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Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.
Microbial parasites are adapted to particular host niches for growth and proliferation. One aspect of parasitism is the ability to engage cells that may serve as suitable hosts. Diversified from an alveolate ancestor, the apicomplexan group of parasites has colonized a wide range of animal cell types. The versatility of the apicomplexan style of parasitism is evidenced also in complicated developmental cycles that typically involve differentiation through distinct host cell niches, often between host species (Sibley, 2004). In humans, infections by apicomplexans cause malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), cryptosporidiosis (Cryptosporidum spp.), and babesiosis (Babesia spp.).
For invasion of a host cell, an extracellular apicomplexan parasite first engages specific surface receptors to form a tight interaction, and then invaginates the host membrane as it burrows into the cytoplasmic space. Specific mechanisms for host cell attachment differ between particular parasite lineages and developmental stages (Carruthers and Tomley, 2008; Paing and Tolia, 2014), and accordingly are primary determinants for host cell tropism. Nevertheless, following attachment, diverse apicomplexans use a similar parasite actin-based mechanism for entry into a broad range of cells (Baum et al., 2006; Sibley, 2010).
In addition to physical mechanisms for cellular adhesion and entry, parasite signal transduction is integral to host cell invasion by apicomplexans. In blood-stage merozoites from Plasmodium species and tachyzoites from Toxoplasma gondii, invasion-related signaling genes are required for exocytosis of micronemes, apicomplexan-specific organelles found at the narrow apical end of the parasite body (Collins et al., 2013; Farrell et al., 2012; Lourido et al., 2010). Microneme secretion, in turn, provides factors for host cell attachment and self-motility by invasive parasite forms (Baum et al., 2006; Carruthers and Sibley, 1999; Huynh and Carruthers, 2006; Singh et al., 2010). Cellular calcium acts as a key second messenger for regulation of microneme exocytosis (Carruthers and Sibley, 1999; Singh et al., 2010), and the expansion of calcium-related genes in Apicomplexa as well as their conservation across species suggests their use for similar parasitic processes (Nagamune and Sibley, 2006; Templeton et al., 2004).
Here, we investigated the function of the apicomplexan homolog of the calcineurin, a protein phosphatase complex central to calcium-responsive signal transduction in other eukaryotes. Calcineurin is also a calcium-regulated phosphatase in Plasmodium (Dobson et al., 1999; Kumar et al., 2004); and we identified the loss of the otherwise well conserved complex from some apicomplexans, an evolutionary signature we hypothesized to be related to signal transduction used for parasitism. Using reverse genetic and chemical-genetic approaches, we show that signaling by calcineurin specifically regulates invasion of erythrocytes by Plasmodium falciparum parasites. Extracellular parasites use calcineurin to attach strongly to cells before host cell entry, and this function is independent of the known role for calcium signaling in microneme secretion. We show that calcineurin in P. falciparum regulates the parasite's ability to recognize and engage diverse host cell receptors, and distinguishes signaling for this primary mode of host cell attachment from a second mode of attachment independent of host cell receptors. Calcineurin functions similarly in Toxoplasma gondii to create strong attachment of extracellular parasites to host cells for invasion. Based on its use for cellular adhesion in various host niches by diverse parasites, we propose that calcineurin underlies an ancestral mechanism used by apicomplexans to expand the range of host cells for invasion.
The diversification of parasitic lifestyles in apicomplexan species might have imposed varying requirements for calcium-based signal transduction. We found that orthologs of likely Plasmodium calcium effectors have been differentially acquired or lost in association with branching of major apicomplexan lineages (Figure 1A; Table S1), a pattern perhaps related to varying use of specific cellular processes among species (Aravind et al., 2000). Though otherwise well conserved in Apicomplexa, many piroplasmid species have lost the genes for both the catalytic CnA subunit and the obligate, regulatory CnB subunit of the calcium-responsive calcineurin protein phosphatase complex (Figure 1A,B; Table S1). Calcineurin regulates calcium-related signal transduction for myriad cellular processes in various eukaryotes, including animals and fungi (Rusnak and Mertz, 2000). We therefore hypothesized that coordinated loss of both calcineurin subunits from some apicomplexans might be related to signaling for parasite-specific, calcium-regulated functions. Consistent with a role in the fully differentiated, invasive forms of apicomplexans, previous studies have shown that CnA and CnB are transcribed at similar points in the cell cycle as other genes used for specialized, parasitic functions in asexual, blood-stage P. falciparum and asexual stage T. gondii (Behnke et al., 2010; Bozdech et al., 2003; Le Roch et al., 2003).
Blood-stage P. falciparum requires cellular calcium for passage of invasive merozoite forms between erythrocytes (Singh et al., 2010). To study a possible function for Plasmodium calcineurin near the time of host cell invasion, we considered the drugs FK506 and cyclosporin A, which are each nanomolar inhibitors of physiological, mammalian calcineurin (Fruman et al., 1992). By contrast, both drugs are toxic to P. falciparum only in the micromolar range (Bell et al., 1994; Singh et al., 2014) (Figure S1A), and have been shown to act potently on parasite targets other than calcineurin (Bell et al., 2006). We found that FK506 and cyclosporin A strongly inhibit general parasite development at multiple stages of the intraerythrocytic cell cycle (Figure S1B), precluding their use for assessment of specific parasitic processes.
To specifically study P. falciparum (Pf) calcineurin, we undertook a genetic approach. Both CnA and CnB are required for the function of other eukaryotic homologs of calcineurin (Klee et al., 1998). We were unable to directly knockout pfcnb, the gene for the parasite CnB, by double crossover homologous recombination at the genetic locus (Maier et al., 2008), suggesting an essential function for calcineurin at the blood-stage. We therefore modified endogenous pfcnb with a 3’ dd-tag to permit conditional protein expression of PfCnB fused to a destabilizing domain (DD) (Figure S1C,D) (Banaszynski et al., 2006; Dvorin et al., 2010a). Expression of the PfCnB-DD protein, when stabilized by the cell-permeable, small molecule Shield-1 (Shld1), increases strongly at the schizont-stage (Figure 1C), when merozoites mature and prepare for invasion of uninfected erythrocytes upon their release from infected cells. As observed previously for PfCnA (Kumar et al., 2005), endogenously expressed PfCnB protein tagged with GFP is predominantly cytoplasmic in schizonts (Figure S1E).
Removal of Shld1 from blood-stage culture reduces cellular levels of PfCnB-DD by >2-fold (Figure 1D; Figure S1F), and inhibits parasite proliferation from one cell cycle to the next cycle by ~70% (Figure 1E). We observed also a strong proliferation defect with knockdown of PfCnA fused to DD (Figure S1G,H), suggesting, as with distant eukaryotic homologs (Fox et al., 2001; Klee et al., 1998), that both subunits of the complex are required for functional calcineurin in parasites.
To determine the timing of action of calcineurin in the blood-stage, we monitored progression through the cell cycle following PfCnB-DD knockdown. In tightly synchronized parasites, knockdown does not influence intraerythrocytic development or the rupture of fully mature schizonts for release of invasive merozoites, but strongly reduces their re-invasion to ring-stage parasites (Figure 1F-H). For the PfCnB-DD-knockdown parasites (No Shld1) that do establish rings, infected erythrocytes develop normally through the ensuing cell cycle (Figure S1I). Shld1-dependent depletion of PfCnA-DD also limits re-invasion to rings without influencing intraerythrocytic development (Figure S1J,K). These data show that the parasite calcineurin complex is a specific regulator of erythrocyte invasion by merozoites.
In Plasmodium merozoites, cellular calcium stimulates microneme release and secretion of invasion ligand proteins onto the apical end of the plasma membrane surface for engagement of erythrocyte receptors (Singh et al., 2010). Because calcium uses specific effectors like PfDOC2.1 to stimulate microneme exocytosis (Farrell et al., 2012), we tested if calcium acts also through calcineurin to stimulate release from this organelle. In contrast to PfDOC2.1, we found that calcineurin knockdown through PfCnB-DD depletion does not limit the regulated exocytosis of the invasion ligand EBA-175 from micronemes (Figure 2A). Similarly, we found that calcineurin does not influence exocytosis of the invasion ligand PfRh2a from rhoptry-necks near the apical end of the parasite; we nevertheless demonstrated a function for PfDOC2.1 upstream of secretion from this organelle (Figure 2B).
In other eukaryotes, FK506 binds to calcineurin to inhibit its phosphatase activity (Klee et al., 1998). While micromolar concentrations of FK506 are required for toxicity toward wild type parasites and do not appear to be specific for calcineurin (Figure S1A,B), we observed that nanomicromolar concentrations of the drug inhibit re-invasion by transgenic PfCnB-DD parasites (IC50 at 0.2 μM Shld1: 8 ± 5 nM, mean ± range, n=2 experiments) (Figure 2C). The potency of low FK506 concentrations toward parasite invasion is thus enhanced by fusion of the PfCnB protein to DD. In contrast to higher concentrations, low FK506 does not influence intraerythrocytic development of PfCnB-DD parasites (Figure S2A). We also observed that the magnitude of inhibition of invasion by low FK506 increases with Shld1 levels in the PfCnB-DD line, demonstrating an association with functional calcineurin in these parasites (Figure 2C; Figure S2B). Taken together, our findings show that low concentrations of FK506 specifically target calcineurin to limit invasion in transgenic PfCnB-DD parasites.
The specific sensitivity of the PfCnB-DD line to low FK506 permits a chemical-genetic approach for assessment of physiological calcineurin that complements and augments Shld1-based knockdown. We found that 100 nM FK506 reduces invasion by PfCnB-DD parasites by up to 95% when combined with Shld1-dependent protein depletion (Figure 2D). The use of FK506 also confirms the independence of calcineurin from regulated exocytosis from micronemes (Figure 2D), demonstrating that calcium-based signaling for host cell invasion extends to functions beyond the mobilization of parasite organelles. The specific chemical-genetic sensitivity of transgenic parasites to FK506 furthermore argues that the biological function of calcineurin is explained by its catalytic phosphatase activity.
A specific role for calcineurin in erythrocyte invasion suggests influence on either merozoite attachment to the erythrocyte surface, or ensuing host cell entry. To separate the two functions, we used the small molecule inhibitor of actin polymerization, cytochalasin D (CytD), to allow erythrocyte attachment by free merozoites but block entry (Miller et al., 1979) (Figure 3A; Figure S3A,B).
We developed a cellular DNA-sensitive, flow cytometry assay to measure attachment to enucleated erythrocytes by singly-nucleated, CytD-treated merozoites released from multinucleate schizonts (Figure 3B). With nucleic acid staining, flow cytometry reveals a cellular population in the CytD-treated samples corresponding to erythrocyte-attached merozoites: these cells are similarly fluorescent to singly-nucleated ring-stage parasites in mock-treated samples (Figure 3B), are strongly reduced in number by the merozoite attachment inhibitor heparin (Figure 3B), and are similar in abundance to merozoite-attached erythrocytes observed by fluorescence microcopy (Figure S3C). The flow cytometry-based approach shows that PfCnB-DD depletion strongly reduces erythrocyte attachment by merozoites in the presence of CytD, similar to the effect on erythrocyte invasion in absence of CytD (Figure 3B; Figure S3C-D). Calcineurin thus primarily regulates erythrocyte attachment by merozoites preceding their actin-dependent entry into host cells.
Despite independence from regulated exocytosis of factors used to engage host cells (Figure 2), a role for calcineurin in erythrocyte attachment (Figure 3) suggests a close functional association with molecules mediating cellular adhesion. To test for functional associations between P. falciparum calcineurin and specific host-parasite interactions, we measured the influence of PfCnB-DD depletion on the potency of inhibitors targeting various aspects of merozoite-erythrocyte attachment (Figure 4).
Following a primary attachment, a merozoite reorients its apical end toward the erythrocyte surface where more specific interactions develop for the parasite to commence host cell entry (Figure 4A). Heparin blocks adhesive events following initial contact including reorientation, likely through interaction with the highly abundant invasion ligand MSP1 (Boyle et al., 2010; Weiss et al., 2015). PfCnB-DD depletion does not significantly change the sensitivity of invasion by parasites to heparin (Figure 4B); calcineurin function is thus not strongly associated with early phases of erythrocyte adhesion affected by the inhibitor.
Plasmodium-specific PfRh and EBL-family invasion ligands at the apical end of the merozoite engage erythrocyte receptors (Figure 4A), and are likely to be primary determinants of the host cell tropism of the parasite (Duraisingh et al., 2008; Otto et al., 2014). The ligand PfRh5 engages the receptor basigin and is essential for erythrocyte invasion by P. falciparum merozoites (Crosnier et al., 2011). We found that knockdown of calcineurin through depletion of PfCnB-DD increases sensitivity to an invasion-inhibitory antibody against basigin by up to ~2.5- fold (Figure 4C), and treatment with FK506 further sensitizes parasites to this antibody (Figure S4A). Calcineurin is thus required for the robust interaction of PfRh5 with the erythrocyte.
Current evidence indicates that besides PfRh5, other PfRh and EBL-family invasion ligands are to a large degree functionally redundant, a feature that affords the P. falciparum merozoite substantial flexibility in its use of erythrocyte receptors for invasion (Duraisingh et al., 2008). To impair invasion mediated by PfRh and EBL invasion ligands, we used protease or neuraminidase to restrict the cognate erythrocyte receptors (Duraisingh et al., 2008). Depletion of PfCnB-DD or PfCnA-DD, but not PfDOC2.1-DD, sensitizes invasion by parasites to these enzymes, increasing their inhibitory effect by as much as ~5-fold (Figure 4D; Figure S4B). When considered with its synergistic interaction also with PfRh5-basigin, our findings strongly suggest that the calcineurin complex is generally required for the function of host receptor-parasite ligand complexes containing PfRh and EBL molecules, and is thus intimately associated with parasite mechanisms for host cell tropism.
Previously, we found that the cytoplasmic domain (CD) of the invasion ligand PfRh2b is physiologically phosphorylated in schizont-stage parasites (Engelberg et al., 2013). In vitro, extract from mature schizont-stage parasites displays calcium-dependent phosphatase activity toward phosphorylated recombinant PfRh2b-CD, perhaps consistent with dephosphorylation by calcineurin (Figure S4C). However, we were unable to demonstrate specific dephosphorylation of the same PfRh2b residue in vivo (Figure S4D).
The invasion ligand AMA1 is used by diverse apicomplexans and constitutes an additional mode of host cell attachment for Plasmodium merozoites (Harvey et al., 2014). In contrast to attachment via PfRh and EBL-family proteins, attachment through AMA1 is thought to be host receptor-independent because the parasite secretes into the host cell membrane the parasite- derived RON complex of proteins, which directly engages AMA1 to create strong host-parasite interaction (Figure 4A) (Riglar et al., 2011; Srinivasan et al., 2011). To test the influence of P. falciparum calcineurin on attachment via AMA1, we combined knockdown of PfCnB-DD with R1, a soluble peptide which directly blocks interaction of AMA1 with the RON2 subunit of the RON complex (Lamarque et al., 2011). We found that PfCnB-DD depletion reduces by up to 2-fold the invasion-inhibitory potency of R1 (Figure 4E). Similarly, either blocking the PfRh5-basigin interaction or enzymatic restriction of erythrocyte receptors dampens the potency of R1 (Figure 4F; Figure S4E,F). Using invasion-inhibitory antibodies, a previous study also reported functional antagonism between AMA1 and PfRh5 (Williams et al., 2012). Host receptor-dependent pathways of attachment are thus distinguished from the AMA1-RON2 system by a requirement for signaling by calcineurin for function.
PfCnB-DD depletion does not influence the sensitivity of parasites to CytD (Figure 4G) and thus erythrocyte entry following attachment (Figure 4A), underscoring the specificity of calcineurin to host cell attachment.
In vivo, blood flow challenges the formation of adhesive cell-cell interactions (Simon and Green, 2005); we hypothesized that for Plasmodium, factors regulating merozoite-erythrocyte interactions might be especially important for invasion in physiological circulation. To test how calcineurin might regulate erythrocyte invasion in such conditions, we continually resuspended PfCnB-DD parasites to produce hydrodynamic forces similar to those observed in human microvasculature (see Supplemental Information) and measured resulting invasion over a range of Shld1 concentrations. Compared to static PfCnB-DD cultures, we identified an elevated requirement for Shld1 in shaking conditions (~10× increase in EC50) and thus functional calcineurin for re-invasion (Figure 4H; Figure S4G). These results both demonstrate that forces similar to what are observed in vivo, can limit the strength of merozoite-erythrocyte attachment for invasion; and that calcineurin stabilizes attachment against such stresses. By contrast, by reducing hematocrit to prolong the time required for productive erythrocyte encounter by merozoites, we found no evidence that calcineurin regulates free merozoite viability when apart from erythrocytes (Figure S4H). Shaking does not influence the sensitivity of parasite invasion to disruption of the AMA1-RON2 interaction by R1 (Figure S4I), showing that calcineurin is related to a specific adhesive mechanism to stabilize erythrocyte attachment against hydrodynamic stresses.
Plasmodium spp. and T. gondii have diverged substantially since descent from a last common ancestor (DeBarry and Kissinger, 2011). Nevertheless, these parasite species share many features in their styles of host cell invasion, including the use of secretory proteins for a specific and strong host cell attachment followed by an active process of intracellular entry (Carruthers and Tomley, 2008).
To study the biological role of calcineurin in the asexual, tachyzoite form of T. gondii (Tg), we initiated a functional analysis of the endogenous tgcna gene encoding the catalytic subunit. We were unable to directly knock out tgcna by standard approaches, and therefore established a line for conditional knockdown (cKD) of endogenous tgcna induced by anhydrous tetracycline (ATc) (Figure S5A,B) (Meissner et al., 2002). ATc reduces TgCnA protein to undetectable levels in these TgCnA-cKD parasites, and strongly diminishes plaque size in infected host cell monolayers (Figure 5A,B).
Knockdown of calcineurin does not influence replication of TgCnA-cKD tachyzoites in host cells (Figure 5C), nor does it limit parasite egress from the infected host cell when intracellular calcium levels are artificially raised with the calcium ionophore A23187 (Figure 5D). When free TgCnA-cKD tachyzoites are added to a fibroblast host cell monolayer, however, knockdown of TgCnA significantly reduces the total number of parasites that are either strongly adhered extracellularly, or invaded into these cells (Figure 5E). Both with and without ATc, roughly 90% of the total parasites observed in association with the monolayer are internalized within the host cell cytoplasm (Figure 5E). Thus, after producing a sufficiently stable attachment, mutant TgCnA-cKD tachyzoites (with ATc) enter host cells with similar efficiency as tachyzoites with higher levels of TgCnA (no ATc). Reduced association and infectivity of mutant TgCnA-cKD parasites is then explained by defects in host cell attachment by the free tachyzoites. A similar assay in which TgCnA-cKD tachyzoites were mixed with a labeled version of the parental strain as an internal control, confirms that calcineurin knockdown reduces host cell association (Figure S5C).
For T. gondii tachyzoites, calcium stimulates invasion and attachment-related functions including gliding motility, extrusion of the conoid structure at the apical end, and exocytosis of micronemes (Carruthers and Sibley, 1999); we observed that calcineurin knockdown in the TgCnA-cKD line does not influence any of these processes (Figure 5F; Figure S5D-F). Calcineurin knockdown does not influence microneme secretion even with high cellular calcium induced by A23187 (Figure 5F; Figure S5F). When considered with our studies in Plasmodium (Figure 2), this finding argues for the conserved independence of apicomplexan calcineurin from the calcium-regulated exocytosis of this organelle.
To further characterize calcineurin in host cell adhesion, we tested how TgCnA knockdown might influence the moving junction (MJ) formed between the invading tachyzoite and host cell. The MJ is established by RON proteins secreted into the host cell subsequent to microneme secretion and is a feature of a late stage of attachment (Alexander et al., 2005). A defect following this step may be marked by host cells with RON protein but no associated tachyzoites (Figure S5G). For a parental parasite line, ~90% of host cells carrying secreted RON proteins remain associated with extracellularly attached or internalized tachyzoites, and knockdown of calcineurin in the TgCnA-cKD line does not strongly influence the frequency of this association (Figure 5G; Figure S5G). Calcineurin thus regulates an adhesive step preceding MJ formation.
We also generated a strain for conditional knockout (cKO) of tgcna (Figure S5H,I) (Andenmatten et al., 2013). Compared to parasites with intact tgcna in the same population of cells, we found that cKO parasites proliferate very slowly for two weeks in host cell monolayers following induced excision of the gene (Figure S5J-L), providing further evidence for a critical function for calcineurin in T. gondii.
For calcineurin from apicomplexans and other eukaryotes, binding by calcium-bound calmodulin (CaM) is necessary for strong activation of phosphatase activity (Figure 1B) (Klee et al., 1998; Kumar et al., 2004). Previous studies have demonstrated apical localization of CaM in both T. gondii tachyzoites and P. falciparum merozoites, and have suggested a role for CaM in parasite invasion (Matsumoto et al., 1987; Pezzella-D'Alessandro et al., 2001). We confirmed apical localization of endogenous TgCaM fused to YFP in both intracellular and extracellular tachyzoites (Figure 5H), and further investigated subcellular localization of fluorescently tagged subunits of calcineurin. We showed that the cytoplasmic pool of both TgCnA and TgCnB in intracellular tachyzoites becomes strongly enriched at the apical end upon their release into the extracellular medium (Figure 5H; Figure S5M). In a strain co-expressing endogenously tagged TgCnA-mCherry and TgCaM-YFP, we observed co-localization of the two proteins at the apical end of extracellular tachyzoites (Figure 5H). These observations are consistent with a mechanism for TgCaM-based recruitment and activation of cytoplasmic calcineurin for calcium-based signaling within the apical complex of the parasite.
Microbes that rely on intracellular parasitism for growth and proliferation must efficiently recognize and enter host cells to survive. While many other eukaryotic parasites rely on host cell uptake for intracellular entry following attachment, apicomplexans link species and stage-specific mechanisms for engagement of diverse host cell types to a conserved, parasite-driven form of invasion (Sibley, 2011). Here, using various genetic and chemical-genetic approaches in P. falciparum and T. gondii, we identified a specific, key function for parasite calcineurin in host cell invasion by apicomplexans: calcineurin is required for the extracellular parasite to strongly attach to the host before intracellular entry. The functional conservation of calcineurin across evolutionary divergent apicomplexan lineages indicates that its appropriation for host cell attachment was an early innovation for a parasitic lifestyle by a common ancestor. The role for calcineurin in a specific form of attachment via host receptors shows a conserved link between species-specific mechanisms determining host cell tropism to a general mode of apicomplexan invasion applied to diverse host cells.
In the evolutionary history of apicomplexans, the ability to physically engage other kinds of cells was a likely pre-requisite for the transition to parasitism by a free-living ancestor. The appropriation of calcineurin for host cell adhesion may then well be one of the most primitive features of these parasites. We showed in blood-stage P. falciparum that calcineurin enhances invasion most strongly when host receptors are limited. In nature, such an ability to overcome restrictions associated with receptor availability is likely to help expand the host cell niche, and by the same token, sustain parasite proliferation in host environments where primary interactions between parasite and target cell are variable. Parasites likely evolved the AMA1-RON2 system of attachment for a similar purpose: the use of a parasite-supplied receptor for an invasion ligand probably helps expand the range of susceptible target cells (Harvey et al., 2014). AMA1-RON2 engagement follows closely in time the development of interactions with host receptors by the parasite (Riglar et al., 2011; Weiss et al., 2015), and our finding of functional antagonism between calcineurin and AMA1-RON2 supports the view that the two systems of attachment share also some degree of functional overlap. The diversity of invasive forms among apicomplexans for various host cell niches may suggest additional species or stage-specific mechanisms for host cell attachment that complement and compensate for calcineurin.
For P. falciparum merozoites, increasing evidence argues that PfRh and EBL invasion ligands are key regulators of host cell tropism (Hayton et al., 2008; Otto et al., 2014). The requirement of calcineurin for the function of host receptor interactions involving PfRh and EBL molecules shows that parasite signal transduction is fundamental to a specific mechanism for host cell recognition by the parasite, independent of invasion ligand release from apical organelles. (Given our findings, a previous report that CsA and FK506 inhibit microneme exocytosis at high concentrations (Singh et al., 2014) might reflect the action of targets secondary to calcineurin.) Previously, the use of inhibitory antibodies showed that PfRh5 synergistically cooperates with the more variably expressed PfRh and EBL-family ligands to promote invasion (Williams et al., 2012), with a more recent study further providing evidence that PfRh5 acts downstream of other PfRh and EBL ligands (Weiss et al., 2015). Our work shows that these invasion ligands are additionally related through a common mechanism of parasite signal transduction involving calcineurin. Calcineurin might thus be exploited as a point of intervention to impair multiple, molecularly distinct interactions of merozoites with erythrocytes. The use of calcineurin by T. gondii tachyzoites for attachment to non-erythrocyte cells further supports the view that apicomplexans have wired the signaling mechanism to diverse host receptor-parasite ligand interactions.
For P. falciparum, infected erythrocytes have developed mechanisms to adhere to epithelial cell receptors when under physiological flow (Crabb et al., 1997), but to our knowledge the influence of hydrodynamic forces on erythrocyte attachment by merozoites has not previously been studied. The ability to tune functional calcineurin levels in the PfCnB-DD line allowed us to determine that forces produced by experimental agitation restrict merozoite-erythrocyte attachment for invasion in blood-stage cultures. A role for calcineurin at a step sensitive to such stresses therefore shows that mechanisms used for high-affinity cellular interactions are coupled directly to target cell recognition. For various apicomplexans, many individual pairwise interactions between a host receptor and parasite ligand are of low affinity (Paing and Tolia, 2014). The challenging conditions of many host environments may well impose requirements for additional cellular mechanisms, such as one we have identified for calcineurin, to strengthen cell-cell adhesion.
Calcineurin is central to signal transduction in diverse eukaryotes. The specific role we have identified for calcineurin in host cell recognition and attachment by Plasmodium and Toxoplasma, shows that apicomplexans have co-opted and specialized the signal transduction pathway for parasitic functions. In a related manuscript, Philip and Waters (2015) show the importance of Plasmodium berghei calcineurin to erythrocyte invasion in vivo in a rodent model, and additionally demonstrate functions in liver-stage invasion by sporozoites and fertilization by sexual-stage male gametes. Calcineurin is thus a pervasive signal transduction strategy for apicomplexan parasitism, extending across evolutionarily diverse lineages and the developmental stages that describe parasite transmission.
We obtained the D10 and 3D7 P. falciparum wild type strains from the Walter and Eliza Hall Institute (Melbourne, Australia), and cultured these parasites as well as derived transgenic P. falciparum in O+ human blood under hypoxic conditions (Trager and Jensen, 1976). Except for the 3D7-based strains used for experiments in Figure S1E and S4D, we created transgenic P. falciparum strains by transfection of plasmids for single crossover integration (Triglia et al., 1998) into D10 parasites. We maintained RH Toxoplasma gondii and its transgenic derivatives in human foreskin fibroblasts (HFF) (Roos et al., 1994). We created strains for anhydrous tetracycline (ATc)-inducible knockdown of TgCnA or conditional knockout of tgcna as previously described (Andenmatten et al., 2013; Morlon-Guyot et al., 2014).
We measured P. falciparum blood-stage proliferation ± 0.2 μM Shld1, essentially as described (Farrell et al., 2012). We measured merozoite attachment or re-invasion in samples following rupture of purified schizonts (± 1 μM CytD) in the presence of ~10-30-fold excess of uninfected erythrocytes under vigorous shaking. To test the effect of orbital shaking on DD-gene influenced erythrocyte invasion, we measured parasitemia following re-invasion in either shaking or static conditions. For the above assays, we measured parasitemia by flow cytometry, following staining of blood-stage samples with SYBR Green I® DNA dye (Invitrogen). We measured proteolytic shedding of merozoite invasion ligands into culture supernatants by densitometric analysis of Western blots. We measured sensitivity of parasites to drugs by light microscopy, flow cytometry or 3H-hypoxanthine uptake. We measured invasion into enzyme-treated erythrocytes by 3H-hypoxanthine uptake (Dvorin et al., 2010b; Fidock et al., 1998).
Plaque assays for parasite growth were performed as previously described (Roos et al., 1994), ± ATc (1 μg/ml). For other assays, TatiΔku80 or TgCnA-cKD parasites were pre-treated ± ATc for ≥48 hrs. In the replication assay, we measured the average number of parasites per vacuole after 24 hrs of infection of a host cell monolayer. We performed the microscopy-based, combined invasion and attachment assay (Farrell et al., 2012); and MIC2 microneme protein secretion assay essentially as described (Carruthers and Sibley, 1999). For the egress assay, we triggered parasite egress with a 5 min treatment with 2 μM A23187 (or DMSO control) at 37°C, and counted intact vacuoles in 10 fields per sample per experiment. For the MJ formation assay, in non-permeabilized samples we counted in random fields the total number of MJs, and determined their association with parasites (Kessler et al., 2008).
We thank Patrick Autissier for assistance with this work, and Drs. Nisha Philip and Andrew P. Waters for careful reading of the manuscript. This study was supported by NIH F32 AI093059 (A.S.P.), Deutsche Forschungsgemeinschaft (K.E.), NIH F32 AI108251 (B.I.C.), American Cancer Society RSG-12-175-01-MPC (M.J.G.), NIH R03 AI107475 (M.J.G.), NIH R21 AI099658 (M.J.G.), NIH R21 AI088314 (M.T.D), and NIH R01 AI091787 (M.T.D.).
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A.S.P. and S.S. designed and performed all experiments, analyzed data, and wrote the manuscript. A.S.P. and R.H.Y.J. performed bioinformatic analysis. K.E. performed in vitro phosphatase assays. A.L.K. and N.E. performed drug assays. B.I.C. performed Western blots. M.G. and C.T.C. assayed subcellular localization. T.W.G., M.J.G., and M.T.D., designed experiments, supervised the study, and wrote the manuscript. All authors edited the manuscript.
Supplemental information includes five Supplemental figures with legends, two Supplemental tables, Extended Experimental Procedures, and Supplemental References.