Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm

doi:10.1016/j.parint.2019.101938

Parasitology International

Volume 72, October 2019, 101938

https://doi.org/10.1016/j.parint.2019.101938 Get rights and content

Abstract

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60–70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

Introduction

Malaria is one of infectious diseases in tropical countries that leads to fatality. Malaria vaccines become important since antimalarial drugs in many parts of the world are faced to some resistance issues. Some vaccines are expected to generate specific antibodies that prevent Plasmodium parasite invasion of the red blood cells. Pre-erythrocytic and erythrocytic stage targeting malaria vaccines i.e. circumsporozoite protein (CSP), apical membrane antigen-1 (AMA-1), and merozoite surface protein 3 (MSP3) have been studied, and now are undergoing clinical trial [1]. To evaluate the antibody generated after MSP3 vaccination, availability of P. falciparum MSP3 recombinant protein is necessary. Besides, the PfMSP3 recombinant protein produced could be used as sero-diagnostic marker to assess malaria transmission in area that entering pre-elimination phase [2].

MSP3 is a soluble protein and expressed on surface of its merozoites by forming a protein complex with MSP1, which is linked to the surface of merozoites by a glycosylphosphatidylinositol anchor. MSP3 function is unclear, many hypothesize that it binds to receptors during invasions and has an ability to form extended oligomers. The MSP3 has three significant regions, alanine heptad repeated region, glutamic acid-rich region and leucine zipper region. The leucine zipper region contributes to the formation of its oligomers together with ¹⁹²YILGW¹⁹⁶ sequence [3]. MSP3-based vaccines induce the protective immunity to P. falciparum [4,5]. MSP3-derived long synthetic peptide showed the significant protective efficacy in the Phase 1b trial [6]. In addition, MSP3 fused with glutamate-rich protein was also investigated, but its vaccine efficacy was low [7]. However, MSP3 was found as a vaccine candidate by wheat germ cell-free system-based immuno profiling even in low endemic area [8].

It was previously reported that MSP3 expressed in Escherichia coli formed amyloid-like fibrils in the same manner as native MSP3 on the surface of merozoites [3]. In addition, its recombinant MSP3 has heme-binding activity. Widely established E. coli-based expression approach provides high-yield heterologous protein production and at relatively low production cost. However, in this way, almost of proteins from eukaryotes are expressed insoluble form due to lack of proper post-translational modification. Insect-based systems including silkworm (Bombyx mori) offer a distinct advantage because of post-translational modification capability including glycosylation, phosphorylation and disulfide bond formation. In addition, protein expression levels in silkworm are relatively high. The recent development of a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid [9], an E. coli and B. mori hybrid shuttle vector, enables to express proteins in silkworm pupae or larvae, reducing the time and eliminating the laborious steps of gene maniplulation [9,10].

Our study aims to express MSP3 of P. falciparum (PfMSP3) in silkworm larvae and purify the recombinant antigens from silkworm larval hemolymph for sero-diagnosis. The purified recombinant PfMSP3 (rPfMSP3) was characterized and its antigenicity was evaluated with enzyme-linked immunoassay (ELISA) and western blot approach using human sera from P. falciparum-infected and non-infected human to explore the possibility of the use of the rPfMSP3 as a serological marker of P. falciparum.

Section snippets

Construction of recombinant bacmid

The MSP3 (25–354 aa from the ORF) cDNA was PCR amplified using polymerase KOD plus NEO (TOYOBO, Tokyo, Japan) with primers as shown in Table 1 from pGEM-pf3D7-XM001347593-MSP3 (Sino Biological, Beijing, China) as per standard protocol for the polymerase enzyme. The amplified cDNA was confirmed by agarose gel electrophoresis and purified by FastGene Gel/PCR Extraction kit (Nippon Genetics, Tokyo, Japan). The purified cDNA was cloned into pFastBac1 (Thermo Fisher Scientific K. K, Tokyo, Japan) at

Purification of rPfMSP3

To express rPfMSP3 in silkworm larvae, its native signal sequence at its N-terminus (1–24 aa) was replaced with that of bombyxin from B. mori. In addition, FLAG tag was added to its C-terminus for the easy purification as shown in schematic representation (Fig. 1a). A recombinant BmNPV bacmid containing rPfMSP3 expression cassette was constructed and injected into silkworm larvae. The hemolymph was collected and rPfMSP3 purified by 40% (saturation) ammonium sulfate treatment, HiTrap Phenyl FF

Discussion

The rPfMSP3 was successfully expressed in silkworm larvae as a secretory protein and purified from silkworm hemolymph. Estimated molecular mass of rPfMSP3 with FLAG tag sequence (not including the bombyxin signal sequence) is approximately 38 kDa, but that of rPfMSP3 expressed in silkworm larvae was 55 kDa. This result suggests that this discrepancy may come from post-translational modifications in silkworms. Therefore, the rPfMSP3 was treated by PNGase F to deglycosylate its N-glycans.

Conclusions

In this study, rPfMSP3 was expressed in silkworm larvae and secreted into its hemolymph by the addition of bombyxin signal sequence. rPfMSP3 was purified from hemolymph using ammonium sulfate precipitation and two-column chromatography. Approximately 234 μg of post-translationally modified rPfMSP3 was obtained from 10 ml of silkworm larval hemolymph. Purified rPfMSP3 had the similar properties to that expressed in E. coli and formed oligomers. In addition, sera from P. falciparum-infected

Acknowledgements

The authors would like to express gratitude to, Puspawati (RS Ratu Zalecha, Banjarmasin), Fridolina Mau (Sumba Tengah), Arta Farmawati (Faculty of Medicine, Public Health and Nursing UGM), and Irena Agustiningtyas in providing sera used in this study. Thanks to Mr. Kenshin Takemura and Ms. Mikiko Hayashitani for their participation in MSP3 purification and transmission electron microscopy experiment.

Ethics approval and consent to participate

Human sera collection was ethically approved by Medical and Health Research Ethics Committee (MHREC), Faculty of Medicine Public Health and Nursing, Universitas Gadjah Mada, Indonesia - DR. Sardjito general Hospital (Ref: KE/FK/0032/EC/2018).

Conflict of interest disclosure

The authors declare no commercial or financial conflict of interest.

References (24)

M. Imam et al.
Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation
J. Biol. Chem.
(2014)
C.W. Tsai et al.
Characterization of a protective Escherichia coli-expressed Plasmodium falciparum merozoite surface protein 3 indicates a non-linear, multi-domain structure
Mol. Biochem. Parasitol.
(2009)
S.B. Sirima et al.
A phase 2b randomized, controlled trial of the efficacy of the GMZ2 malaria vaccine in African children
Vaccine
(2016)
H. Sakamoto et al.
Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission
Parasitol. Int.
(2018)
T. Motohashi et al.
Efficient large-scale protein production of larvae and pupae of silkworm nuclear polyhedrosis virus (BmNPV) bacmid system
Biochem. Biophys. Res. Commun.
(2005)
V.K. Deo et al.
Display of Neospora caninum surface protein related sequence 2 on Rous sarcoma virus-derived gag protein virus-like particles
J. Biotechnol.
(2013)
B.R. Burgess et al.
Dissection of merozoite surface protein 3, a representative of a family of Plasmodium falciparum surface proteins, reveals an oligomeric and highly elongated molecule
J. Biol. Chem.
(2005)
H. Mirahmadi et al.
Soluble recombinant merozoite surface antigen-142kDa of Plasmodium vivax: an improved diagnostic antigen for vivax malaria
J. Microbiol. Methods
(2016)
P. Sonaimuthu et al.
Detection of human malaria using recombinant Plasmodium knowlesi merozoire surface protein-1 (MSP-1₁₉) expressed in Escherichia coli
Exp. Parasitol.
(2015)
B.L. Rice et al.
The origin and diversification of the merozoite surface protein 3 (msp3) multi-gene family in Plasmodium vivax and related parasites
Mol. Phylogenet. Evol.
(2014)

A. Outtara et al.

Vaccines against malaria

Clin. Infec. Dis.

(2015)

K. Kassegne et al.

Contribution of Plasmodium immunomics: potential impact for serological testing and surveillance of malaria

Expert Rev. Proteom.

(2018)

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Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm

Abstract

Introduction

Section snippets

Construction of recombinant bacmid

Purification of rPfMSP3

Discussion

Conclusions

Acknowledgements

Ethics approval and consent to participate

Conflict of interest disclosure

J. Biol. Chem.

Mol. Biochem. Parasitol.

Vaccine

Parasitol. Int.

Biochem. Biophys. Res. Commun.

J. Biotechnol.

J. Biol. Chem.

J. Microbiol. Methods

Exp. Parasitol.

Mol. Phylogenet. Evol.

Vaccines against malaria

Clin. Infec. Dis.

Contribution of Plasmodium immunomics: potential impact for serological testing and surveillance of malaria

Expert Rev. Proteom.