Diversity pattern of Plasmodium knowlesi merozoite surface protein 4 (MSP4) in natural population of Malaysia

Autoři: Md Atique Ahmed aff001;  Ahmed Saif aff002;  Fu-Shi Quan aff001
Působiště autorů: Department of Medical Zoology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea aff001;  Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Najran University, Najran, Saudi Arabia aff002;  Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute, School of Medicine, Graduate school, Kyung Hee University, Seoul, Republic of Korea aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224743


Human infections due to the monkey malaria parasite Plasmodium knowlesi are increasingly being reported from Malaysia. The parasite causes high parasitaemia, severe and fatal malaria in humans thus there is a need for urgent measures for its control. The MSP4 is a potential vaccine candidate, which is well studied in Plasmodium falciparum and Plasmodium vivax; however, no study has been conducted in the orthologous gene of P. knowlesi. In this study, we investigated the level of polymorphisms, haplotypes, natural selection and population structure of full-length pkmsp4 in 32 clinical samples from Malaysian Borneo along with 4 lab-adapted strains. We found low levels of polymorphism across the gene with exon I showing higher diversity than the exon II. The C- terminal epidermal growth factor (EGF) domains and GPI-anchored region within exon II were mostly conserved with only 2 non-synonymous substitutions. Although 21 amino acid haplotypes were found, the frequency of mutation at the majority of the polymorphic positions was low. We found evidence of negative selection at the exon II of the gene indicating existence of functional constraints. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. High population differentiation values were observed within parasite populations originating from Malaysian Borneo (Kapit, Sarikei and Betong) and laboratory-adapted strains obtained from Peninsular Malaysia and Philippines indicating distinct population structure. This is the first study to genetically characterize the full-length msp4 gene from clinical isolates of P. knowlesi from Malaysia and thus would be very useful for future rational vaccine studies. Further studies with higher number of samples and functional characterization of the protein will be necessary.

Klíčová slova:

Borneo – DNA sequence analysis – Haplotypes – Malarial parasites – Malaysia – Natural selection – Plasmodium – Vaccines


1. Pinheiro MM, Ahmed MA, Millar SB, Sanderson T, Otto TD, Lu WC, et al. Plasmodium knowlesi genome sequences from clinical isolates reveal extensive genomic dimorphism. PLoS One. 2015;10(4):e0121303. Epub 2015/04/02. doi: 10.1371/journal.pone.0121303 25830531.

2. Amir A, Cheong FW, de Silva JR, Liew JWK, Lau YL. Plasmodium knowlesi malaria: current research perspectives. Infection and drug resistance. 2018;11:1145–1155. doi: 10.2147/IDR.S148664 30127631.

3. World Health Organisation (2017) World Malaria Report.

4. Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, et al. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet. 2004;363(9414):1017–1024. doi: 10.1016/S0140-6736(04)15836-4 15051281.

5. Barber BE, William T, Grigg MJ, Menon J, Auburn S, Marfurt J, et al. A prospective comparative study of knowlesi, falciparum, and vivax malaria in Sabah, Malaysia: high proportion with severe disease from Plasmodium knowlesi and Plasmodium vivax but no mortality with early referral and artesunate therapy. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2013;56(3):383–397. doi: 10.1093/cid/cis902 23087389.

6. Barber BE, William T, Jikal M, Jilip J, Dhararaj P, Menon J, et al. Plasmodium knowlesi malaria in children. Emerg Infect Dis. 2011;17(5):814–820. Epub 2011/05/03. doi: 10.3201/eid1705.101489 21529389; PubMed Central PMCID: PMC3321776.

7. Daneshvar C, Davis TM, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PC, et al. Clinical and laboratory features of human Plasmodium knowlesi infection. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2009;49(6):852–860. doi: 10.1086/605439 19635025.

8. William T, Menon J, Rajahram G, Chan L, Ma G, Donaldson S, et al. Severe Plasmodium knowlesi malaria in a tertiary care hospital, Sabah, Malaysia. Emerg Infect Dis. 2011;17(7):1248–1255. doi: 10.3201/eid1707.101017 21762579.

9. Willmann M, Ahmed A, Siner A, Wong IT, Woon LC, Singh B, et al. Laboratory markers of disease severity in Plasmodium knowlesi infection: a case control study. Malar J. 2012;11:363. doi: 10.1186/1475-2875-11-363 23110615.

10. Assefa S, Lim C, Preston MD, Duffy CW, Nair MB, Adroub SA, et al. Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi. Proc Natl Acad Sci U S A. 2015;112(42):13027–13032. doi: 10.1073/pnas.1509534112 26438871

11. Ahmed MA, Fong MY, Lau YL, Yusof R. Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo. Malar J. 2016;15:241. doi: 10.1186/s12936-016-1294-6 27118390

12. Ahmed MA, Lau YL, Quan FS. Diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium knowlesi in Malaysia. Malar J. 2018;17(1):274. doi: 10.1186/s12936-018-2423-1 30053885.

13. Yusof R, Ahmed MA, Jelip J, Ngian HU, Mustakim S, Hussin HM, et al. Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia. Emerg Infect Dis. 2016;22(8):1371–1380. doi: 10.3201/eid2208.151885 27433965.

14. Fowkes FJ, Richards JS, Simpson JA, Beeson JG. The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: A systematic review and meta-analysis. PLoS medicine. 2010;7(1):e1000218. doi: 10.1371/journal.pmed.1000218 20098724.

15. Takala SL, Coulibaly D, Thera MA, Batchelor AH, Cummings MP, Escalante AA, et al. Extreme polymorphism in a vaccine antigen and risk of clinical malaria: implications for vaccine development. Science translational medicine. 2009;1(2):2ra5. doi: 10.1126/scitranslmed.3000257 20165550.

16. Marshall VM, Silva A, Foley M, Cranmer S, Wang L, McColl DJ, et al. A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain. Infection and immunity. 1997;65(11):4460–4467. 9353020.

17. Gilson PR, Nebl T, Vukcevic D, Moritz RL, Sargeant T, Speed TP, et al. Identification and stoichiometry of glycosylphosphatidylinositol-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Molecular & cellular proteomics: MCP. 2006;5(7):1286–1299. doi: 10.1074/mcp.M600035-MCP200 16603573.

18. Wang L, Richie TL, Stowers A, Nhan DH, Coppel RL. Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam. Infection and immunity. 2001;69(7):4390–4397. doi: 10.1128/IAI.69.7.4390-4397.2001 11401978.

19. de Silva HD, Saleh S, Kovacevic S, Wang L, Black CG, Plebanski M, et al. The antibody response to Plasmodium falciparum Merozoite Surface Protein 4: comparative assessment of specificity and growth inhibitory antibody activity to infection-acquired and immunization-induced epitopes. Malar J. 2011;10:266. doi: 10.1186/1475-2875-10-266 21920045.

20. Kedzierski L, Black CG, Coppel RL. Characterization of the merozoite surface protein 4/5 gene of Plasmodium berghei and Plasmodium yoelii. Molecular and biochemical parasitology. 2000;105(1):137–147. PMID: 10613706. doi: 10.1016/s0166-6851(99)00178-4 10613706

21. Wang L, Black CG, Marshall VM, Coppel RL. Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum. Infection and immunity. 1999;67(5):2193–2200. 10225874.

22. Perraut R, Varela ML, Joos C, Diouf B, Sokhna C, Mbengue B, et al. Association of antibodies to Plasmodium falciparum merozoite surface protein-4 with protection against clinical malaria. Vaccine. 2017;35(48 Pt B):6720–6726. doi: 10.1016/j.vaccine.2017.10.012 29042203.

23. Martinez P, Suarez CF, Gomez A, Cardenas PP, Guerrero JE, Patarroyo MA. High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4). Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2005;5(4):354–361. doi: 10.1016/j.meegid.2004.12.001 16168942.

24. Putaporntip C, Jongwutiwes S, Ferreira MU, Kanbara H, Udomsangpetch R, Cui L. Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene. Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2009;9(5):821–826. doi: 10.1016/j.meegid.2009.04.017 19409511.

25. Benet A, Tavul L, Reeder JC, Cortes A. Diversity of Plasmodium falciparum vaccine candidate merozoite surface protein 4 (MSP4) in a natural population. Molecular and biochemical parasitology. 2004;134(2):275–280. doi: 10.1016/j.molbiopara.2003.12.005 15003847.

26. Ahmed MA, Quan F- S. Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D). bioRxiv. 2019:537621. doi: 10.1101/537621

27. Ahmed AM, Pinheiro MM, Divis PC, Siner A, Zainudin R, Wong IT, et al. Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members. PLoS neglected tropical diseases. 2014;8(8):e3086. doi: 10.1371/journal.pntd.0003086 25121807.

28. Ahmed MA, Chu KB, Vythilingam I, Quan FS. Within-population genetic diversity and population structure of Plasmodium knowlesi merozoite surface protein 1 gene from geographically distinct regions of Malaysia and Thailand. Malar J. 2018;17(1):442. doi: 10.1186/s12936-018-2583-z 30497496.

29. Almagro Armenteros JJ, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nature biotechnology. 2019;37(4):420–423. doi: 10.1038/s41587-019-0036-z 30778233.

30. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451–1452. doi: 10.1093/bioinformatics/btp187 19346325.

31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution. 2011;28(10):2731–2739. doi: 10.1093/molbev/msr121 21546353.

32. Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evolutionary bioinformatics online. 2007;1:47–50. 19325852.

33. Wang L, Marshall VM, Coppel RL. Limited polymorphism of the vaccine candidate merozoite surface protein 4 of Plasmodium falciparum. Molecular and biochemical parasitology. 2002;120(2):301–303. doi: 10.1016/s0166-6851(01)00457-1 11897136.

34. Ahmed MA, Fauzi M, Han ET. Genetic diversity and natural selection of Plasmodium knowlesi merozoite surface protein 1 paralog gene in Malaysia. Malar J. 2018;17(1):115. doi: 10.1186/s12936-018-2256-y 29540177.

Článek vyšel v časopise


2019 Číslo 11