Abstract
Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.
Introduction
The Plasmodium life cycle alternates between a mosquito and a mammalian host, which involves invasive and replicative stages. When a mosquito probes for the blood meal in an infected human, it ingests the gametocytes, which develop further into gametes that fuse to form the zygote. The zygote transforms into ookinetes, producing hundreds to thousands of oocysts in the mosquito midgut. Following oocyst rupture, sporozoites are released in the hemolymph and further invade the salivary gland (Klug & Frischknecht, 2017; Douglas et al, 2015). Sporozoites transform into liver stages after transmission to the human host, forming thousands of merozoites that initiate the erythrocytic cycle (Ripp et al, 2021; Prudêncio et al, 2006). Sporozoites are highly motile cells and rely on gliding motility to travel through different host species. The gliding motility is powered by the inner membrane complex (IMC) anchored actomyosin machinery termed the glideosome (Baum et al, 2008; Kono et al, 2013). The motor is located in the IMC, separating the flattened alveolar sacs and parasite plasma membrane (PPM) (Gould et al, 2011). The gliding-associated proteins (GAPs) tether MyoA to the IMC and hold the PPM and the IMC together (Boucher & Bosch, 2015). These transmembrane proteins connect the submembrane motor to the extracellular environment (King, 1988)
Plasmodium sporozoites can invade target cells in the mosquito and the mammalian hosts and many proteins that have been implicated during this process are expressed specifically in sporozoites. Thrombospondin-related anonymous protein (TRAP) was found to be the first protein involved in sporozoite gliding motility and host cell invasion. TRAP mutant sporozoites failed to invade the salivary gland, and hemolymph sporozoites were nonmotile (Sultan et al, 1997). TRAP cytoplasmic tails incorporate a C-terminal tryptophan residue that is crucial for interaction with aldolase that connects with an actin-myosin-based motor (Heintzelman, 2015; Buscaglia et al, 2003). S6 is also a TRAP family adhesion, and by disruption, its role has been implicated in parasite adhesion and gliding motility (Steinbuechel & Matuschewski, 2009). The other proteins involved in parasite motility and host cell invasion include MAEBL (Kariu et al, 2002; Saenz et al, 2008), TLP (Heiss et al, 2008), the rhoptry-resident proteins TRSP (Labaied et al, 2007), RON4 (Giovannini et al, 2011), GEST (Talman et al, 2011), TREP/UOS3 (Mikolajczak et al, 2008; Combe et al, 2009), the GPI-anchored circumsporozoite protein (CSP) of the sporozoite and the small solute transporter PAT (Kehrer et al, 2016) and claudin-like apicomplexan microneme protein (CLAMP) (Loubens et al, 2023). The individual functions of these proteins are known. However, how they interact with each other to coordinate gliding motility and invasion is poorly understood.
These proteins are specific to sporozoites; however, several overlapping proteins are known that function in both merozoites and sporozoites. Proteins such as GAP-40, −45, and −50 together with myosin A tail domain interacting protein (MTIP) cluster MyoA with IMC (Poulin et al, 2013; Daher & Soldati-Favre, 2009). In P. falciparum, the peripheral protein GAP45 is myristoylated and palmitoylated, possibly required for membrane targeting (Rees-Channer et al, 2006). GAP45 in Toxoplasma gondii is involved in the recruitment of the motor complex (Frénal et al, 2010). PfGAP50 is a transmembrane protein that anchors the invasion machinery in the inner membrane complex (Baum et al, 2006; Yeoman et al, 2011). In T. gondii, GAP45 and GAP50 form a complex with MyoA and its light chain, MLC1.
While the glideosome and many surface proteins have been studied and found essential for gliding motility and invasion, the role of gliding-associated proteins is unexplored. To identify a novel GAP, we performed bioinformatic studies on S14, whose transcripts were highly upregulated in sporozoites in a suppressive subtraction hybridization study (Kaiser et al, 2004). Similar to GAP45 and −50, we found that S14, while not containing transmembrane domains or signal peptides, is predicted to be secreted via the nonclassical pathway. It also contains a predicted palmitoylation signal, possibly indicating its membrane targeting (Table S1). In this study, we investigated the role of S14 in the P. berghei life cycle. We demonstrate that S14 is an IMC protein interacting with the glideosome-associated proteins MTIP and GAP45. We disrupted the gene and found that S14 is essential for sporozoite gliding motility, host cell invasion, and malaria transmission.
Results
In silico studies show that S14 is secreted via a nonclassical pathway
A study comparing transcriptome differences between sporozoites and merozoites using suppressive subtraction hybridization found several genes highly upregulated in sporozoites and named them ‘S’ genes (Kaiser et al, 2004). We narrowed it down to a candidate named S14, which lacked signal peptide and transmembrane domains. In silico studies revealed that PbS14 is a protein conserved in all Plasmodium species and has no similarity in other organisms (Figure S1). The identity matrix showed that the PbS14 protein is highly conserved among all Plasmodia (Table S2). To understand its possible function, we analyzed several sporozoite-specific proteins. We found that gliding-associated proteins GAP45 and GAP50 show similar properties and contain a palmitoylation signal, secreted via the nonclassical pathway and localized to the inner membrane complex (Rees-Channer et al, 2006). Next, we performed bioinformatic analysis to check whether S14 is targeted to the inner membrane by a nonclassical secretion pathway. We analyzed the presence of transmembrane domains, prediction of palmitoylation signals, and interactions as previously described (Boucher and Bosch, 2015). We found that S14, while not containing transmembrane domains or signal peptides, is predicted to be secreted via the nonclassical pathway. It also contains a predicted palmitoylation signal, possibly indicating its membrane targeting (Table S1). The nonclassical secretion pathway, which depends on palmitoylation and myristoylation, has been identified in most eukaryotes (Rabouille et al. 2012), including P. falciparum (Moskes et al. 2004).
S14 is expressed and localized on the membrane of sporozoites
We started our study by validating the transcripts of S14 in different stages of the parasites using quantitative real-time PCR. We found that S14 was predominantly expressed in midgut and salivary gland sporozoites (Figure 1A). To further investigate whether S14 transcripts are translated, we endogenously tagged the S14 gene with 3XHA-mCherry using double crossover homologous recombination (Figure S2A). The correct site-specific integration was confirmed by diagnostic PCR (Figure S2B). We initiated the mosquito cycle and found that the C-terminal tag did not affect parasite development throughout the life cycle stages (Figure S2C and S2D). We monitored live mCherry expression throughout the parasite life cycle stages. S14-3XHA-mCherry expression was observed in sporozoites but not in the blood and liver stages (Figure 1B and C). Analysis of the mCherry pattern on sporozoites revealed localization on the membrane of the sporozoites (Figure 1D). Furthermore, IFA with anti-CSP and anti-mCherry antibodies confirmed S14 localization on the membrane (Figure 1E). Expression of the S14-3XHA-mCherry fusion protein (∼66.5 kDa) was also confirmed by Western blotting using an anti-mCherry antibody. The appearance of an extra band with higher molecular weight in immunoblotting was possibly due to the palmitoylation of S14 (Figure 1F). Similar electromobility shifts of GAP45 due to the palmitoylation have been reported (Frénal et al, 2010). These results indicate that S14 is a sporozoite-specific membrane protein.
(A) The gene expression of S14 was analyzed using quantitative real-time PCR, which revealed the highest expression in sporozoites. The expression of S14 was normalized to the PbHsp70 transcript. BS; blood stages, Schz; schizonts, MG Spz; midgut sporozoites, SG Spz; salivary gland sporozoites, LS; liver stages. (B) Live microscopy image of midgut oocysts expressing the mCherry reporter. (C) mCherry-expressing salivary gland sporozoites. (D) Salivary gland sporozoites expressing S14-3XHA-mCherry on the membrane. (E) Confirmation of S14 expression on the membrane of the sporozoites by coimmunostaining with surface protein marker anti-CSP antibody. (F) Western blot analysis of S14-3XHA-mCherry salivary gland sporozoite lysates. The blot was first probed with an anti-mCherry antibody, then stripped and reprobed with an anti-CSP antibody.
S14 KO sporozoites egress from the oocyst normally while failing to invade the salivary gland
To investigate the role of S14 in the P. berghei life cycle, we disrupted the gene by double-crossover homologous recombination (Figure S3A). The drug-resistant parasites expressing GFP indicated successful transfection (Figure S3B). We obtained clonal lines of the KO parasites by limiting dilution of the parasites, integration, and the absence of WT locus was confirmed by diagnostic PCR (Figure S3C). Finally, two S14 KO clonal lines were confirmed by Southern blotting, which showed the modified locus in the KO parasites (Figure S3D). The S14-complemented parasite line was also generated to check the specificity of the phenotype (Figure S3E). First, a new S14 KO parasite with yFCU was generated, and then an S14 expression cassette consisting of the 5’UTR, ORF, and 3’UTR was amplified and transfected into S14 KO (yFCU) parasites. After recombination, the WT locus was amplified in S14 comp parasites (Figure S3F). Next, we checked the propagation of the asexual intraerythrocytic cycle of KO parasites, which was comparable to that of WT GFP parasites (Figure S4). To analyze the phenotype of S14 KO parasites in mosquito stages, we transmitted them to mosquitoes by allowing them to probe for a blood meal. We observed the mosquito midgut and salivary glands on day 14 and day 19 post-blood meal. We found that oocyst formation and development of sporozoites were comparable to those of WT GFP parasites (Figure 2A-D). However, no sporozoite-associated GFP signals were observed in salivary glands, and the number of salivary gland sporozoites per mosquito was severely reduced (Figure 2E and F). Genetic complementation of the KO parasites restored salivary gland sporozoite numbers to a level similar to WT GFP parasites (Figure 2F). To investigate whether the KO sporozoites failed to egress from the oocyst or could not invade the salivary gland. We counted the sporozoite numbers in the mosquito hemolymph. We found a higher accumulation of hemolymph sporozoites in KO-infected mosquitoes than in WT GFP-infected mosquitoes (Figure 2G), suggesting that mutant sporozoites failed to invade the salivary gland. These results indicate that mutant sporozoites egress from the oocysts normally but fail to invade salivary glands.
(A) Live microscopy images of the mosquito midgut showing oocysts. (B) The number of oocysts in WT GFP and S14 KO parasites was not significantly different (P= 0.952, one-way ANOVA). (C) Live microscopy images of sporulating oocysts of WT GFP and S14 KO parasites. (D) Midgut oocyst sporozoite number, no significant difference (P= 0.943, one-way ANOVA). (E) Dissected salivary glands showed GFP-expressing sporozoites in WT GFP, while no GFP-expressing sporozoites were observed in S14 KO. (F) Salivary gland sporozoite numbers, negligible sporozoites in S14 KO, a significant difference (***P<0.0008, one-way ANOVA). Complementation restored the sporozoites number in S14 KO parasites at the WT GFP levels, no significant difference (P= 0.434, Student’s t test) (G) S14 KO and WT GFP hemolymph sporozoites were collected on the indicated days post blood meal, and sporozoite numbers were quantified. We found a comparable number of sporozoites on day 15, with no significant difference (P=0.621). There was a higher accumulation of hemolymph sporozoites in S14 KO on days 17-23 with a significant difference from WT GFP (day 19 *P=0.0187, day 17 ***P<0.0001, days 21 and 23 ***P=0.0006, one-way ANOVA).
S14 is essential for malaria transmission
To evaluate the ability of salivary gland sporozoites to transmit malaria, infected mosquitos were allowed to inoculate sporozoites in C57BL/6 mice by enabling them to probe for the blood meal. We found that mice inoculated with WT GFP or S14 comp sporozoites became patent on day 3, whereas S14 KO sporozoites failed to initiate blood-stage infection (Table 1). Next, we checked whether this in vivo infectivity defect was due to negligible salivary gland sporozoite load or mutant sporozoites failed to infect mice. For this, we intravenously inoculated C57BL/6 mice with 5,000 hemolymph sporozoites and found that all the mice inoculated with KO sporozoites failed again to initiate blood-stage infection (Table 1). To determine the stage-specific defect, we performed in vitro assays. First, we infected HepG2 cells with hemolymph sporozoites and observed EEF development at 40 hpi. In three independent experiments, we found EEFs in culture inoculated with WT GFP sporozoites but not in the KO-infected cells (Figure 3A and S5A). Next, we checked the invasion capacity of S14 KO hemolymph sporozoites using double immunostaining, which differentiates outside vs inside sporozoites (Rénia et al, 1988). S14 KO sporozoites showed a complete failure to invade hepatocytes (Figure 3B and S5B), explaining the inability to establish an infection in mice. Next, we performed a development assay to check the mutant sporozoite transformation ability into a bulb-like structure. For this, we incubated the mutant sporozoites in a transformation medium for 4 h. We found that S14 KO sporozoites retain the ability to transform into bulb-like structures (Figure 3C and D). These data demonstrate that S14 KO sporozoites lost infectivity to mammalian hosts due to the inability of sporozoites to invade cells.
(A) HepG2 cells infected with hemolymph sporozoites were immunostained, and EEF numbers were quantified. No EEFs were observed in S14 KO parasites, a significant difference (***P<0.0001, one-way ANOVA). (B) Quantification of sporozoites inside vs outside in invasion assay. All sporozoites were found to be outside in S14 KO parasites, a significant difference (***P<0.0001, one-way ANOVA). (C) S14 KO and WT GFP hemolymph sporozoites transformed into bulbs after incubation for 4 h in an activation medium. (D) Quantifying sporozoite transformation into bulbs, no significant difference (P=0.419, one-way ANOVA).
C57BL/6 mice were inoculated with WT GFP, S14 KO or S14 comp sporozoites by mosquito bite or intravenous injection. The appearance of parasites in the blood was confirmed by making Giemsa-stained blood smear.
S14 localizes on the inner membrane and is essential for parasite gliding motility
Invasion-deficient S14 KO sporozoites indicate that either S14 interacts with host receptors or cannot generate power to invade cells. As S14 lacks a signal sequence and transmembrane domain, we analyzed whether S14 is present on the outer or inner membrane. We treated S14-3XHA-mCherry hemolymph sporozoites with Triton X-100 to remove the outer membrane. The Triton X-100-treated and untreated sporozoites were immunostained with anti-CSP and anti-mCherry antibodies. The CSP signal was lost in Triton X-100-treated sporozoites, whereas the S14-3XHA-mCherry signal was retained (Figure 4A). Western blotting confirmed the IFA result, which detected the mCherry signal in Triton X-100-treated sporozoites (Figure 4B). To further confirm the inner membrane localization of S14, we generated antibodies against two IMC proteins MTIP (Bergman et al, 2003) and GAP45 (Gaskins et al, 2004), and performed IFA. The MTIP and GAP45 signals were retained in Triton X-100-treated sporozoites and colocalized with the mCherry signal (Figure 4A). This result indicates that S14 is present within the inner membrane of sporozoites. Plasmodium parasites actively invade host cells, powered by gliding motility (Frénal et al, 2017). Next, we checked the gliding motility of WT GFP and S14 KO hemolymph sporozoites. WT GFP sporozoites glided normally, whereas S14 KO sporozoites were found to be nonmotile (Figure 4C). We counted the sporozoites associated with or without trails to quantify the percentage of gliding sporozoites. Approximately 53% of WT GFP sporozoites were associated with trails, whereas no trails associated with sporozoites were observed in S14 KO (Figure 4D). These data demonstrate that S14 is an IMC protein that powers the sporozoite’s gliding motility.
(A) Triton X-100-treated sporozoites retained mCherry staining, which colocalized with MTIP and GAP45, while CSP staining was lost, suggesting that S14 is present within the inner membrane of the sporozoites. (B) Triton X-100-treated and untreated sporozoites were denatured in SDS-PAGE sample buffer and resolved on SDS-PAGE. The blot was probed with an anti-mCherry antibody, then stripped and reprobed with an anti-CSP antibody. Detection of the mCherry signal in Triton X-100-treated sporozoites confirmed its presence on the inner membrane. S14 Tr; S14-3XHA-mCherry transgenic parasite. (C) The WT GFP and S14 KO hemolymph sporozoites were allowed to glide for one hour, and the CSP trail left was revealed using a biotin-CSP antibody followed by streptavidin-FITC. All S14 KO sporozoites were nonmotile. (D) Gliding was quantified by counting the CSP trials, and a significant difference was observed (***P<0.0003, one-way ANOVA).
In silico docking revealed the interaction of S14 with GAP45 and MTIP
The gliding phenotype of the S14 KO and its localization on IMC prompted us to investigate the association of S14 with glideosome-associated proteins. We chose two IMC-localized proteins, MTIP (Bergman et al, 2003) and GAP45 (Gaskins et al, 2004), for the interaction studies. We started with bioinformatic studies to check the interaction of S14 with MTIP and GAP45. Structural models of MTIP, GAP45 and S14 were obtained and docking was performed (Figure S6). The ClusPro Docking server binding energy of MTIP-GAP45, S14-GAP45, and S14-MTIP were −1216 Kcal/mol, −898.6 Kcal/mol, and −1024.6 Kcal/mol, respectively. Further, we performed the docking using the HDOCK server for the receptor-ligand interface (Figure S7). The set cut-off confidence score was 0.5 and the confidence score for MTIP-GAP45, S14-GAP45 and S14-MTIP interactions were 0.6400, 0.6976 and 0.7235, respectively. These results indicated that MTIP-GAP45 and S14-GAP45 would possibly bind and S14-MTIP are likely to bind. Furthermore, we analyzed protein-protein interaction using HADDOCK server and obtained top Z score clusters were submitted to PDBe_PISA, which revealed interacting residues interface through hydrogen bonding and salt bridge residues. The receptor-ligand interface residues less than 3Å were visualized using Pymol (Figure 5). The respective receptor-ligand interface are given in Tables S3 and S4. The binding energies and residue interface studies indicate that S14 interact with GAP45 and MTIP.
In silico docking studies using HDOCK server showing binding of P. berghei S14-MTIP, MTIP-GAP45 and S14-GAP45. A list of receptor-ligand interface are given in Table S4. Images were visualized using Pymol 2.5.4 software. (A) S14-MTIP interacting receptor ligand residues (228A CYS-4A GLN, 230A ASN-142A GLU, 231A TYR-4A GLN, 233A CYS-4A GLN, 235A ALA-1A MET). (B) MTIP-GAP45 interacting receptor ligand residues (7A MET −97A ASP, 10A PHE-101A THR, 149A ALA-105A LEU, 152A ASP-108A SER, 255A THR-107A LEU, 157A GLY-112A THR). (C) S14-GAP45 interacting receptor ligand residues (231A TYR-135A LYS, 233A CYS-134A TYR, 237A VAL-133A THR, 246A TYR-130A ILE, 248A TYR-131A TYR).
S14 interacts with GAP45 and MTIP in Yeast two-hybrid assay
To further confirm the in-silico interaction results, yeast two-hybrid assay was performed. For this, P. berghei S14 was cloned into pAS2, and the MTIP and GAP45 genes were cloned into pGAD-C1 yeast two-hybrid vectors. The plasmid containing the S14 gene was cotransformed with MTIP or GAP45 genes in the S. cerevisiae strain PJ69-4A. Interaction studies were performed using LacZ and HIS3 as reporters and revealed a positive interaction of S14 with the MTIP and GAP45 proteins, as these cells were able to grow on SD-Trp-Leu-His plates containing 10 and 25 mM 3-AT (Figure 6). Furthermore, these cells also gave blue color on plates containing X-gal (Figure 6), indicating a positive interaction of the S14 protein with the MTIP and GAP45 proteins. In contrast, a negative control containing two unrelated proteins could not grow on 3-AT plates and did not give a blue color on the X-gal plate. This data confirmed the interaction of S14 with GAP45 and MTIP.
The S14, MTIP, and GAP45 genes were cloned in a yeast two-hybrid vector. The interaction was analyzed using LacZ as a reporter gene on SD-trp-leu plates containing X-gal and the his3+ marker as a reporter gene on SD-trp-leu plates lacking histidine. 3AT was used to prevent any leaky expression of the his3 marker gene.
S14 coordinate gliding motility without affecting IMC and surface protein expression and localization
Plasmodium sporozoites exhibit a substrate-dependent gliding motility for which surface and IMC proteins are employed. We analyzed the expression and organization of two IMC proteins, MTIP and GAP45, and two surface proteins, CSP and TRAP in S14 KO sporozoites. Immunostaining revealed an intact IMC and surface organization in S14 KO sporozoites (Figure 7). This data indicate that S14 performs gliding-specific function and does not affect the organization of IMC and surface proteins.
WT GFP and S14 KO hemolymph sporozoites were spotted on a 12-well slide and air-dried. Immunostaining with anti-GAP45, anti-MTIP, anti-CSP, and anti-TRAP antibodies revealed similar expression and localization in WT GFP and S14 KO sporozoites.
Discussion
This study identified a novel Plasmodium protein, S14, which lacks signal peptide and transmembrane domains. However, it contains a palmitoylation signal, secreted via the nonclassical pathway and localized to the inner membrane complex. We found that S14 is a gliding-associated protein with a core function in gliding motility and host cell invasion. Deletion of S14 resulted in the accumulation of a higher number of sporozoites in hemolymph, which failed to invade the salivary gland and hepatocytes. The inability of S14 KO sporozoites to invade the host cell was due to impaired gliding motility. Furthermore, we show that S14 interacts with the glideosome-associated proteins GAP45 and MTIP. Overall, these results indicate a central role for S14 in coordinating gliding motility and invasion of Plasmodium sporozoites.
We propose that S14 works with GAP45 and MTIP, facilitating gliding motility. It is possible that S14 connects GAP45 and MTIP or could act entirely independently. Both scenarios are possible because the details of this complex are not known. Similar to S14, several parasite proteins play a role in the gliding motility and invasion of both mosquito and human hosts, such as surface protein TRAP (Sultan et al., 1997) and claudin-like apicomplexan microneme protein (CLAMP) (Loubens et al, 2023); however, MAEBL was found to be important for attachment to the salivary gland surface and did not affect sporozoite motility and infectivity to the vertebrate host (Kariu et al, 2002). S14 is not a surface protein with an extracellular domain, and its host cell invasion defect was due to impaired gliding. Cellular transmigration and host cell invasion are prerequisites for gliding motility. Several sporozoite proteins, such as SPECT, SPECT2, and PLP1, differ from S14, as they invade the host cell usually with a deficiency in cell traversal (Ishino et al, 2004, 2005b; Risco-Castillo et al, 2015). Surface proteins such as P36 and P52 show normal gliding motility and cell traversal and play a role in host cell invasion by interacting with host cell receptors (Ishino et al, 2005a; Manzoni et al, 2017; Arredondo et al, 2018).
The parasite gliding machinery consists of the atypical myosin MyoA, MTIP, and ELC1, the glideosome-associated proteins GAP40 and GAP45, and the transmembrane protein GAP50 (Bergman et al, 2003; Frénal et al, 2010). This MyoA interacts with F-actin, which connects with surface proteins through aldolase (Sultan et al, 1997; Jewett & Sibley, 2003; Huynh & Carruthers, 2006). In T. gondii, GAP45 plays a vital role in maintaining the close association of the IMC to the plasma membrane (Frénal et al, 2010). GAP45 also interacts with GAP50 through its C-terminal region, supporting its function as the anchor of the motor complex in the IMC. We selected GAP45 and MTIP for interaction studies with S14 because MyoA was lost upon the downregulation of MTIP. Furthermore, MTIP was found to be reduced in GAP45 knockdown (Sebastian et al, 2012).
Deletion of S14 resulted in the accumulation of a higher number of sporozoites in hemolymph, indicating the dispensable role of S14 during the egress of sporozoites from oocysts. The conditional deletion of GAP45 during P. falciparum asexual blood stages revealed its role in the invasion but not in egress. These results indicate that a functional motor complex is not required for egress from RBCs, which plays a critical role in invasion (Perrin et al, 2018). GAP40 and GAP50 and members of the GAPM family play critical roles in the biogenesis of IMCs during intracellular replication. Parasites lacking GAP40 or GAP50 start replication but fail to complete it, implicating a structural role in maintaining the stability of the developing IMC during replication (Harding et al, 2016). It was shown that IMC is critical for the anchorage and stabilization of the glideosome (Opitz & Soldati, 2002) and is required during the invasion of the host cell (Bargieri et al, 2013; Egarter et al, 2014; Togbe et al, 2008; Meissner et al, 2013). We hypothesize that S14 possibly plays a structural role and maintains the stability of IMC required for the activity of motors during gliding and invasion. S14 deletion does not affect GAP45, MTIP, CSP and TRAP expression and localization, suggesting that it performs motor-related functions only. These results indicate that the S14-associated IMC complex possibly exists in sporozoites and coordinates gliding motility (Figure 8); however, the link between S14, GAP45, and MTIP requires further investigation.
The sporozoite membrane is shown with surface proteins TRAP and CSP. We show that S14 interacts with MTIP and GAP45 and coordinates gliding motility by connecting these two proteins.
Material and methods
Parasites, Mosquitos, and Mice
Plasmodium berghei ANKA (MRA 311) and P. berghei ANKA GFP (MRA 867 507 m6cl1) were obtained from BEI resources, USA. Anopheles stephensi mosquitos were reared at 28°C and 80% relative humidity and kept under a 12 h light/dark cycle as previously described (Gupta et al, 2020). Swiss albino and C57BL/6 mice were used for parasite infections. All animal procedures were approved by the Institutional Animal Ethics Committee at CSIR-Central Drug Research Institute, India (IAEC/2013/83).
Protein association prediction for PbS14
Using the “guilt-by-association” principle of prediction, we chose to study the following properties of existing glideosome components along with S14: (1) Classical pathway secretion using the signal peptide (SignalP). (2) Nonclassical pathway secretion (SecretomeP). (3) Presence of transmembrane domains (TMHMM). (4) Presence of a potential palmitoylation site (CSS-Palm). (5) Known associations from the literature. This is a similar association prediction method as employed by the STRING database. These properties were chosen on the rationale that the presence of these signals ascertains protein targeting to different cellular membranes. Associating these characteristics with the known glideosome proteins and their interactions (Boucher & Bosch, 2015), we could select a few glideosome components to check for physical interactions using in-silico studies and yeast two-hybrid assay.
S14 expression analysis by RT‒qPCR
For the absolute quantification of S14 transcripts, a standard was generated by amplifying a 120 bp fragment within the S14 ORF (PBANKA_0605900) using primers 1001/1002 (primers are given in Table S5). The amplified product was cloned into the pCR 2.1-TOPO vector. For the normalization of transcripts, Hsp70 was used (Choudhary et al, 2019). Total RNA was isolated from blood stage schizonts (Schz), liver stages (LS), midgut (MG), and salivary gland (SG) sporozoites using TRIzol reagent (Takara Bio, Japan) and an RNA isolation kit (Genetix, India) following the manufacturers’ instructions. cDNA was prepared by reverse transcription using a Superscript cDNA synthesis kit. Real-time PCR was carried out using SYBR green reagent (Takara Bio, Japan), and the ratio of transcript numbers of S14 and Hsp70 was used to determine the copy number.
Generation of S14-3XHA-mCherry transgenic parasites
For the endogenous tagging of S14 with 3XHA-mCherry, two fragments, F1 (0.74 kb) and F2 (0.65 kb), were amplified using 1010/1011 and 1005/1006 and cloned into plasmid pBC-3XHA-mCherry-hDHFR at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts as previously described (Janse et al, 2006). Correct 5’ and 3’ site-specific integration was confirmed by diagnostic PCR using primers 1007/1392 and 1215/1008, respectively (primers are given in Table S5). The clonal lines were obtained by limiting dilution of the parasites and analyzed for expression and localization.
Generation of S14 KO and complemented parasites
P. berghei S14 (PBANKA_0605900) was disrupted by double-crossover homologous recombination. For this, two fragments, F3 (0.73 kb) and F4 (0.637 kb), were amplified using P. berghei ANKA genomic DNA with primers 1003/1004 and 1005/1006, respectively. The fragments F3 and F4 were cloned sequentially into the pBC-GFP-hDHFR vector at XhoI/ClaI and NotI/AscI, respectively, and finally linearized with XhoI/AscI and transfected into P. berghei schizonts (Janse et al, 2006). The drug-resistant GFP-expressing parasites were confirmed for 5’ and 3’ site-specific integrations using primers 1007/1225 and 1215/1008, respectively (primers are given in Table S5). To generate an S14 KO complemented parasite line, another S14 KO parasite line was generated with the hDHFR:yFCU selection cassette. A fragment consisting of the S14 5’UTR, ORF, and 3’UTR was amplified using primers 1003/1006 and transfected into S14 KO parasites. Parasites containing restored S14 loci were selected by negative selection using a 5-fluorocytosine (5-FC) drug (Sigma, USA) as previously described (Srivastava & Mishra, 2022). The clonal lines were obtained by limiting dilution of the parasites, and the absence of S14 ORF was confirmed using primers 1010/1011. Furthermore, two clonal lines were also confirmed by Southern blotting as described previously (Narwal et al, 2022). Fragment F3 was used as a probe to detect the band in Southern blot.
Phenotypic characterization of S14 KO parasites
The S14 KO clonal lines were first analyzed for asexual blood-stage propagation, and for this, 200 µl of iRBCs with 0.2% parasitemia was intravenously injected into a group of mice. Parasitemia was monitored daily by Giemsa staining of blood smears. Next, we initiated infection with KO parasites in Anopheles stephensi mosquitos as previously described (Narwal et al, 2022). On days 14 and 19, midgut and salivary glands were observed for infection, and sporozoite numbers were counted. The hemolymph sporozoites were collected and counted as previously described (Mastan et al, 2017).
In vivo infectivity
To determine the in vivo infectivity of KO sporozoites, C57BL/6 mice were either infected by mosquito bite or by intravenously injecting hemolymph sporozoites. For the bite experiment, ten mosquitos per cage were used. The appearance of parasites in the blood was observed by making Giemsa-stained blood smears.
In vitro infectivity of sporozoites
The in vitro infectivity of sporozoites was tested by infecting HepG2 cells as previously described (Narwal et al, 2022). Fifty thousand cells/well were seeded in 48-well plates containing sterilized coverslips pretreated with collagen. Hemolymph sporozoites (10,000 sporozoites/well for the invasion assay or 5,000 sporozoites/well for EEF development) were added to the HepG2 monolayers, and the plate was centrifuged at 310 g for 4 min and incubated at 37°C in a CO2 incubator. The culture was fixed using 4% PFA at 1 hpi for the invasion assay and 40 hpi for the EEF development assay.
Transformation of sporozoites into early EEFs without host cells
WT GFP and S14 KO hemolymph sporozoites were incubated in a medium containing DMEM with 2 mM L-glutamine, 4.5 g/liter glucose, and supplemented with 10% FBS (Sigma, USA), 500 U/ml penicillin‒streptomycin, (Thermo Fisher Scientific, USA) and 1.25 μl/ml fungizone as previously described (Kaiser et al, 2003). The sporozoites were incubated at 37°C in a CO2 incubator for 4 hrs and then fixed using 4% PFA.
Generation of anti-MTIP and anti-GAP45 antibodies
Affinity-purified polyclonal rabbit antibodies against P. berghei MTIP and GAP45 were developed by GenScript Inc., Piscataway, NJ, against the peptide sequences CVNKDDRKIYFDEKS and CHKYENDSDKLETGS, respectively.
Triton X-100 membrane extraction
Sporozoites (3 ×104) were collected and treated with 1.0% Triton X-100 diluted in PBS and incubated on ice for 30 min as previously described (Bergman et al, 2003). After incubation, sporozoites were spun at 13,800 × g for 20 min at 4°C. Both treated and untreated sporozoites were washed three times with PBS and fixed with 2% paraformaldehyde diluted in PBS or resuspended.
Sporozoite gliding motility assay
To quantify sporozoite gliding motility, a glass eight-well chamber slide was coated with 10 μg/ml anti-CSP antibody in PBS overnight, and the assay was performed as described previously (Stewart & Vanderberg, 1988). Hemolymph sporozoites collected in 3% BSA/DMEM were added at 5,000/well and incubated for 1 h at 37°C in a CO2 incubator. After incubation, sporozoites were fixed with 4% PFA, blocked using 3% BSA/PBS, and incubated with biotin-labeled anti-CSP antibody, and signals were revealed using streptavidin-FITC (Invitrogen, USA). Trails associated with sporozoites were counted using a Nikon 80i fluorescence microscope.
Immunofluorescence assay
Fixed sporozoites were washed with PBS, permeabilized using 0.1% Triton X-100 for 15 min at room temperature, and then blocked with 1% BSA/PBS for 1 hr at room temperature. Further sporozoites were incubated with anti-mCherry developed in rabbit (Novus Biologicals, USA), anti-CSP mouse monoclonal (Yoshida et al, 1980), anti-MTIP, anti-GAP45 or anti-TRAP (Mishra et al, 2023) antibodies. The signals were revealed using Alexa Fluor 594-conjugated or Alexa Fluor 488-conjugated antibodies (diluted 1:1,000; Invitrogen, USA). For the staining of EEFs, fixed cultures were washed with PBS, and staining with anti-UIS4 antibody (Mueller et al, 2005) was performed as previously described (Narwal et al, 2022). Nuclei were stained with Hoechst 33342 (Sigma‒Aldrich, USA) and mounted using Prolong diamond anti-fade reagent (Invitrogen, USA). The images were acquired using a confocal laser scanning microscope with a UPlanSAPO 100x/1.4 oil immersion objective (Olympus BX61WI) at 4X magnification.
Western blot analysis
Sporozoites were pelleted by centrifuging at 14,000 rpm for 4 min and resuspended in Laemmli buffer. Immunoblotting was performed as previously described (Narwal et al, 2022). Briefly, samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane (Bio-Rad, USA), and blocked with 1% BSA. The blot was incubated with an anti-mCherry (diluted 1:1,000, Novus Biologicals, USA) or anti-CSP (Yoshida et al, 1980) antibodies. The membrane was washed and incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (diluted 1:5,000, Amersham Biosciences, United Kingdom). The signals were detected using ECL chemiluminescent substrate (Thermo Scientific, USA) in a ChemiDoc XRS+ System (Bio-Rad, USA).
Bioinformatic approaches for interrogating protein-protein interactions
To check the interaction of S14 (PBANKA_0605900) with MTIP (PBANKA_1459500) and GAP45 (PBANKA_1437600), in-silico docking was performed. The PDB structures of S14, MTIP and GAP45 are not available, therefore, to obtain their model structure, amino acids sequences were submitted to trRosetta server (https://yanglab.nankai.edu.cn/trRosetta). Obtained structural models were validated using SAVE webserver (https://saves.mbi.ucla.edu/) for Quality factors and Ramachandran plots acceptibility. Models with quality factor above 95% were further processed for the protein-protein interaction studies. ClusPro server (https://cluspro.bu.edu/home.php) was used for initial protein-protein docking studies to predict MTIP-GAP45, MTIP-S14 and S14-GAP45 interaction according to their binding energy. Further, PDB files of the models were submitted to the HDOCK server (http://hdock.phys.hust.edu.cn) for their interaction studies using default parameters. The model with the highest docking score was further used for visualization by Pymol 2.5.4 software (http://www.pymol.org/pymol) (Schrödinger & DeLano).
Yeast two-hybrid interaction studies
The P. berghei S14 gene was amplified using primers 2058/2059 and cloned into the Gal4 binding domain-containing vector pAS2 for yeast two-hybrid interaction studies. The MTIP and GAP45 genes were amplified using primer pairs 2060/2061 and 2062/2063, respectively, and cloned into the pGAD-C1 vector in frame with the Gal4 activation domain. These plasmids were cotransformed into S. cerevisiae strain PJ69-4A (Clontech, USA), which contains the lacZ gene from E. coli encoding β-galactosidase and the HIS3 selectable marker as reporter genes. Interaction studies were performed on SD-leu-trp plates containing X-gal or SD-leu-trp-his plates containing 3-aminotriazole (3-AT). The appearance of blue color on X-gal plates and growth on plates containing 10 and 25 mM 3-AT in the absence of histidine indicates a positive interaction.
Statistical analysis
Statistical analysis was done using two-tailed, unpaired Student’s t test or one-way ANOVA in GraphPad Prism software.
Data and Materials Availability
All data are available within this manuscript, and raw data are available from the corresponding author upon reasonable request. Materials generated in this study are available from the corresponding author on request.
Author contributions
AG, AV and SM conceived the idea, designed and performed the experiments, and analyzed the data. SKN and RG performed the experiments. N completed the in-silico protein-protein interaction studies. SG and SA performed the yeast two-hybrid interaction studies. SM wrote the manuscript, and all the authors have read and approved the manuscript.
Conflict of interest
The authors declare that they have no conflicts of interest.
Acknowledgments
We thank Dr. Pratik Narain Srivastava for performing initial bioinformatic studies. We thank BEI Resources, USA, for the parasite strains and plasmids and Dr. Kota Arun Kumar (University of Hyderabad, India) for the pBC-3XHA-mCherry-hDHFR vector. We thank Dr. Photini Sinnis (Johns Hopkins University, USA) for the anti-UIS4 antibody. We acknowledge the THUNDER (BSC0102) and MOES (GAP0118) Intravital and Confocal microscopy facility of CSIR-CDRI. The Council of Scientific and Industrial Research, University Grants Commission, and Indian Council of Medical Research, Government of India research fellowships supported AG, AV, N, RG, SG and SKN. The Ramalingaswami Fellowship grant supported the work (BT/RLF/Re-entry/20/2012). This manuscript is CDRI communication no. 144/2023/SM.
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