserved disulphide bonds between cysteine residues on the gamete surface. The 6-Cys family members P48/45, P47, P230 are all definitively evidenced to mediate gamete adhesion, whereas HAP2 requires the correct formation of multiple crucial disulphide bridges to enable membrane fusion. It cannot be discounted that PDI-Trans may in some way catalyse disulphide bond rearrangement in one of these transmission-essential proteins, exposing key residues critical for fertility-based function. Given the ability of the PDI-Trans knockout described here to undergo normal levels of gametogenesis, function post-activation, but pre-gamete fusion seems likely.
The results described here clearly indicate that PDI-Trans is a potential target for anti-malarial compounds or vaccines to successfully inhibit malarial transmission. The generation of novel TBIs to reduce disease burden is a key component of the current anti-malarial strategy, and it is widely accepted that to achieve eradication, it will be necessary to use interventions that inhibit the transmission of parasites from humans to mosquitoes [2]. We demonstrate that bacitracin reversibly inhibits malarial transmission with high efficacy, and additionally, that antibodies targeting PDI-Trans on the surface of the sexual stages of the parasite mediate significant transmission blocking immunity. It should be noted that bacitracin is already an FDA approved compound, traditionally used clinically against gram-positive compounds. This example illustrates the potential value of re-purposing drugs with observed efficacy against non-malarial species. Previous studies examining the anti-malarial efficacy of bacitracin only examined impact on asexual growth, where no effect was demonstrated [64]. The data here provides further evidence that potent anti-malarial transmission blocking efficacy can be achieved by targeting the male (micro) gamete. Previously described compounds effective against the process of fertilization (methylene blue and atovaquone) are effective in blocking transmission, as are antibodies against multiple male gamete-surface proteins [21-27]. To target the sexual stages of the malaria parasite further, a deeper understanding of transmission and specifically, the mechanism of fertilization within Plasmodium is advantageous, and offers the potential for the development of new, effective interventions. More broadly, PDI-like proteins are expressed across multiple taxa, and species, including in a wide range of organisms of veterinary and clinical importance [31,46]. Given the ability of both anti-PDI-Trans compounds and antibodies to block malarial transmission described here, and the proven role of PDI function in the regulation of infection across multiple species, it is not unreasonable to suggest that further studies may want to examine the possibility of targeting PDI proteins/functions using specifically designed novel anti-malarial drugs or vaccines.
Experimental procedures
General parasite maintenance
General parasite maintenance was carried out as described in [60]. Briefly, P. berghei parasites were maintained in 6–8-week-old female Tuck Ordinary (TO) mice (Harlan) by serial mechanical passage (up to a maximum of eight passages). If required, hyper-reticulosis was induced three days before infection by treating mice intraperitoneally (i.p) with 200 μl phenylhydrazinium chloride (PH; 6 mg/ml in PBS; ProLabo UK). Mice were infected i.p. and infections were monitored using Giemsa-stained tail blood smears as described previously [70].
Generation and analysis of transgenic parasite lines
PDI-Trans-GFP
To examine the expression and localization of PDI-Trans, the PDI-Trans-GFP transgenic line was created, introducing a C-terminal GFP tag to the native by single homologous recombination. The targeting construct pPDI-Trans-GFP was constructed using the backbone of the EGFP-tagging vector p277 [62]. The terminal 1527 bp of the PDI gene (PBANKA_082030) was synthesized (IDT) to remove internal ApaI site and introduce unique AvrII site within the gene and flanking KpnI and ApaI sites to the amplicon. This block was cloned in frame into ApaI/KpnI sites of p277, resulting in pPDI-Trans -GFP. For transfection, this construct was linearized at a unique AvrII site within the PDI sequence.
Parasites were transfected using the Nucleofector device (Amaxa Biosystems) as described previously [62]. Integration of the DNA constructs into the chromosome was confirmed by PCR flanking a region upstream from the 5′ integration site into the EGFP sequence (oligo 35; 5′-GCATGTGCGATTGTATTGGG-3; oligo 14; 5′-ACGCTGAACTTGTGGCCG-3′) and the presence of the DHFR selection cassette (oligo 91 5’-TTCGCTAAACTGCATCGT −3’; oligo 92 5’-GTACTTAATGCCTTTCTCCT-3’). Oligos against the Pbs25 gene (PBANKA_0051500) were used as positive control (oligosF1: 5′-CAACTTAGCATAAATATAATAATGCGAAAGTTACCGTGG-3′;F25′-CCATCTTTACAATCACATTTATAAATTCCATC-3′). GFP expression in transfected, drug resistant parasites were confirmed by fluorescence microscopy. Two independent clones were obtained from two independent transfections, demonstrating identical phenotypes and GFP expression.
ΔPDI-Trans
To examine the function of PBANKA_082030 the ΔPDI-Trans transgenic line was generated. The plasmid was designed and constructed by PlasmoGEM (PlasmoGem ID PbGEM-239637) using recombinase-mediated engineering followed by a Gateway® mediated exchange [39,63]. Prior to transfection the construct was digested by NotI to release the P. berghei insert from its vector backbone. Parasites were transfected using the Nucleofector device (Amaxa Biosystems) as described previously [62]. Integration of the DNA constructs into the chromosome was confirmed by PCR region flanking 5’ of the modified target locus and 3’ DHFR selection cassette (oligo 72; 5′-ACGTGCATGTGCGATTGTATTGGGT −3; oligo 9; 5′-CTTTGGTGACAGATACTAC −3′) and the absence of the wildtype locus (oligo 69 5’-ATGGGAAACTATACTTATATATATATTTTTTTCA −3’; oligo 70 5’-TTATAAATCAGAATTTTCTTCTCCTTC −3’). Two independent clones were obtained from two independent transfections, demonstrating statistically indistinguishable phenotypes.
ΔPDI-Trans-Comp
For the complementation construct the clonal knockout line was injected into mice and mice were treated with 5-Fluorocytosine (5FC) nucleoside analog (Sigma) drinking water, 1.5 mg/ml to recycle the Hdhfr-yfcu marker. The subsequent marker free line was subjected to dilution cloning to achieve a pure population of marker free parasites. Following this the full length endogenous PBANKA_082030 gene was transfected on top using the artificial chromosome library clone mapping to PBANKA_082030 from PlasmoGEM (clone ID PbAC02-74d11) as described previously (Figure S4) [37, 62].
RT-PCR
P. berghei RNA was isolated from gametocyte deficient strain 2.33, activated or inactivated gametocytes, ookinetes and sporozoites from wild type P. berghei 2.34 strain using Trizol reagent (Invitrogen). cDNA synthesis was performed using Prime script kit from (Clonetech). PCR reactions were set up to amplify sections of PDI-Trans ORF (Forward 5’-ATGGGAAACTATACTTATATATATATTTTTTTCA-3’; and reverse 5’-CTACATATTTATCGACATCTCCAA-3’). The expected RT amplicon was 481 bp. The ubiquitously expressed α-tubulin gene PB300720.00.0 was amplified for each sample to ensure amplifiability of cDNA from respective RNA samples (Forward, 5′-CCAGATGGTCAAATGCCC-3′; Reverse, 5′-CTGTGGTGATGGCCATGAAC-3′). The expected products were 435 bp (cDNA). Thirty RT–PCR cycles were carried out with denaturation for 1 min at 94°C, annealing for 45 secs at 50°C, and extension for 1.5 min at 68°C, and products were visualized on a 0.8% agarose gel.
Direct Feeding Assay (DFA)
Routine maintenance of P. berghei was carried out as described above. Prior to challenge, mice were PH treated, and 3 days later infected i.p. with 106 P. berghei ANKA 2.34 or ΔPDI-Trans parasites. Three-days post-infection, animals were anesthetized, and >50 female Anopheles stephensi mosquitoes allowed to blood feed on each mouse. Twenty-four hours later, unfed mosquitoes were removed. Mosquitoes were maintained on 8% (w/v) fructose, 0.05% (w/v) p-aminobenzoic acid at 19-22 °C and 50-80% relative humidity. Day 14 post-feeding, mosquito midguts were dissected and oocyst intensity and prevalence observed by standard phase microscopy and recorded. Reduction in oocyst intensity and prevalence in knockout mice were calculated with respect to wild type controls.
In Vitro Ookinete Conversion Assay (IVOA)
PH-treated mice were injected with 5×107 parasites i.p. On day 3 or 4 of infection, parasitaemia was counted on a Giemsa-stained tail blood smear and exflagellation of male gametocytes was checked by addition of a drop of exflagellation medium to a drop of tail blood. Hosts observed to have exflagellating parasites were exsanguinated by cardiac puncture and each 20 μl of blood taken up in 450 μl ookinete medium. Individual cultures were then added to pre-prepared 24 well plates (Nunc) and incubated for 24h at 19°C. Cultures were harvested after 24h by centrifugation (500×g, 5min), washed once in 100 μl ookinete medium, and the pellet taken up in 50 μl ookinete medium containing Cy3-conjugated Pbs28 mAb clone 13.1 (1:500). Ookinetes and macrogametocytes were then immediately counted by fluorescence microscopy. Ookinete conversion rates were calculated as described previously [19]. In bacitracin experiments harvested parasites were added to ookinete medium containing a range of Bacitracin (Sigma Aldrich: #B0125) concentrations and either left in or washed and put into fresh medium 30 min after drug treatment. In antibody experiments harvested parasites were added to ookinete medium containing anti-PDI-Trans rabbit sera or anti-UPC10 (negative control). In each set of experiments results were collated from three separate experiments and inhibition expressed as the percentage reduction in ookinete conversion with respect to wild type parasites, samples with no bacitracin or the anti-UPC10 control.
Crosses
At day 3 post infection of phenylhydrazine treated mice, infected with parasites with either ΔPDI-Trans, Δnek4, Δmap2 and wt were harvested by heart puncture and mixed at a 1:1 ratio in ookinete medium. After 24 h, ookinete conversion assays were performed by incubating samples with 13.1 antibody (antibody against Pb28 conjugated with Cy3). The proportion of ookinetes to all 13.1-positive cells (unfertilised macrogametes and ookinetes) was established, counting fields at 60 × magnification. Experiments were performed in biological triplicate [40,41].
PDI activity assay
PDI activity was measured in a microplate PDI inhibitor screening assay kit from Abcam (ab139480). Briefly, ΔPDI-Trans ΔPDI-Trans Comp and wild-type gametocytes were purified [72]. Both activated and non-activated gametocytes of each parasite line were used in the assay and the PDI-catalyzed reduction of insulin in the presence of Dithiothreitol resulting in the formation of insulin aggregates which bind avidly to the red-emitting fluorgenic PDI detection reagent were measured on Tecan, Infinite M200 Pro. The background media signal for each sample was subtracted and PDI activity was calculated as a percent relative to the positive control (human recombinant PDI). Experiments were performed in triplicate.
Standard Membrane Feeding Assay (SMFA)
P. berghei
Female An. stephensi (SDA 500 strain) were starved for twenty-four hours and then fed on heparinized P. berghei infected blood using standard membrane feeding methods [60]. For each feed, 350 µl of P. berghei ANKA 2.34 infected blood containing asexual and sexual stages of the parasite was mixed with 150 µl of PBS containing either antibody to yield final concentration of 500 μg/ml or drug at 0.3, 1 and 3 mM. Mosquitoes were handled, maintained and analyzed as described above. Reductions in oocyst intensity and prevalence was calculated with respect to control feeds as described in [4].
P. falciparum
Mature gametocytes of P. falciparum (NF54) were produced in vitro as described previously [65] with slight modifications. Briefly, mature gametocyte cultures (0.5 to 2 % final gametocytaemia) were fed for 15-20 min at room temperature to An. gambiae mosquitoes through an artificial membrane kept at 37 °C. For each feed 300 µl of mature P. falciparum gametocytes were mixed with bacitracin at a concentration of 3 mM. Engorged mosquitoes were housed in pots at 26°C and 60–80% relative humidity. On days 7-9, midguts were dissected and the results analyzed as outlined in the above P. berghei section.
Antibody production
Synthetic peptide to PDI-Trans (VSDDFAKKVNHLTHC) was produced, conjugated to KLH and used to raise polyclonal rabbit antisera (Genscript, USA). Resulting sera was IgG purified and validated by Genscript via ELISA.
Microscopy
Immunofluorescence assay (IFA)
PDI-Trans-GFP parasites were assessed by IFA for the presence of GFP tag with anti-GFP, Roche at a dilution of 1:500. Signal was detected by Alexa Fluor 488-labelled goat, anti-mouse IgG (Molecular Probes) at 1:500. Rabbit antibodies to PDI-Trans were assessed by IFA on wild-type P. berghei ANKA 2.34 gametocytes and ookinetes at a dilution of 1:500. Signal was detected by Alexa Fluor 488-labelled goat, anti-rabbit IgG (Molecular Probes) at 1:500. Parasites were cultured and IFAs were performed as described previously [4]. Slides were visualized under x60 objective magnification using a fluorescence microscope (EVOSFL Cell Imaging System, Life Technologies).
Live imaging
PDI-Trans-GFP parasites were examined for GFP signal by live microscopy. Parasites were cultured and allowed to settle on glass slides before microscopy. Slides were visualized under X40 objective magnification using a fluorescence microscope (Leica DMR).
Statistical Analysis
Statistical analysis was performed using Graphpad Prism. For DFA, SMFA and DMFA, significance was assessed using Mann–Whitney U (to examine differences in intensity) and Fisher’s exact probability tests (to examine differences in prevalence). Parametric ELISA tests were assessed using t-test. P values < 0.05 were considered statistically significant (*** = <0.0001, ***= 0.001, **= 0.001-0.01, *= 0.01-0.05).
Ethical Statement
All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act (PPL 70/8788) and approved by the Imperial College AWERB. The Office of Laboratory Animal Welfare Assurance for Imperial College covers all Public Health Service supported activities involving live vertebrates in the US (no. A5634-01).
Conflicts of Interest
The authors are not aware of any conflicts of interest arising from this work.
Acknowledgements
This work was funded by the MRC (New Investigator Research Grant; award number MR/N00227X/1) A.M.B thanks PATH-MVI for funding. Funders had no role in study design, data collection and interpretation, of the decision to submit the work for publication. We gratefully acknowledge Mark Tunnicliff for mosquito production and Tibebu Habtewold for advice regarding P. falciparum infections.
Footnotes
↵2 Lead Contact
References
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