Research paper
Genetic diversity in the C-terminus of merozoite surface protein 1 among Plasmodium knowlesi isolates from Selangor and Sabah Borneo, Malaysia

https://doi.org/10.1016/j.meegid.2017.06.019Get rights and content

Highlights

  • P. knowlesi malaria is significant in Southeast Asia.

  • This study aims to determine the sequence variation in P. knowlesi MSP-142.

  • Pk-msp-142 gene was amplified by PCR and directly sequenced. Haplotype diversity (Hd) and nucleotide diversity (л) were studied among the isolates.

  • There was relatively high genetic variation among P. knowlesi isolates. A total of nine different haplotypes related to amino acid alterations were found at 13 positions and the Pk-MSP-119 sequence was found to be more conserved than Pk-msp-133.

  • There was evidence of negative selection in Pk-msp-42 as well as the 33kDa and 19kDa fragments by comparing the rate of non-synonymous versus synonymous substitutions.

Abstract

Plasmodium knowlesi, a malaria parasite of macaques, has emerged as an important parasite of humans. Despite the significance of P. knowlesi malaria in parts of Southeast Asia, very little is known about the genetic variation in this parasite. Our aim here was to explore sequence variation in a molecule called the 42 kDa merozoite surface protein-1 (MSP-1), which is found on the surface of blood stages of Plasmodium spp. and plays a key role in erythrocyte invasion. Several studies of P. falciparum have reported that the C-terminus (a 42 kDa fragment) of merozoite surface protein-1 (MSP-142; consisting of MSP-119 and MSP-133) is a potential candidate for a malaria vaccine. However, to date, no study has yet investigated the sequence diversity of the gene encoding P. knowlesi MSP-142 (comprising Pk-msp-119 and Pk-msp-133) among isolates in Malaysia. The present study explored this aspect. Twelve P. knowlesi isolates were collected from patients from hospitals in Selangor and Sabah Borneo, Malaysia, between 2012 and 2014. The Pk-msp-142 gene was amplified by PCR and directly sequenced. Haplotype diversity (Hd) and nucleotide diversity (л) were studied among the isolates. There was relatively high genetic variation among P. knowlesi isolates; overall Hd and л were 1 ± 0.034 and 0.01132 ± 0.00124, respectively. A total of nine different haplotypes related to amino acid alterations at 13 positions, and the Pk-MSP-119 sequence was found to be more conserved than Pk-msp-133. We have found evidence for negative selection in Pk-msp-42 as well as the 33 kDa and 19 kDa fragments by comparing the rate of non-synonymous versus synonymous substitutions. Future investigations should study large numbers of samples from disparate geographical locations to critically assess whether this molecule might be a potential vaccine target for P. knowlesi.

Introduction

Malaria, caused by protists of the genus Plasmodium, is transmitted by mosquitoes and remains one of the most important parasitic diseases in the world, with > 3 billion people at risk of infection and about 200 million human cases of malaria occurring each year (WHO, 2015). The annual malaria-associated mortality is approaching a staggering 43, 8000 people, with mortality primarily in children of less than five years of age (WHO, 2015). The recent recognition of Plasmodium knowlesi, a zoonotic malaria parasite, as one of the causative agents of human malaria has made the abatement of global incidence of this disease more challenging (Singh et al., 2004, Cox-Singh et al., 2008).

Plasmodium knowlesi is transmitted by mosquitoes to humans from two major monkey reservoir hosts, the long-tailed (Macaca fascicularis) and pig-tailed (M. nemestrina) (Knowles and Das Gupt, 1932, Garnham, 1966). To date, human-to-human transmission has not been reported. The parasite is unique in that it has a short life cycle of 24 h, enabling a rapid progression of disease (Daneshvar et al., 2009, Cox-Singh et al., 2010). Importantly, it can be very virulent in human patients, associates with high parasitaemia, and can cause severe complications and death (Cox-Singh et al., 2008, Cox-Singh et al., 2010). Although globally the prevalence and incidence of human infection with P. knowlesi are much less than those caused by P. falciparum and P. vivax (80–95%) (Stępień, 2014), recent reports of severe and fatal consequences of knowlesi malaria in humans (Cox-Singh et al., 2010, Lau et al., 2011) highlight the public importance of this simian parasite, particularly in Malaysia. In 2016, P. knowlesi comprised 69% of the malaria cases reported in Malaysia (Ministry of Health, Malaysia, unpublished reports).

A study conducted by William et al. (2014) described the changing epidemiology of malaria in Sabah Borneo, and suggested a more than ten-fold increase in incidence of P. knowlesi infection in humans (from 59 in 2004 to 703 in 2011, 815 in 2012 and 996 in 2013). Besides Malaysian Borneo (Singh et al., 2004, Cox-Singh et al., 2008, Daneshvar et al., 2009, Lau et al., 2011, William et al., 2011) and Peninsular Malaysia (Cox-Singh et al., 2008, Kantele et al., 2008, Vythilingam et al., 2008, Lee et al., 2010), P. knowlesi infection in humans has also been reported in Southeast Asian regions such as Singapore (Ng et al., 2008, Ong et al., 2009, Jeslyn et al., 2011), Thailand (Putaporntip et al., 2009, Sermwittayawong et al., 2012), Myanmar (Zhu et al., 2006, Jiang et al., 2010), the Philippines (Luchavez et al., 2008), Indonesia (Figtree et al., 2010, Sulistyaningsih et al., 2010), Vietnam (Eede et al., 2009) and Cambodia (Khim et al., 2011).

Despite the significance of P. knowlesi malaria in these geographical regions, there is limited information about the genetic variation within the parasite (Putaporntip et al., 2013). Recent multilocus microsatellite genotyping of P. knowlesi from diverse regions of Malaysia indicated the presence of three major subpopulations of P. knowlesi, including two divergent clusters of human cases in Malaysian Borneo (associated with long-tailed macaques and pig-tailed macaques) and a third cluster in humans in Peninsular Malaysia, with most of the infections from wild long-tailed macaques sampled in Kelantan (Divis et al., 2017). Our focus here was to explore sequence variation in an immunogenic molecule called the merozoite surface protein-1 (MSP-1), which is found on the surface of blood stages of Plasmodium spp. and plays a key role in erythrocyte invasion (Holder and Freeman, 1984, Holder et al., 1992). Consequently, MSP-1 has been recognised as a vaccine candidate (Holder, 2009).

However, Plasmodium MSP-1 is known to exhibit sequence diversity among isolates, which might be the result of selective pressure by host immune responses (Tanabe et al., 1987, Putaporntip et al., 2002, Miahipour et al., 2012). In Plasmodium species, msp-1 gene encodes a 190 kDa precursor protein which undergoes a two-step proteolytic cleavage during merozoite maturation. It is cleaved into four major fragments of 83-, 42-, 38-, 30-kDa (MSP-183, MSP-142, MSP-138 and MSP-130), which remain on the merozoite surface as a glycosylphosphatidylinositol-anchored complex. Before erythrocyte invasion, the MSP-142 fragment undergoes a second cleavage, resulting in the 33- and 19-kDa (MSP-133 and MSP-119) fragments, the latter of which remains on the surface as the merozoite (Blackman et al., 1991, Blackman et al., 1996), and MSP-142, which has been reported to be highly antigenic (Cheong et al., 2013). MSP-142 and MSP-119 fragments have received attention as immunogens, given that antibodies directed against MSP-142 and MSP-119 were shown to interrupt merozoite invasion in vitro (Patino et al., 1997, Nwuba et al., 2002). Sequence variation in the central repeat region of MSP-142 of P. falciparum (see Mehrizi et al., 2008, Zamani et al., 2009, Pan et al., 2010, Viputtigul et al., 2013) and P. vivax (see Dias et al., 2011, Kang et al., 2012) has been relatively well studied, but nothing is known about the structure, function or genetic variation in MSP-1 in P. knowlesi. In the present study, we explored, for the first time, sequence variation in P. knowlesi MSP-142 (designated Pk-MSP-142) from Peninsular Malaysia and Sabah Borneo, Malaysia.

Section snippets

Collection of blood samples

Genomic DNAs (n = 12) from blood samples from confirmed knowlesi malaria patients were subjected to molecular analysis. Samples were collected in Selangor (n = 7), state of peninsular Malaysia, and in Sabah (n = 5), state of Malaysia Borneo. For samples from Selangor, filter blood spots were collected between 2012 and 2014 from Kuala Kubu Bharu Hospital (n = 5), Selayang Hospital (n = 1) and Hospital Sungai Buloh (n = 1). For samples from Sabah, whole blood samples were collected from humans in 2014 at

Results and discussion

High quality C-terminal sequences of Pk-msp-142 (940 bp) were obtained for 12 isolates (seven from Selangor, Peninsular Malaysia, and five from Sabah Borneo). This fragment contained a region coding for a protein sequence of 313 amino acids. Analysis and comparison at the nucleotide level against the reference sequence for P. knowlesi strain H (GenBank accession no. XM_002258546) displayed nucleotide alterations at 37 positions among the isolates in the 940 bp region. P. knowlesi msp-133 (Pk-msp-1

Acknowledgements

Funding from the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) of Australia, Melbourne Water Corporation (R.B.G. et al.) and University of Malaya/Ministry of Higher Education High Impact Research (UM.C/625/1/HIR/MOHE/MED/23), University of Malaya (PG056-2013A) (Y.A.L.L. et al.) are gratefully acknowledged. Support from the Endeavour program (ID 4731-2015) is also gratefully acknowledged (NJY).

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