Plasmodium Sporozoites Research Articles

Plasmodium sporozoite phospholipid scramblase interacts with mammalian carbamoyl-phosphate synthetase 1 to infect hepatocytes

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate.

A Mosquito AgTRIO Monoclonal Antibody Reduces Early Plasmodium Infection of Mice.

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. Passive immunization of mice with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers significant protection against Plasmodium infection of mice. Furthermore, passive transfer of both AgTRIO antiserum and an anti-circumsporozoite protein monoclonal antibody provides synergistic protection. In this study, we generated monoclonal antibodies against AgTRIO to delineate the regions of AgTRIO associated with protective immunity. Monoclonal antibody 13F-1 markedly reduced Plasmodium infection in mice and recognized a region (VDDLMAKFN) in the carboxyl terminus of AgTRIO. 13F-1 is an IgG2a isotype monoclonal antibody, and the Fc region is required for protection. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Limited Plasmodium sporozoite gliding motility in the absence of TRAP family adhesins

BackgroundPlasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated.MethodsFour transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive–negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice.ResultsThe major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement.ConclusionsThe individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility.Graphical

Malaria parasite liver stages

Malaria parasite liver stages

Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of C\xf4te d\u2019Ivoire

BackgroundAlthough malaria and Anopheles mosquito vectors are highly prevalent in Côte d’Ivoire, limited data are available to help understand the malaria vector density and transmission dynamics in areas bordering the country. To address this gap, the Anopheles mosquito species diversity, the members of the Anopheles gambiae complex and the transmission of malaria were assessed in four health districts along the borders of Côte d’Ivoire.MethodsFrom July 2016 through December 2016 and July 2017 through December 2017, adult Anopheles mosquitoes were collected in four health districts of Côte d’Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. The collected mosquitoes were identified morphologically at species level and the members of the An. gambiae complex were separated using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l. and Anopheles nili specimens were analysed for malaria Plasmodium parasite detection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks.ResultsA total of 281 female Anopheles were collected in Aboisso, 754 in Bloléquin, 1319 in Odienné and 2443 in Ouangolodougou. Seven Anopheles species were recorded including An. gambiae s.l. (94.8–99.1%) as the main vector, followed by An. funestus s.l. (0.4–4.3%) and An. nili (0–0.7%). Among An. gambiae s.l., Anopheles coluzzii represented the predominant species in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae sensu stricto (s.s.) was the major species in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0%; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus s.l., Plasmodium falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus s.l. specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria incidence was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared to that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585).ConclusionAnopheles vector species diversity, abundance and Plasmodium sporozoite infection were high within the health districts along the borders of the country of Côte d’Ivoire, resulting in high malaria transmission among the local populations. Anopheles gambiae s.l. and An. funestus s.l. were found to be highly infected with Plasmodium in the health districts of Bloléquin and Odienné where higher malaria incidence was observed than the other districts. This study provides important information that can be used to guide Côte d’Ivoire National Malaria Control Programme for vector control decision-making, mainly in districts that are at the country borders.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay.

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Conservation of S20 as an Ineffective and Disposable IFNγ-Inducing Determinant of Plasmodium Sporozoites Indicates Diversion of Cellular Immunity.

Despite many decades of research to develop a malaria vaccine, only one vaccine candidate has been explored in pivotal phase III clinical trials. This candidate subunit vaccine consists of a portion of a single Plasmodium antigen, circumsporozoite protein (CSP). This antigen was initially identified in the murine malaria model and shown to contain an immunodominant and protective CD8+ T cell epitope specific to the H-2Kd (BALB/c)-restricted genetic background. A high-content screen for CD8+ epitopes in the H2Kb/Db (C57BL/6)-restricted genetic background, identified two distinct dominant epitopes. In this study, we present a characterization of one corresponding antigen, the Plasmodium sporozoite-specific protein S20. Plasmodium berghei S20 knockout sporozoites and liver stages developed normally in vitro and in vivo. This potent infectivity of s20(-) sporozoites permitted comparative analysis of knockout and wild-type parasites in cell-based vaccination. Protective immunity of irradiation-arrested s20(-) sporozoites in single, double and triple immunizations was similar to irradiated unaltered sporozoites in homologous challenge experiments. These findings demonstrate the presence of an immunogenic Plasmodium pre-erythrocytic determinant, which is not essential for eliciting protection. Although S20 is not needed for colonization of the mammalian host and for initiation of a blood infection, it is conserved amongst Plasmodium species. Malarial parasites express conserved, immunogenic proteins that are not required to establish infection but might play potential roles in diverting cellular immune responses.

Lessons Learned for Pathogenesis, Immunology, and Disease of Erythrocytic Parasites: Plasmodium and Babesia.

Malaria caused by Plasmodium species and transmitted by Anopheles mosquitoes affects large human populations, while Ixodes ticks transmit Babesia species and cause babesiosis. Babesiosis in animals has been known as an economic drain, and human disease has also emerged as a serious healthcare problem in the last 20–30 years. There is limited literature available regarding pathogenesis, immunity, and disease caused by Babesia spp. with their genomes sequenced only in the last decade. Therefore, using previous studies on Plasmodium as the foundation, we have compared similarities and differences in the pathogenesis of Babesia and host immune responses. Sexual life cycles of these two hemoparasites in their respective vectors are quite similar. An adult Anopheles female can take blood meal several times in its life such that it can both acquire and transmit Plasmodia to hosts. Since each tick stage takes blood meal only once, transstadial horizontal transmission from larva to nymph or nymph to adult is essential for the release of Babesia into the host. The initiation of the asexual cycle of these parasites is different because Plasmodium sporozoites need to infect hepatocytes before egressed merozoites can infect erythrocytes, while Babesia sporozoites are known to enter the erythrocytic cycle directly. Plasmodium metabolism, as determined by its two- to threefold larger genome than different Babesia, is more complex. Plasmodium replication occurs in parasitophorous vacuole (PV) within the host cells, and a relatively large number of merozoites are released from each infected RBC after schizogony. The Babesia erythrocytic cycle lacks both PV and schizogony. Cytoadherence that allows the sequestration of Plasmodia, primarily P. falciparum in different organs facilitated by prominent adhesins, has not been documented for Babesia yet. Inflammatory immune responses contribute to the severity of malaria and babesiosis. Antibodies appear to play only a minor role in the resolution of these diseases; however, cellular and innate immunity are critical for the clearance of both pathogens. Inflammatory immune responses affect the severity of both diseases. Macrophages facilitate the resolution of both infections and also offer cross-protection against related protozoa. Although the immunosuppression of adaptive immune responses by these parasites does not seem to affect their own clearance, it significantly exacerbates diseases caused by coinfecting bacteria during coinfections.

Identification of the Glycan Binding Profile of Human and Rodent Plasmodium Sporozoites

The transmission of Plasmodium spp. sporozoites to the mammalian host is the first step in the initiation of the mosquito-borne disease known as malaria. The exact route of transmission from the bloodstream to the liver is still not clearly elucidated, and identification of the host glycan structures bound by the sporozoites may inform as to which host cells are involved. Here, we provide a comprehensive analysis of the glycan structures that sporozoites from the human pathogen, P. falciparum, and the rodent pathogen, P. yoelii, recognize and bind. Glycan array analysis was used to profile the glycans bound by the sporozoites, and the binding affinities of these sporozoite-glycan interactions were then determined by surface plasmon resonance. Data showed that the different Plasmodium spp. bind different classes of glycans. P. falciparum was observed to bind to glycans with terminal N-acetylgalactosamine (GalNAc) or Galactose (Gal) linked to a GalNAc, and the highest-affinity observed was with the GalNAc monosaccharide (12.5 nM). P. yoelii bound glycosaminoglycans, mannosyl glycans, Gal linked to N-acetylglucosamine structures, and the αGal epitope. The highest-affinity interaction for P. yoelii was with the αGal epitope (31.4 nM). This is the first study to identify the key host glycan structures recognized by human and rodent Plasmodium spp. sporozoites. An understanding of how Plasmodium sporozoites interact with the specific glycan structures identified here may provide further insight into this infectious disease that could help direct the design of an effective therapeutic.

A guide to investigating immune responses elicited by whole-sporozoite pre-erythrocytic vaccines against malaria.

In the last few decades, considerable efforts have been made toward the development of efficient vaccines against malaria. Whole-sporozoite (Wsp) vaccines, which induce efficient immune responses against the pre-erythrocytic (PE) stages (sporozoites and liver forms) of Plasmodium parasites, the causative agents of malaria, are among the most promising immunization strategies tested until present. Several Wsp PE vaccination approaches are currently under evaluation in the clinic, including radiation- or genetically-attenuated Plasmodium sporozoites, live parasites combined with chemoprophylaxis, or genetically modified rodent Plasmodium parasites. In addition to the assessment of their protective efficacy, clinical trials of Wsp PE vaccine candidates inevitably involve the thorough investigation of the immune responses elicited by vaccination, as well as the identification of correlates of protection. Here, we review the main methodologies employed to dissect the humoral and cellular immune responses observed in the context of Wsp PE vaccine clinical trials and discuss future strategies to further deepen the knowledge generated by these studies, providing a toolbox for the in-depth analysis of vaccine-induced immunogenicity.

Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India

BackgroundEfforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here.MethodsMembrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively.ResultsLaboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load.ConclusionsOptimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

Prime-Trap immunization with protective liver stage antigen, MIF-4G and Kb17 peptide, induces liver CD8 TRM cells and protection against Plasmodium berghei malaria

Abstract Exposure to Plasmodium sporozoites (spz), the causative agent of malaria, induces anti-disease immunity, but fails to protect against repeated Plasmodium infections. In contrast, multiple immunizations with radiation-attenuated Plasmodium spz (RAS) induce sterile lasting protection in humans, non-human primates, and murine models. The mechanisms of protective immunity are multifactorial and CD8 T cells are considered sine qua none of protection. The Plasmodium berghei (Pb) RAS-induced protection in C57Bl/6 mice correlates with a network of liver stage antigens (LS Ags)-activated liver CD8 T cell subsets including resident memory (RM) CD8 TRM cells. A protective Pb LS Ag, termed MIF-4G, induced liver CD8 T cells that recognize H-2Kb-restricted epitope, Kb17. One of the current efforts to improve malaria vaccines focuses on maximizing the induction of liver CD8 TRM cells by Prime-Trap immunization regimen. Here we asked if priming with DNA-MIF-4G followed by trapping with Adenovirus serotype 5 (Ad5) vectored-Kb17 would induce Kb17-specific liver CD8 TRM cells as well as protection against Pb spz challenge. Liver Kb17-specific CD8 TRM cells (CD69+CXCR6+) exceeded the number of these cells in the spleens, 27% vs 2%, respectively. Protection measured by liver parasite burden was CD8 T cell dependent. In contrast, prime:boost immunization with DNA/Ad5 MIF-4G induced only 8% liver CD8 TRM cells, as did control immunization with DNA/Ad5 empty vector. Utilizing LS Ags and the corresponding CD8 T cell-inducing epitopes in Prime-Trap immunization lends itself for further explorations as an ancillary approach to malaria vaccine regimens where liver CD8 TRM may be crucial in preventing virulent blood stages by targeting LS parasites.

Intravital imaging experiments track CD8 T cells searching for Plasmodium liver stages, but do the imaging frequency and duration impact the detection of CD8 T cell bias?

Abstract Vaccine-induced CD8 T cells can provide sterilizing protection by killing Plasmodium sporozoites in mice, but how do they find those parasites? We recently showed that most liver-localized CD8 T cells search for infected hepatocytes randomly, with some cells displaying bias toward the infection site only when there is already a CD8 T cell cluster present. Our initial intravital microscopy experiments minimized tissue damage from laser exposure by using low imaging frequency (volume/1.5–2min) and were fairly lengthy (1–2h). In these experiments, activated CD8 T cells displayed Brownian-like turns and slow average calculated speeds of 1–3 um/min. Recently, we performed new experiments in which we recorded the movement of CD8 T cells at a higher frequency (volume/0.2min) at the cost of shorter experiments (30min). Interestingly, observed speeds ranged between 5 and 18 um/min, suggesting that the calculated speed of CD8 T cells depends on the details of the experiment. Further testing found that imaging frequency impacts the estimated average speed per cell: reducing the imaging frequency from 0.2min to 2min can reduce the average calculated cell speed 5-fold, partially explaining the discrepancy between our datasets’ speeds. Nonetheless, the new data still suggest that some CD8 T cells display attraction towards the infection site only when there is a cluster of CD8 T cells already present. Taken together, our results highlight the impact of details of intravital imaging experiments on inferred parameters of T cell movement in complex environments.

Prevalence of asymptomatic P. falciparum gametocyte carriage among school children in Mbita, Western Kenya and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence.

Background: Asymptomatic Plasmodium falciparum gametocyte carriers are reservoirs for sustaining transmission in malaria endemic regions. Gametocyte presence in the host peripheral blood is a predictor of capacity to transmit malaria. However, it does not always directly translate to mosquito infectivity. Factors that affect mosquito infectivity include, gametocyte sex-ratio and density, multiplicity of infection (MOI), and host and vector anti-parasite immunity. We assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and to further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: P. falciparum parasite infections were detected by RDT (Rapid Diagnostic Test) and microscopy among schoolchildren (5-15 years old). Blood from 37 microscopy positive gametocyte carriers offered to laboratory reared An. gambiae s.l. mosquitoes. A total of 3395 fully fed mosquitoes were screened for Plasmodium sporozoites by ELISA. P. falciparum was genotyped using 10 polymorphic microsatellite markers. The association between MOI and gametocyte density and mosquito infection prevalence was investigated. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) ( p-value < 0.001) compared to females 26.72% (657/2459). The microscopic gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3-3.4], P = 0.002) as compared to children aged 10-15 years. After offering gametocyte positive blood to An. gambiae s.l. by membrane feeding assay, our results indicated that 68.1% of the variation in mosquito infection prevalence was accounted for by gametocyte density and MOI (R-SQR. = 0.681, p<0.001). Conclusions: We observed a higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI significantly predicted mosquito infection prevalence.

Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species‐specific barrier

Malaria infection starts with the injection of Plasmodium sporozoites into the host’s skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species‐specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre‐clinical screening of next‐generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down‐selection of candidates prior to human vaccine trials.

Prevalence of asymptomatic P. falciparum gametocyte carriage among school children in Mbita, Western Kenya and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence.

Background: Asymptomatic Plasmodium falciparum gametocyte carriers are reservoirs for sustaining transmission in malaria endemic regions. Gametocyte presence in the host peripheral blood is a predictor of capacity to transmit malaria. However, it does not always directly translate to mosquito infectivity. Factors that affect mosquito infectivity include, gametocyte sex-ratio and density, multiplicity of infection (MOI), and host and vector anti-parasite immunity. We assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and to further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: P. falciparum parasite infections were detected by RDT (Rapid Diagnostic Test) and microscopy among schoolchildren (5-15 years old). Blood from 37 microscopy positive gametocyte carriers offered to laboratory reared An. gambiae s.l. mosquitoes. A total of 3395 fully fed mosquitoes were screened for Plasmodium sporozoites by ELISA. P. falciparum was genotyped using 10 polymorphic microsatellite markers. The association between MOI and gametocyte density and mosquito infection prevalence was investigated. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) ( p-value < 0.001) compared to females 26.72% (657/2459). The microscopic gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3–3.4], P = 0.002) as compared to children aged 10-15 years. After offering gametocyte positive blood to An. gambiae s.l. by membrane feeding assay, our results indicated that 68.1% of the variation in mosquito infection prevalence was accounted for by gametocyte density and MOI (R-SQR. = 0.681, p < 0.001). Conclusions: We observed a higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI significantly predicted mosquito infection prevalence.

Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

Despite the critical role of Plasmodium sporozoites in malaria transmission, we still know little about the mechanisms underlying their development in mosquitoes. Here, we use single-cell RNA sequencing to characterize the gene expression profiles of 16,038 Plasmodium berghei sporozoites isolated throughout their development from midgut oocysts to salivary glands, and from forced salivation experiments. Our results reveal a succession of tightly regulated changes in gene expression occurring during the maturation of sporozoites and highlight candidate genes that could play important roles in oocyst egress, sporozoite motility, and the mechanisms underlying the invasion of mosquito salivary glands and mammalian hepatocytes. In addition, the single-cell data reveal extensive transcriptional heterogeneity among parasites isolated from the same anatomical site, suggesting that Plasmodium development in mosquitoes is asynchronous and regulated by intrinsic as well as environmental factors. Finally, our analyses show a decrease in transcriptional activity preceding the translational repression observed in mature sporozoites and associated with their quiescent state in salivary glands, followed by a rapid reactivation of the transcriptional machinery immediately upon salivation.

In-depth proteomic analysis of Plasmodium berghei sporozoites using trapped ion mobility spectrometry with parallel accumulation-serial fragmentation.

Sporozoites of the malaria parasite Plasmodium are transmitted by mosquitoes and infect the liver for an initial and obligatory round of replication, before exponential multiplication in the blood and onset of the disease. Sporozoites and liver stages provide attractive targets for malaria vaccines and prophylactic drugs. In this context, defining the parasite proteome is important to explore the parasite biology and to identify potential targets for antimalarial strategies. Previous studies have determined the total proteome of sporozoites from the two main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, which infects rodents. Another murine malaria parasite, P. berghei, is widely used to investigate the parasite biology. However, a deep view of the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage of the highly sensitive timsTOF PRO mass spectrometer, combined with three alternative methods for sporozoite purification, to identify the proteome of P. berghei sporozoites using low numbers of parasites. This study provides a reference proteome for P. berghei sporozoites, identifying a core set of proteins expressed across species, and illustrates how the unprecedented sensitivity of the timsTOF PRO system enables deep proteomic analysis from limited sample amounts.

Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites

In the malaria-causing parasite’s life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite’s development in the mosquito.