Open Access

Plasmodium berghei Cap93, a novel oocyst capsule-associated protein, plays a role in sporozoite development

  • Hanae Sasaki1,
  • Harumi Sekiguchi2,
  • Makoto Sugiyama3 and
  • Hiromi Ikadai2Email author
Contributed equally
Parasites & Vectors201710:399

https://doi.org/10.1186/s13071-017-2337-8

Received: 25 April 2017

Accepted: 16 August 2017

Published: 25 August 2017

Keywords

Plasmodium berghei oocyst PbCap93 Sporozoite Knocked out parasite

Abstract

Background

Disruption of Plasmodium oocyst capsule protein (PbCap380), and an oocyst wall interior protein, circumsporozoite protein, results in sporozoites not being formed, despite the formation of oocysts, and prevents malaria transmission. Therefore, these key oocyst capsule-associated proteins are responsible for the development of the oocyst capsule and play an important role in the later growth and maintenance of sporozoites. We attempted to discover novel oocyst capsule-associated proteins and analyze their functions by assuming that such proteins will be strategically important targets for preventing malaria transmission. A putative, novel oocyst capsule-associated protein, known as PbCap93, was determined from the PlasmoDB database, and we aimed to create a knockout parasite of the PbCap93 gene to analyze its functions in the mosquito stage.

Results

To investigate the kinetics of PbCap93 protein expression, we labelled the asexual stage and mosquito stage parasites with anti-PbCap93 antibodies using IFAT. PbCap93 was detected in oocysts on day 15 after infection, though it was not detected in sporozoites of ruptured oocysts. PbCap93 localizes interior to the oocyst capsule alone without localization to the sporozoite plasma membrane. To gain further insight regarding PbCap93 function, we disrupted the gene in P. berghei parasites. Between 14 and 15 days after receiving a parasite-laden blood meal, 100 midguts were dissected from mosquitoes that received either wild-type (WT) or knocked out (KO) parasites. For WT parasites, the oocyst infection rate was 50%, whereas, for KO parasites, the infection rate was 16.7%. The average number of oocysts per midgut was 12 for the WT parasites compared with 0.8 for the KO parasites. Furthermore, KO parasite oocysts were significantly smaller than WT parasite oocytes. Using transmission electron microscopy, we observed that the electron density of the PbCap93-KO oocyst capsule was lower than that of the WT oocyte capsule.

Conclusions

We posited that the PbCap93 protein is secreted from sporoblasts within the oocysts until sporozoites are formed. PbCap93 constructs the interior of the oocyst capsule or part of the plasma membrane and affects sporozoite differentiation. Further studies are warranted to understand the mechanism of oocyst formation.

Background

Along with acquired immune deficiency syndrome and tuberculosis, human malaria is one of the major infectious diseases in the world [1, 2]. The disease affects 270 million people, with 627,000 deaths per year. Mortality primarily affects children aged ≤5 years [3, 4]. Malaria is caused by the protozoan parasites, Plasmodium spp. in human blood and are transmitted human-to-human via insect vectors, the Anopheles mosquitoes. When an Anopheles mosquito bites a host, the parasites in their sporozoite form enter the blood stream and ultimately migrate to the host liver. In the liver, sporozoites develop into merozoites that are released into the blood circulation and then start a cycle of asexual reproduction. In blood, only a small portion of the parasites exist as gametocytes. When a host is bitten by a female Anopheles mosquito, gametocytes present in the host blood are ingested into the mosquito midgut, where the gametocytes differentiate into male and female gametes, which in turn form zygotes after fertilization. They then differentiate into motile ookinetes. The ookinetes migrate and traverse the midgut epithelium and lodge under the basement membrane to differentiate into oocysts at approximately 24–30 h after blood ingestion. Several thousands of sporozoites form within each oocyst, from which they are released approximately 2 weeks after blood ingestion [59]. The released sporozoites then migrate and invade the salivary glands of the mosquito, thereby enabling the transmission when this mosquito bites another host.

In Plasmodium, parasite differentiation in the mosquito is complex and undergoes severe losses at the oocyst stage. Of hundreds of ookinetes that form in the midgut, only a few differentiate into oocysts [1015]. In the developing oocyst, nuclear division occurs in the absence of cell division resulting in a syncytium containing thousands of nuclei embedded in a common cytoplasm. The oocyst is surrounded by an inner plasma membrane and a rigid outer capsule [1618]. The oocyst capsule makes sporozoite formation and its growth inside the capsule possible [1821]. It is conjectured that the formation of the oocyst capsule stabilizes the growth of oocysts, thereby enabling their long-lasting presence within the mosquito’s hemocoel. Previous studies have revealed that deletion of an oocyst capsule surface P. berghei protein gene (PbCap380), and of an oocyst capsule interior protein gene, circumsporozoite protein (CSP), results in interruption of sporozoite differentiation [16, 2225]. Therefore, these key oocyst capsule-associated proteins are not only responsible for the development of the oocyst capsule but also play an important role in their later growth and maintenance of sporozoites. In other words, they are essential for the survival, proliferation, and transmission of Plasmodium parasites.

In the present study, we identify and characterize a novel oocyst capsule-associated protein 93 of Plasmodium berghei (PbCap93), PbANKA_0905200.

Methods

Bioinformatics

The PbCap93 (PbANKA_0905200) genomic sequences used in this study were retrieved from PlasmoDB (http://​www.​plasmodb.​org). All Plasmodium orthologues are encoded by a single exon (Additional file 1).

Mice, parasites, and mosquitoes

For P. berghei infections, 6- to 8-week old male BALB/c mice (SLC, Japan) were infected with wild-type P. berghei (ANKA strain 2.34). Anopheles stephensi (STE2 strain) mosquitoes were maintained at 27 °C and 80% relative humidity with a 14/10 h light/dark cycle in an insectary and fed 10% (w/v) sucrose solution.

PbCap93 gene expression analysis

To study PbCap93 expression during the P. berghei life-cycle, using the Trizol reagent (Thermo Fisher Scientific, MA, USA), total RNA was isolated from blood samples obtained during the asexual stage of P. berghei-infected mouse at 10% parasitemia and from those obtained during the mosquito stages at 30 min, 1 day, 3 days, 5 days, 10 days, 15 days and 17 days after infection. cDNA was synthesized using the ReverTra Ace kit (Toyobo, Osaka, Japan). PCR was performed using gene-specific primers PbCap93 (PbCap93-F: 5′-CCT GCT TCT TCA ACA GAT TAT AAT G-3′ and PbCap93-R: 5′-GGA TTG TTT GAA ATC GAA TCG AAA G-3′), PbCap380 (PbCap380-F: 5′-GAA ATC ACC ATT TAA TTT CTC CAA TGG GT-3′ and PbCap380-R: 5′-TGT AGT TCG AAA AGG ATG GTT TTG ATT GT-3′ [26]), CHT1 (CHT1-F: 5′-GAT TGG GAA CCA AAT GGA AGT TTT AAC-3′ and CHT1-R: 5′-GAT ACA CAT GAT AAT GCT GCA TTT GAT G-3′), and CSP (CSP-F: 5′-GAC CCA GCA CCA CCA AAC GCA AAT G-3′ and CSP-R: 5′-CCT TGT GGT GGT GCT GGG TCA TTT G-3′ [27]). Plasmodium 18S ribosomal RNA (18S rRNA) (18S rRNA-F: 5′-AAG CAT TAA ATA AAG CGA ATA CAT CCT TAC-3′ and 18S rRNA-R: 5′-GGA GAT TGG TTT TGA CGT TTA TGT G-3′) was used for normalization [28].

Immunization and anti-PbCap93 serum

Polyclonal antibodies were raised against the PbCap93 protein (amino acids 379–392) (Additional file 1) and PbCSP (PBANKA_040320; amino acids 133–148) and used in this study (Eurofins Genomics Inc., Tokyo, Japan).

Indirect immunofluorescence assay

To detect PbCap93 in oocysts, infected midguts were fixed in acetone/methanol for 1 h and then blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Midguts were incubated with rabbit anti-PbCap93 sera (1:200) at 37 °C for 60 min, washed three times with PBS, and then incubated with Alexa fluor 488 goat-conjugated anti-rabbit immunoglobulin G (IgG) (1:500, Thermo Fisher Scientific) at 37 °C for 30 min. Pre-immune serum from the same rabbit was used as a control. Oocysts were observed after staining with anti-PbCap380 [26] (1:400) and anti-PbCSP (1:400) antibodies and detected with Alexa fluor 568 goat-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific). Plasmodium berghei sporozoites in oocysts and salivary glands were fixed in acetone/methanol and processed as described above.

Confocal microscopy

Midguts were dissected 10 days after infection and processed as described above. Paraffin-embedded P. berghei-infected midgut tissue sections were deparaffinized using xylene and graded alcohols. The slides were incubated in 10% BSA in PBS for 1 h and then incubated overnight with primary antibodies at room temperature. Rabbit antibodies against PbCap93 diluted in 0.1% BSA in PBS (1:400) were used as primary antibodies. Preimmune rabbit sera (diluted 1:100) were used as negative controls. After washing three times with PBS, the slides were incubated with Alexa fluor 488 goat-conjugated anti-rabbit IgG (1:800 dilution) (Thermo Fisher Scientific). Antibodies against PbCap380 and PbCSP were used for oocyst double-labelling experiments and were detected using Alexa fluor 568 goat-conjugated anti-rabbit secondary antibodies. P. berghei nuclei were counterstained with SlowFade Diamond Antifade Mountant with DAPI (4′, 6-diamidino-2-phenylindole) (Thermo Fisher Scientific). Sections were observed by confocal microscopy (LSM 710, Carl Zeiss Inc., Oberkochen, Germany).

Generation of PbCap93-KO parasites

PbCap93 was knocked out (KO) using double-crossover homologous recombination technology as previously described [29]. For targeted disruption of the PbCap93 locus, a disruption plasmid was generated by amplification of a PCR fragment using primers P93F (5′-GAT GTT ATG TGG TAT GTT CCA GA-3′) and P93R (5′-CAT TTT GGA AAT GTG TAA TGC TCA-3′) and P. berghei genomic DNA as a template. The gene was cloned into the pCR-BluntII-TOPO vector (Thermo Fisher Scientific), resulting in the plasmid pCap93. Subsequently, pCap93 was digested using Acc I. Digested pCap93 was inserted into the hdhfr expression cassette [30]. pCap93 (10 μg) was linearized with PvuII and electroporated into cultured P. berghei schizonts using Nucleofector II. Transfected parasites were intravenously injected into male BALB/c mice and treated with pyrimethamine (70 μg/ml) 24 h later via drinking water. Infected blood was collected to examine the integration of the disruption cassette by PCR using integration-specific primers P93R and DHFR-F1 (5′-CTT CTC TGT GTA TTA ATA TTG T-3′) and P93F and DHFR-R1 (5′-CTA TCA ATT ATT TCC CGT GG-3′). Parasites were cloned by limiting dilution.

Phenotypic analysis of PbCap93-KO parasites

Development of wild-type (WT) and PbCap93-KO parasites in mice was assessed by injecting a known number of parasites in BALB/c mice. Eight mice each were injected with 1 × 106 PbCap93-KO parasites, whereas the other seven were injected with 1 × 106 WT parasites. Parasitemia was monitored daily via Giemsa-stained blood smears. For 10% parasitemia, gametocytaemia (gametocytes per 500 red blood corpuscles) and gametocyte sex ratio (in 300 mature gametocytes) were determined by Giemsa-stained tail blood smears. Exflagellation of male gametocytes was quantified as previously described [31, 32]. Briefly, 2 μl of gametocyte-infected blood was obtained from the tail vein and mixed immediately with 38 μl of complete ookinete culture medium. The mixture was placed under a coverslip at RT, and 5 min later, exflagellation centres were counted over the next 10 min using a phase contrast microscope. The ability of parasites to differentiate into gametocytes and from male gametes (exflagellation) was assessed. Infected mice were fed to A. stephensi mosquitoes, and oocysts (days 14–15) were microscopically examined. For each mouse, up to 30 mosquitoes were dissected 14–15 days after feeding. The midguts were stained with 0.5% mercurochrome (Merck Millipore, MA, USA). Oocysts were counted to determine the prevalence of infection (number of infected mosquitoes) and intensity of infection (number of oocysts per positive midgut) [33].

Transmission electron microscopy

Mosquito midguts at 15 days post-infection were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.4) at 4 °C for 2 h. After rinses, samples were post-fixed at RT in buffered 1% osmium tetroxide for 2 h and then dehydrated in ethanol and propylene oxide series and embedded in epoxy resin. Ultrathin sections were cut using an Ultracut N (Reichert-Nissei, Tokyo, Japan), stained using uranyl acetate followed by lead citrate. Sections were examined using a H-7650 transmission electron microscope (Hitachi Ltd., Tokyo, Japan).

Statistical analyses

A Student’s t-test was used to statistically compare the groups (parasitemia, gametocytaemia, and oocyst size). The intensity of infection (number of oocysts/midguts) was analyzed using the Mann–Whitney U-test. For these statistical analyses, GraphPad Prism software (GraphPad Software, Inc., CA, USA) was used. P < 0.05 was considered statistically significant.

Results

Identification of the gene encoding P. berghei capsule-associated protein 93 (PbCap93)

To select candidates for novel oocyst capsule-associated proteins, we used a P. bergehi in PlasmoDB database (http://plasmodb.org/plasmo/). The following criteria were used: “expressed only in the oocyst stage,” “possessing transmembrane domains,” and “conserved proteins with unknown function”. Using these criteria, we identify PbCap93 (PBANKA_0905200), which is highly homologous with other protozoa. A total of 65 genes were found only to be expressed in the oocyst stage, of which 14 genes have transmembrane domains and of these, five genes that encoded novel conserved proteins with unknown functions. Finally, among these five genes, those having high homology with other protozoa were selected.

PbCap93 has orthologues in every sequenced Plasmodium species. The P. berghei-predicted amino acid sequence was determined to be > 69% identical to the P. chabaudi- (89.3%), P. yoelii- (92.5%), P. falciparum- (69.9%), P. knowlesi- (69.0%), and P. vivax-predicted sequences (77.0%) (Fig. 1; PlasmoDB accession numbers: PCHAS_0706400, PY17X_0906600, PF3D7_1143800, PKNH_0941700, and PVX_092795, respectively). The C-terminal half of the orthologues is more similar than the N-terminal half (Additional file 1).
Fig. 1

Subcellular localization of PbCap93. Immunofluorescence assays of oocysts at 15 days post-infection (upper panels) and sporozoite (lower panels) with PbCap93 (green) and PbCap380 (red) antibodies, stained with the DAPI nuclear stain (blue), and showing differential interference contrast (DIC), as indicated. Scale-bars: 10 μm

PbCap93 is expressed during oocyst development

To investigate the kinetics of PbCap93 protein expression, we labelled asexual stage and mosquito stage parasites with anti-PbCap93 antibodies by IFAT. PbCap93 was not detected in blood stage but was detected in oocysts on day 15 after infection (Fig. 1). Quantitative RT-PCR (qRT-PCR) analysis indicated that PbCap93 is expressed during oocyst development, particularly in late oocysts but its expression diminished by day 17 when the sporozoites within the oocysts matured (Fig. 2). The antibody did not detect the protein in oocysts sporozoites or salivary gland sporozoites. (Fig. 1).
Fig. 2

Quantification of PbCap93 mRNA abundance. Quantitative RT-PCR was performed using cDNA prepared from blood stage parasites at 10% parasitemia (10%) and midguts dissected at different times after infection (30 min to 17 days, d). Experiments were performed in triplicate, and data shown are from one representative experiment. Four independent biological replicates were performed for each experimental condition

PbCap93 localizes internally to the oocyst capsule and may be associated with the oocyst capsule

PbCap380 is a surface protein of the oocyst capsule [24]. PbCSP, the most abundant sporozoite plasma membrane protein is also localized on the inner face of the oocyst capsule [23, 25, 34]. Confocal microscopy of day 10 oocysts using anti-PbCap93, anti-PbCap380, and anti-PbCSP antibodies indicated that PbCap93 co-localizes with PbCSP and PbCap380 (Fig. 3). Moreover, PbCap93 was not detected in sporozoites of oocysts and salivary glands (Fig. 1). Therefore, our data suggested that PbCap93 localizes to the oocyst capsule alone without localizing to the sporozoite plasma membrane.
Fig. 3

PbCap93 localizes to the oocyst capsule interior. Confocal microscopy of a 15 days post-infection oocyst. The parasite was double labelled with antibodies against PbCap380 (red: upper panel), PbCSP (red: lower panel), and PbCap93 (green). The right panels show a higher magnification of the area in the merged image. PbCap93 appears to be located internally relative to PbCap380 and co-localized to CSP

Production of PbCap93-KO parasites

To gain further insight into PbCap93 function, we disrupted the gene in P. berghei parasites (Fig. 4a). Independent clonal parasite lines generated from the transfection were confirmed for gene disruption by insertion-specific PCR. To ascertain that the drug-resistance gene hDHFR was successfully inserted into the KO parasites, we performed PCR using the following three primer combinations: P93F-P93R, P93F-DHFR-R1, and DHFR-F1-P93R. As a result, we confirmed amplification products with their expected size from the P93F-P93R, P93F-DHFR-R1, and DHFR-F1-P93R primer combinations, respectively. In WT parasites, the P93F-P93R primer combination yielded an amplification product measuring 1066 bp, whereas the other two primer combinations did not produce any amplification products (Fig. 4a, b). Moreover, Southern blot analysis confirmed that the drug-resistance gene was successfully inserted into the targeted P93F–P93R region and semi-quantitative RT-PCR ascertained that no mRNA was being expressed. Consequently, the PbCap93-KO was successfully generated (Fig. 4c, d).
Fig. 4

Characterization of PbCap93 KO parasites. a Schematic of targeted disruption of the PbCap93 gene. The targeting vector (middle) containing a selectable marker gene was integrated into the PbCap93 gene locus (top) by double crossover. This recombination event resulted in the disruption of the PbCap93 gene and conferred pyrimethamine resistance (bottom). b PCR analysis of wild-type (WT) and PbCap93-KO parasites. Integration-specific PCR primer sets were used: P93F and P93R, P93F and DHFR-R1, and P93R and DHFR-F1. c Genomic Southern blot hybridization of WT and PbCap93-KO parasites. Parasite genomic DNA was digested with SpeI/HpaI and hybridized with the probe indicated by a solid bar (blue and red) in a. By integration of the targeting construct, the size of detected fragments increased from 2.5 to 3.5 kbp (blue) and 3.5 kbp (red). d Semi-quantification of PbCap93 mRNA abundance. Semi-quantitative RT-PCR was performed using cDNA prepared from midguts of mosquitoes infected with either WT or PbCap93-KO parasites at 10 days post-infection

PbCap93 is not required for asexual growth but is crucial for oocyst development

PbCap93 is expressed in the mosquito stages of parasite development, and it is expected not to be required for asexual growth in mice. When mice were infected with either PbCap93-KO parasites or WT parasites, no significant changes were observed by day 14 (Fig. 5). Furthermore, when blood smears of KO parasite-infected and WT parasite-infected mice were compared by Giemsa staining, no morphological differences were apparent between KO and WT parasites (data not shown). Neither gametocytaemia nor female/male gametocyte ratio was affected (Table 1, Fig. 6).
Fig. 5

Comparison of parasitemia in mice infected with either WT or PbCap93-KO parasites. Comparison of parasitemia in mice intravenously infected with WT (red) or PbCap93-KO (blue) parasites. All mice were infected by intravenously injecting 106 blood-stage parasites. Number of mice analyzed: n = 8 (WT) and n = 7 (KO)

Table 1

Ratio of gametocytemia in 10% parasitemia

Parasite

♀-gametocytes/infected RBC

♂-gametocytes/infected RBC

 

Mean ± SD

Mean ± SD

WT

7.4 ± 3.9

1.4 ± 1.0

KO

7.1 ± 2.4

1.5 ± 0.7

Abbreviations: KO knocked out, RBC red blood cell, WT wild-type, SD standard deviation

Fig. 6

The proportion of female and male gametocyte in mice infected with either WT or PbCap93-KO parasites. The proportion of female (white) and male (black) gametocytes in mice intravenously infected with WT and PbCap93-KO parasites. Number of gametocytes analyzed: n = 316 (WT) and n = 271 (KO)

Between 14 and 15 days after receiving a parasite-laden blood meal, 100 midguts and 102 midguts were dissected from A. stephensi mosquitoes that received WT or KO parasites, respectively. For WT parasites, the oocyst infection rate was 50%, with 50 midguts harbouring oocysts. For KO parasites, on the other hand, the infection rate was 16.7%, with only 17 midguts harbouring oocysts (Fig. 7). The average number of oocysts per midgut was 12 for the WT parasites compared with 0.8 for the KO parasites. Meanwhile, KO parasite oocysts were significantly smaller than WT parasite oocytes (Fig. 8).
Fig. 7

PbCap93 is important for oocyst development. Oocyst number per midgut of mosquitoes infected with either WT or PbCap93-KO parasites. Horizontal bars represent medians. Data from three independent replicates. % mosquitoes infected: proportion of mosquitoes carrying at least one oocyst

Fig. 8

Representative oocyst size and numbers in WT and PbCap93-KO parasites. Diameters (μm) of 14 days post-infection WT and PbCap93-KO oocysts (arrowheads). *P < 0.01. Scale-bar: 20 μm

Subsequently, we examined by real-time PCR the expression dynamics of PbCap380 and CSP in mosquitoes infected with WT parasites. As a result, mRNA abundance of both genes peaked between 10 and 15 days post-infection. Similarly, we also observed dynamics of PbCap380 and CSP genes in mosquitoes infected with PbCap93-KO parasites, and we found that PbCap380 and CSP were substantially suppressed at 10 and 15 days post-infection (Fig. 9).
Fig. 9

Quantification of PbCap380 and CSP mRNA in PbCap93-KO parasites. qRT-PCR was performed using cDNA prepared from midguts dissected after infection (10 and 15 days post-infection). Experiments were performed in triplicate, and data shown are from one representative experiment. Four independent biological replicates were performed for each experimental condition

By transmission electron microscopy, we observed that the electron density of the PbCap93-KO oocyst capsule is lower than that of WT parasites. Accordingly, KO oocysts had fewer matrix granules (Fig. 10, MG), which were abundant in the WT oocyst capsule. Moreover, the cross-section structures of sporozoites in KO oocysts varied significantly in size and cell structure, with some taking an amorphous shape, compared with sporozoites in WT oocysts (Fig. 10). Some sporozoites of KO parasites were observed outside the oocyst (Fig. 10, oSP).
Fig. 10

Ultrastructure of oocyst development in PbCap93-KO parasites. Left panels: Low power ultrastructural images of oocyst development at 15 days post-infection. Right panels: Details of the oocyst capsule from the area outlined by red squares on the left. The cleft enlarges, dividing the oocyst cytoplasm into the intermediate sporoblast form (SB) and budding sporozoite (SP). Some sporozoites are outside the oocyst (oSP). Small tufts (T) of material appear on the inner capsule (C) and matrix granules (MG). Scale-bars: 1 μm

Discussion

The oocyst capsule is in contact with the basal lamina of the mosquito midgut epithelium. The oocyst capsule helps to fend off defensive responses by the mosquito. It is intriguing how this supposedly rigid structure enlarges during the dramatic growth of the oocyst. This may happen by intercalation of newly synthesized proteins or by protein addition at one specific growth site. The deficiency of a known oocyst wall surface protein, PbCap380, and an oocyst capsule interior protein, CSP, results in sporozoites not being formed despite the formation of oocysts [16, 2225]. Therefore, these key oocyst capsule-associated proteins are not only responsible for the development of the oocyst capsule but also play an important role in their later growth and maintenance of sporozoites. In the present study, we sought to search for novel oocyst capsule-associated proteins, and analyze their functions on the assuming that such proteins will prove to be important targets for preventing the transmission of malaria.

PbCap93 is expressed in oocysts but not by blood stage parasites, and PbCSP appears to be integrated into the oocyst capsule alone without association with sporozoite plasma membrane. We gleaned that PbCap93 is probably secreted from sporoblasts within the lining of the oocyst capsule. Therefore, the fewer number of protein granules observed within the oocysts was attributed to knocking out of PbCap93. This phenomenon was considered to be due to the abnormal differentiation of sporoblasts, which made it difficult for sporozoites to secrete PbCap93. Accordingly, the lack of PbCap93 constituting the oocyst capsule led to the low electron density of the oocyst capsule. PbCap380 is a protein expressed in the exterior oocyst capsule. It is abundantly expressed at the later stage of oocyst formation [24]. However, based on the assessment of mRNA abundance, PbCap380 expression is regulated in PbCap93-KO parasites. This may have resulted in the insufficient composition of the oocyst capsule, thereby resulting in the observed lower electron density.

CSP is expressed during sporozoite division at the oocyst formation [23, 25]. Because CSP mRNA abundance was almost undetectable in PbCap93-KO oocysts, sporozoite division and maturation may have been adversely affected, with some shape deformities observed in sporozoites within the oocysts. Therefore, our data suggested that PbCap93 localizes to the oocyst capsule interior, into the inner face of the oocyst capsule, implying that the PbCap93 protein participates in the maturation of sporozoites in oocysts and/or at the time of capsule rupture.

Conclusions

We posited that the PbCap93 protein is secreted from sporoblasts within the oocysts until sporozoites are formed. In addition, PbCap93 constructs the interior of the oocyst capsule or part of the plasma membrane and affects sporozoite differentiation. Further studies are needed to determine the potential role of the PbCap93 protein and the underlying mechanism of oocyst formation.

Abbreviations

CSP: 

Circumsporozoite protein

KO: 

Knocked out

PbCap93: 

oocyst capsule-associated protein 93 of Plasmodium berghei

WT: 

wild-type

Declarations

Acknowledgements

Plasmodium berghei and anti-PbCap380 antibody used in this work were kindly provided by Dr. Marcelo Jacobs-Lorena, Johns Hopkins University, MD, US. Anopheles stephensi (STE2 strain) was provided by MR4. This work would not have been possible without assistance from Mai Tanaka, support staff in our laboratory. We thank Dr. Marcelo Jacobs-Lorena for helpful discussions on particular aspects of this work.

Funding

This study was supported by the JSPS KAKENHI Grant Number JP25450430, 17H04073, the Takeda Science Foundation (to HI) and The Grant-in-Aid from METI of Japan.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Authors’ contributions

All authors contributed equally to this manuscript. All authors read and approved the final manuscript.

Ethics approval

All procedures were executed in accordance with a protocol approved by the Kitasato University Animal Care and Use Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Hokusan Co. Ltd.
(2)
Laboratory of Veterinary Parasitology, School of Veterinary Medicine, Kitasato University
(3)
Laboratory of Veterinary Anatomy, School of Veterinary Medicine, Kitasato University

References

  1. Breman JG, Egan A, Keusch GT. The intolerable burden of malaria: a new look at the numbers. Am J Trop Med Hyg. 2001;64:4–7.View ArticleGoogle Scholar
  2. Greenwood B, Mutabingwa T. Malaria in 2002. Nature (London). 2002;415:670–2.View ArticleGoogle Scholar
  3. World Health Organization. Severe falciparum malaria. Trans R Soc Trop Med Hyg. 2000;94(Suppl. 1):S1–90.Google Scholar
  4. World Health Organization. World malaria report 2015. 2015Google Scholar
  5. Arrighi RB, Hurd H. The role of Plasmodium berghei ookinete proteins in binding to basal lamina components a transformation into oocysts. Int J Parasitol. 2001;32:91–8.View ArticleGoogle Scholar
  6. Canning EU, Sinden RE. The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology. 1973;67:29–40.View ArticlePubMedGoogle Scholar
  7. Ghosh A, Edwards MJ, Jacobs-Lorena M. The journey of the malaria parasite in the mosquito: hopes for the new century. Parasitol Today. 2000;16:196–201.View ArticlePubMedGoogle Scholar
  8. Howells RE, Davies EE. Post-meiotic nuclear divisions in the oocyst of Plasmodium berghei. Trans R Soc Trop Med Hyg. 1971;65:421.View ArticlePubMedGoogle Scholar
  9. Vanderberg J, Rhodin J. Differentiation of nuclear and cytoplasmic fine structure during sporogonic development of Plasmodium berghei. J Cell Biol. 1967;32:C7–10.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, et al. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science. 1986;234:607–10.View ArticlePubMedGoogle Scholar
  11. Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog. 2006;2:e52.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Povelones M, Waterhouse RM, Kafatos FC, Christophides GK. Leucine-rich repeat protein complex activates mosquito complement in defense against Plasmodium parasites. Science. 2009;324:258–61.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Pringle G. A quantitative study of naturally-acquired malaria infections in Anopheles gambiae and Anopheles funestus in a highly malarious area of East Africa. Trans R Soc Trop Med Hyg. 1996;60:626–32.View ArticleGoogle Scholar
  14. Sinden RE. A biologist’s perspective on malaria vaccine development. Hum Vaccin. 2010;6:3–11.View ArticlePubMedGoogle Scholar
  15. Sinden RE. Molecular interactions between Plasmodium and insect vectors. Cell Microbiol. 2002;4:713–24.View ArticlePubMedGoogle Scholar
  16. Aldrich C, Magini A, Emiliani C, Dottorini T, Bistoni F, Crisanti A, et al. Roles of the amino terminal region and repeat region of the Plasmodium berghei circumsporozoite protein in parasite infectivity. PLoS One. 2012;7:e32524.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Sinden RE, Strong K. An ultrastructural study of the sporogonic development of Plasmodium falciparum in Anopheles gambiae. Trans R Soc Trop Med Hyg. 1978;72:477–1.View ArticlePubMedGoogle Scholar
  18. Terzakis JA, Sprinz H, Ward RA. The transformation of the Plasmodium gallinaceum oocyst in Aedes aegypti mosquitoes. J Cell Biol. 1967;34:311–26.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Adini A, Krugliak M, Ginsburg H, Li L, Lavie L, Warburg A. Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages. Mol Biochem Parasitol. 2001;117:161–8.View ArticlePubMedGoogle Scholar
  20. Adini A, Warburg A. Interaction of Plasmodium gallinaceum ookinetes and oocysts with extracellular matrix proteins. Parasitology. 1999;119:331–6.View ArticlePubMedGoogle Scholar
  21. Aikawa M. Parasitological review. Plasmodium: the fine structure of malarial parasites. Exp Parasitol. 1971;30:284–320.View ArticlePubMedGoogle Scholar
  22. Coppi A, Natarajan R, Pradel G, Bennett BL, James ER, Roggero MA, et al. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J Exp Med. 2011;208:341–56.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Nagasawa H, Procell PM, Atkinson CT, Campbell GH, Collins WE, Aikawa M. Localization of circumsporozoite protein of Plasmodium ovale in midgut oocysts. Infect Immunol. 1987;55:2928–32.Google Scholar
  24. Srinivasan P, Fujioka H, Jacobs-Lorena M. PbCap380, a novel oocyst capsule protein, is essential for malaria parasite survival in the mosquito. Cell Microbiol. 2008;10:1304–12.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Wang Q, Fujioka H, Nussenzweig V. Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium. Cell Microbiol. 2005;7:1616–26.View ArticlePubMedGoogle Scholar
  26. Srinvasan P, Abraham EG, Ghosh AK, Valenzuela J, Ribeiro JM, Dimopoulos G, et al. Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation. J Biol Chem. 2004;279:5581–7.View ArticleGoogle Scholar
  27. Akinosoglou KA, Bushell ES, Ukegbu CV, Schlegelmilch T, Cho JS, Redmond S, et al. Characterization of Plasmodium developmental transcriptomes in Anopheles gambiae midgut reveals novel regulators of malaria transmission. Cell Microbiol. 2015;17:254–68.View ArticlePubMedGoogle Scholar
  28. Kumar KA, Oliveira GA, Edelman R, Nardin E, Nussenzweig V. Quantitative Plasmodium sporozoite neutralization assay (TSNA). J Immunol Methods. 2004;292:157–64.View ArticlePubMedGoogle Scholar
  29. Janse CJ, Ramesar J, Waters AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006;1:346–56.View ArticlePubMedGoogle Scholar
  30. Yuda M, Iwanaga S, Shigenobu S, Kato T, Kaneko I. Transcription factor AP2-sp and its target genes in malarial sporozoites. Mol Microbiol. 2010;75:854–63.View ArticlePubMedGoogle Scholar
  31. Arreaza G, Corredor V, Zavala F. Plasmodium yoelii: quantification of the exoerythrocytic stages based on the use of ribosomal RNA probe. Exp Plasitol. 1991;72:103–5.View ArticleGoogle Scholar
  32. Kawamoto F, Alejo-Blanco R, Fleck SL, Kawamoto Y, Sinden RE. Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum. Mol Biochem Parasitol. 1990;42:101–8.View ArticlePubMedGoogle Scholar
  33. Mlambo G, Coppens I, Kumar N. Aberrant sporogonic development of Dmc1 (a meiotic recombinase) deficient Plasmodium berghei parasites. PLoS One. 2012;7:1–13.View ArticleGoogle Scholar
  34. Hamilton AJ, Davies CS, Sinden RE. Expression of circumsporozoite proteins revealed in situ in the mosquito stages of Plasmodium berghei by the Lowicryl-immunogold technique. Parasitology. 1988;96:273–80.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2017

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement