Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Eric Tzyy Jiann Chong ,Joveen Wan Fen Neoh ,Tiek Ying Lau ,Kek Heng Chua ,Yvonne Ai-Lian Lim,3 ,Ping-Chin Lee,4✉

1Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

2Department of Biomedical Science, Faculty of Medicine Building, University of Malaya, 50603 Kuala Lumpur, Malaysia

3Centre of Excellence for Research in AIDS (CERiA), University of Malaya, 50603 Kuala Lumpur, Malaysia

4Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

ABSTRACT Objective:To determine the genetic diversity of Plasmodium (P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

KEYWORDS: Plasmodium knowlesi;S-type small subunit ribosomal RNA;Genetic diversity;Natural selection;Haplotype

1.Introduction

The COVID-19 pandemic has lessened access to healthcare services,especially in the African regions.This has directly contributed to a 69 000 increase in malaria deaths,raising the malaria mortality rate to 15 deaths per 100 000 population in 2020[1,2].Although human malaria cases caused by the fivePlasmodiumspecies,namelyPlasmodium(P.)malariae,P.falciparum,P.knowlesi,P.ovale,andP.vivax,gradually decreased from 2 000 to 2019,the situation worsened in 2021,with an estimated 247 million malaria cases and 619 thousand malaria deaths being reported across the 84 endemic countries[3].Malaysia has reported zero indigenous malaria cases since 2018,but it is estimated that about 1.34 million of the Malaysian population are at risk of contracting malaria[3].

The Southeast Asian regions,including Malaysia,are severely impacted by the deadlyP.knowlesiparasites,a simianPlasmodium[4,5].From 2013 to 2017,P.knowlesicases accounted for 77.1% of malaria cases in Sabah and Sarawak,Malaysian Borneo,and 40.3% in Peninsular Malaysia[6].There are several strategies to estimate the spread of these parasites in the country,one of which is to understand the genetic diversity ofP.knowlesigenes.

Many studies have been conducted to understand the genetic diversity of selected genes ofP.knowlesiisolates from Malaysian Borneo and Peninsular Malaysia,such as circumsporozoite protein(csp),cytochrome b,gamma protein regionⅡ,and merozoite surface protein 1[7-12].However,the genetic diversity of the small subunit ribosomal RNA (SSU rRNA) gene in Malaysia’sP.knowlesiisolates is rarely reported.TheSSU rRNAgene encodes the SSU rRNA molecules of ribosomes,which are responsible for translating mRNA to functional proteins inPlasmodiumspecies.In parasitology,theSSU rRNAgene can be used as a biomarker to detect the presence of blood-stage parasites and is always a preferred target for identifying and determining evolutionary relationships ofPlasmodiumspecies in infected samples through a phylogenetic approach[13,14].TheSSU rRNAgene could also be utilized to differentiatePlasmodiumparasites undergoing asexual multiplication in the erythrocytes from those in the sexual stages,which helps in understanding the clinical manifestations of the disease.

TheSSU rRNAgenes appear to be multicopy per haploid genome and are located on different chromosomes.InP.knowlesi,the A-typeSSU rRNAgene isoforms are annotated on chromosomes 3 and 10,and they are expressed during the asexual blood stages in the vertebrate host.On the other hand,the S-typeSSU rRNAgene is mapped to chromosome 13 of the parasite’s genome[15,16].During the sexual stage ofP.knowlesiparasites,the merozoites differentiate into microgametocytes (male) and macro gametocytes (female),and the S-typeSSU rRNAgene is expressed in this stage to ensure their survival.The S-typeSSU rRNAis often considered more interesting since it can provide insights into the parasite's reproductive biology and transmission dynamics.

Despite the extensive use of theSSU rRNAgene in various diagnostic and evolutionary studies,information on the genetic diversity of the S-typeSSU rRNAgene inP.knowlesiisolates remains scarce,particularly in Malaysia,where this parasite has a severe impact.Therefore,this study aims to understand the genetic diversity of the S-typeSSU rRNAgene inP.knowlesiisolates from Sabah,Malaysian Borneo and Peninsular Malaysia,including aspects of natural selection and haplotypes.

2.Material and methods

2.1.P.knowlesi samples collection and DNA extraction

Peripheral blood samples were collected from 39 malaria patients infected withP.knowlesiparasites in different areas of Sabah,Malaysian Borneo,including Telupid (n=9),Keningau (n=6),Nabawan (n=3),Tambunan (n=5),and Tenom (n=6),as well as in Peninsular Malaysia (n=10) from 2008 to 2013 for this study (Table 1).The presence of theP.knowlesiparasites in the collected blood samples was previously verified using the Giemsa stain and crossvalidated using the PlasmoNexTMdiagnostic system[17].DNA was extracted from all the blood samples using a procedure previously described[18].

Table 1.Blood samples infected with Plasmodium knowlesi were collected from different areas in Sabah,Malaysian Borneo,and Peninsular Malaysia.

2.2.S-type SSU rRNA gene amplification using polymerase chain reaction

Polymerase chain reaction (PCR) amplification of the S-typeSSU RNAgene was performed usingPlasmodium-specific primers as previously described[19,20].In brief,a PCR was carried out in a 20 µL reaction mixture containing 1× of GoTaq Buffer (Promega,Madison,USA),2 mM of MgCl2,0.2 mM of dNTP mixtures,0.25 µM of each primer (rPLU5 and Pmk8),1 unit of Taq DNA polymerase(Promega,Madison,USA),and 100 ng of extracted DNA as a template.The PCR condition was set at 94 ℃ for 4 min,followed by 35 cycles of 94 ℃ for 30 s,55 ℃ for 1 min,and 72 ℃ for 1 min,and a final extension step at 72 ℃ for 5 min.The PCR products(≈1 080 bp) were electrophoresed and analysed in 1% agarose gel stained with ethidium bromide.

2.3.Cloning of the PCR amplicons and sequencing

The QIAquick Gel Extraction Kit (Qiagen,Hilden,Germany)was used to isolate the 39 successfully amplified PCR amplicons from agarose gel,and the purified PCR products were cloned into a pCRTM4-TOPO® TA vector using the TOPOTMTA CloningTMKit(Invitrogen,Waltham,USA).The ligated vectors were transformed intoEscherichia(E.)coliTOP10 strain using a heat-shock approach.The desired plasmid containing the S-typeSSU rRNAfragment was isolated using the QIAprep Spin Miniprep Kit (Qiagen,Hilden,Germany).Sanger sequencing utilizing the M13 forward and reverse sequencing primers was performed using the Applied Biosystems 3500 Series Genetic Analyzer (Thermo Fisher Scientific,Waltham,USA) according to the manufacturer's instructions.

2.4.SSU rRNA sequences alignment and phylogenetic tree construction

A total of 46P.knowlesi SSU rRNAsequences,including the 39 sequences from this study (S1) and seven S-type and A-type sequences retrieved from the Genbank database (S-type strain from human hosts: DQ350255,DQ350263,and LR 701173;S-type strain fromMacaca fascicularhost: DQ 350270;A-type from human hosts:AY327549 and AY 327557;A-type fromMacaca fascicularishost:DQ641518) were aligned using the CLUSTAL-W tool in Molecular Evolutionary Genetics Analysis 11 (MEGA 11) software[21].TheP.coatneyiSSU rRNAsequence (KC662442) was used as an outgroup.A phylogenetic tree was constructed using a Neighbour-joining method with bootstrap replicates of 1 000 to test the robustness and reliability of the tree.

2.5.Natural selection and haplotype analyses of the S-type SSU rRNA sequences

The aligned S-typeSSU rRNAsequences of theP.knowlesiisolates were trimmed,and a portion of the sequences (total length=939 bp) was subjected to natural selection and haplotype analyses by comparing to the referenceP.knowlesistrain H (LR701173).Polymorphisms in the S-typeSSU rRNAsequences were determined using the DnaSP v6 software[22].Data such as the average number of pairwise nucleotide differences (K),number of haplotypes(h),haplotype diversity (Hd),and nucleotide diversity (π) were obtained.A comprehensive analysis of the π was performed on a sliding window of 100 bases with a step size of 25 bp to estimate the stepwise diversity of the S-typeSSU rRNAsequences.The neutral theory of evolution was tested with Tajima's D as well as Fu and Li's D and F using the tools implemented in the DnaSP v6 software.The median-joining method in NETWORK v10 software (available at:https://www.fluxus-engineering.com/sharenet.htm) was utilized to generate a networking linkage of the S-typeSSU rRNAhaplotypes forP.knowlesiisolates.

Figure 1.Phylogenetic tree of SSU rRNA sequences constructed using a Neighbour-joining method in MEGA 11.A total of 46 S-type and A-type SSU rRNA sequences of Plasmodium knowlesi and one Plasmodium coatneyi SSU rRNA sequence as an outgroup were included in this phylogenetic tree.The number at the nodes indicates the percentage support of 1000 bootstrap replicates.

2.6.Ethical approval

Ethical approval of this study was obtained from the Medical Research and Ethics Committee of the University of Malaya Medical Centre with a reference number: 709.1.

3.Results

3.1.Phylogenetic tree analysis of the SSU rRNA sequences

Figure 2.Nucleotide segregating in the portion of the S-type SSU rRNA gene of Plasmodium knowlesi in this study.The sliding window plot with a window length of 100 bp and a step size of 25 bp for the number of segregating sites within the S-type SSU rRNA sequences was generated using DnaSP v6.

The phylogenetic tree revealed a distinct separation between the A-type and S-typeSSU rRNAsequences (Figure 1).When considering the S-typeSSU rRNAsequences,there is no evidence of a geographical cluster amongP.knowlesiisolates in Sabah,Malaysian Borneo,and Peninsular Malaysia.Additionally,there is no distinction betweenP.knowlesiisolates from humans andM.fascicularishosts.

3.2.Genetic analysis of the S-type SSU rRNA sequences

The genetic analysis of the 39 S-typeSSU rRNAsequences in this study revealed that the average number of pairwise nucleotide differences (K) was 5.768 when compared to the S-typeSSU rRNAsequence ofP.knowlesistrain H (LR701173),which served as the reference.The overall nucleotide diversity (π) and haplotype diversity (Hd) were 0.006±0.001 and 0.897±0.002,respectively.A comprehensive analysis of the π,using a sliding window length of 100 bp and a step size of 25 bp,revealed that the diversity ranged from 0.001 to 0.010.The nucleotide position at 482-581 bp was the most conserved region among the aligned S-typeSSU rRNAsequences,whereas the highest peak of nucleotide diversity was observed within 382-481 bp (Figure 2).

3.3.Natural selection and haplotype analyses

In natural selection analysis,Tajima’s D was calculated to be-2.406 (P<0.01),while Fu and Li’s D and F were -5.103 (P<0.02)and -4.938 (P<0.02),respectively.All the negative values in Tajima's D and Fu and Li's D and F suggest an expansion in population size with purifying selection.

This study identified several novel single nucleotide polymorphisms within the aligned S-typeSSU rRNAnucleotide sequences (Figure 3).These sequences were further classified into 26 different haplotypes.Notably,half of the haplotypes were shared among theP.knowlesiisolates from various areas of Sabah,Malaysian Borneo (haplotype 9).However,the median-joining network analysis revealed no haplotype sharing between theP.knowlesiisolates from Sabah,Malaysian Borneo,and Peninsular Malaysia (Figure 4).

Figure 3.Nucleotide sequence polymorphisms in the S-type SSU rRNA gene of Plasmodium knowlesi isolates from different areas in Sabah,Malaysian Borneo,and Peninsular Malaysia.Polymorphic nucleotide sites are listed for each haplotype,and the total number of sequences for each haplotype is listed in the rightend column.Nucleotides matched to those of the reference Plasmodium knowlesi strain H sequence (accession ID: LR701173) are marked by dots.

Figure 4.Median-joining network of haplotypes (h=26) for the S-type SSU rRNA gene of Plasmodium knowlesi isolates between different areas in Sabah,Malaysian Borneo,and Peninsular Malaysia.The radius of the circle corresponds to the total number of samples for each haplotype,and different colors represent each sampling site.Numbers along the lines are the polymorphism sites between two haplotypes.

4.Discussion

TheSSU rRNAgene is widely used in molecular approaches to identify bacterial strains and determine parasite species.In malaria research,theSSU rRNAgene is particularly valuable for differentiatingPlasmodiumspecies,especially between theP.knowlesiandP.malariaeparasites that are exceptionally challenging to distinguish under microscopy examinations.Despite being a highly acceptable biomarker for species identification,reports on the genetic diversity of theSSU rRNAgene inP.knowlesistudies are rare.Therefore,this study assessed the genetic diversity targeting the S-typeSSU rRNAgene ofP.knowlesiisolates from Sabah,Malaysian Borneo and Peninsular Malaysia,including the natural selection and haplotype aspects to address this issue.

The phylogenetic tree in this study divided theSSU rRNAsequences of theP.knowlesiisolates into two distinct parts according to their sexual types.While previous reports have shown transcription switching between stage-specific rRNA genes inP.berghei[23],it is worth investigating whetherP.knowlesiparasites have the ability to modify their genetic contents and selectively regulate the transcription activities when they exist in different sexual types within different hosts or environments to ensure their survival.Considering recent findings on the differential response of male and femalePlasmodiummature gametocytes to antimalarial drugs[24],understanding this aspect will have significant implications for drug development by clarifying whether certain antimalarial drugs are only effective in treatingP.knowlesiat specific sexual stages.

The average number of pairwise nucleotide differences (K) in the S-typeSSU rRNAgene is higher than that of the cytochromebgene but smaller than that of thecspgene,as previously reported inP.knowlesiisolates from Malaysia[8,9].However,the S-typeSSU rRNAgene exhibits lower overall nucleotide diversity (π),indicating that it is considerably more conserved compared to thecspand cytochromebgenes.In addition,the negative values in Tajima's D and Fu and Li's D and F suggest that the S-typeSSU rRNAgene is under a purifying effect,which is supported by previous studies[25,26].These characteristics make the S-typeSSU rRNAgene a promising target for comparative studies to investigate evolutionary relationships and determine the common ancestry among differentP.knowlesiisolates.

Haplotype sharing of the S-typeSSU rRNAgene was observed only between theP.knowlesiisolates collected from different areas in Sabah,Malaysian Borneo (haplotype 9 and haplotype 14).However,none of these haplotypes was identified among theP.knowlesiisolates from Sabah,Malaysian Borneo,and Peninsular Malaysia in this study.This could be partly explained by the existence of the South China Sea acting as a geographical barrier that inhibits genetic exchange betweenP.knowlesiisolates from Sabah,Malaysian Borneo,and Peninsular Malaysia.This is supported by a previous study that identified spatially distinct clusters ofP.knowlesiisolates between Malaysian Borneo and Peninsular Malaysia using multilocus microsatellite genotyping[27].Nevertheless,a recent whole-genome analysis ofP.knowlesiisolates from Malaysian Borneo and Peninsular Malaysia revealed substantial genome-wide divergence[26].

One limitation of this study is that the primers used could only amplify a portion of theSSU rRNAgene.As a result,determining the genetic diversity and natural selection of the completeSSU rRNAgene was not possible.Additionally,this study exclusively focuses onP.knowlesiisolates from Sabah,Malaysian Borneo,and Peninsular Malaysia.Notably,no comparison of genetic diversity and natural selection of theSSU rRNAgene with other countries(such as Thailand and Indonesia),which are also experiencing a high number ofP.knowlesiincidences,was conducted.Therefore,future studies should consider these limitations in their study design.

In conclusion,this study highlights that the genetic diversity of the S-typeSSU rRNAgene is conserved among theP.knowesiisolates in Sabah,Malaysian Borneo and Peninsular Malaysia,and with purifying effect.This finding establishes it as a promising target for comparative studies to determine the evolutionary relationships and common ancestry ofP.knowlesispecies.The data of this study is beneficial for conservation and environmental management and public health officials,especially in understanding the transmission ofP.knowlesiin Malaysia.

Conflict of interest statement

The authors declare no conflict of interests.

Funding

This study was supported by the Ministry of Higher Education,Malaysia (FRGS0322-SG-1/2013) and Universiti Malaysia Sabah(GUG0521-2/2020).

Authors’contributions

TYL,KHC,YALL,and PCL contributed to the study’s concepts and design.JWFN performed the literature search.JWFN and TYL conducted experimental studies.Data acquisition involved JWFN,TYL,KHC,YALL,and PCL.Data analysis was conducted by ETJC and JWFN.ETJC was involved in statistical analysis and manuscript preparation.Manuscript editing and review were conducted by ETJC,KHC,YALL,and PCL.PCL is the guarantor of this study.ETJC and JWFN contributed equally to this study.All authors approved the final version of this manuscript for submission.