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RESEARCH ARTICLE

Analysis of polymorphisms and selective pressures on ama1gene in Plasmodium knowlesi

isolates fromSabah, Malaysia

CHUEN YANG CHUA1, PING CHIN LEE2, TIEK YING LAU1*

*Corresponding author at: Biotechnology Research Institute, Universiti Malaysia Sabah,

88400 Kota Kinabalu, Sabah, Malaysia;

e-mail address: sophialau@ums.edu.my

Biotechnology Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah,

Malaysia. 2Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu,

Sabah, Malaysia.

Abstract

The apical membrane antigen-1 (AMA-1) of Plasmodium spp. is a merozoite surface antigen

that is essential for the recognition and invasion of erythrocytes. Polymorphisms occurring in this

surface antigen will cause major obstacles in developing effective malaria vaccines based on AMA-1.

The objective of this study was to characterize ama1 gene in Plasmodium knowlesiisolates from

Sabah. DNA was extracted from blood samples collected from Keningau, Kota Kinabaluand Kudat.

The Pkama1 gene was amplified using nested PCR and subjected to bidirectional sequencing.

Analysis of DNA sequence revealed that most of the nucleotide polymorphisms were synonymous

and concentrated in domain I of PkAMA-1. 14 haplotypes were identified based on amino acid

variations and haplotype K5 was the most common haplotype.dN/dS ratiosimplied that purifying

selection was prevalent in Pkama1 gene. Fu & Li’s D and F valuesfurther provided evidence of

negative selection acting on domain II of Pkama1. Low nucleotide diversity was also detected for the

Pkama1 sequences, which is similar to reports on Pkama1 from Peninsular Malaysia and Sarawak.

The presence of purifying selection and low nucleotide diversity indicated that domain II of Pkama1

can be used as a target for vaccine development.

1. Introduction

Malaria is regarded as one of the world’s most widespread and deadly disease, with an

infection rate of 500 million cases and more than one million deaths reported annually (Lim et al.,

2010). It is prevalent in tropical and subtropical regions such as Malaysian Borneo because of the

rainfall, warm temperatures and stagnant waters, which provide ideal habitats for mosquito larvae.

The Anopheles mosquito acts as the insect vector that transmits the causative agents of malaria,

Plasmodium parasites. Currently there are sixPlasmodium species that are known to infect humans,

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namely P. falciparum, P. vivax, P. malariae, P. ovalecurtisi, P. ovalewallikeriand most recently, P.

knowlesi(White, 2007).

Plasmodium knowlesiis the fifth Plasmodium parasite that is capable of natural human

infections and it is widespread throughout Southeast Asia, with cases of naturally acquired P.

knowlesiinfections in humans being reported (Singh et al., 2004). The high rates of replication

observed in Plasmodium knowlesihave resulted in extensive erythrocyte destruction in infected

humans and consequently, a mortality rate that is comparable to malaria caused by P. falciparum

(William et al., 2011). The high parasitaemia associated with P. knowlesiinfections has also been

attributed to the severity of malaria caused by this species (Daneshwaret al., 2009).Conventional

microscopic examination involving the diagnosis ofP. knowlesiinfections has proven to be a difficult

task for many laboratory technicians due to its identical morphology with other Plasmodium

parasites, such as P. falciparum and P. malariae(Lee et al., 2009).

The recognition and invasion of erythrocytes by Plasmodium parasites are central to the

whole process of the parasitic infection and it is dependent on several surface molecules on the

parasites’ surface, such as merozoite surface protein-1 (MSP-1), Duffy binding protein (DBP),

circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA-1). AMA-1 is a micronemal

protein that is commonly found in apicomplexan parasites. It was first identified as an invariant

merozoite surface antigen derived from a single gene found in Plasmodium knowlesi(Deans, 1984).

Trigliaet al. (2000) studied the gene substitution of ama1 and they found that the parasite’s ability to

invade erythrocytes was affected when the ama1 gene was disrupted. This finding validated the

importance of AMA-1 in the erythrocytic invasion process of the Plasmodium parasites.

On the genomic level, there are widespread diversities in the gene encoding for AMA-1

protein in Plasmodium spp., and they are mainly due to single nucleotide polymorphisms (SNPs)

(Takalaet al., 2009). Escalante et al. (2001) described the population distribution of polymorphism in

ama1 gene and suggested that the polymorphic patterns observed are results of diversifying

selection to avoid the binding of inhibitory antibodies. One example of the selective pressures

influencing ama1 allelic diversity includes the human immune system (Polley& Conway, 2001).

However, the genetic diversity was not evenly distributed across the ama1 gene, where most of

them are concentrated in the region encoding for the ectodomain of AMA-1 protein. Genetic

diversity is less prevalent in other regions of the ama1 gene due to the structural and functional

constraints (Mahajan et al., 2005).

The whole coding region of ama1 gene can be sub-divided into several regions that encode

for prosequence, ectodomain, transmembrane region and cytoplasmic tail of the AMA-1 protein

(Remarque et al., 2008). The ectodomain segment can be divided into Domain I, Domain II and

Domain III, which are defined as disulfide-bonded structures of the AMA-1 protein (Hodder et al.,

1996). These domains have been shown to display varying patterns of nucleotide diversity between

Plasmodium species. The polymorphic sites and nucleotide diversity is more prevalent in Domain I of

P. falciparum (Mardaniet al., 2012) and P. vivax (Putaporntipet al., 2009). Positive natural selection,

on the other hand, is more prevalent in Domain I of P. falciparum (Abouieet al., 2013) and Domain II

of P. vivax (Dias et al., 2011). Although the concentration of polymorphism and selective pressures

has been elucidated forPkama1 in Sarawak (Faber et al., 2015) and Peninsular Malaysia (Fong et al.,

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2015), such studies have yet to carried out in the regions around Sabah.Hence, this study will be

beneficial for a better understanding of polymorphic patterns and selective pressures acting on ama1

gene of P. knowlesi.

The knowlesimalaria reported in Malaysia is less widespread and accounts for only 5% of

malaria cases in Malaysia (WHO, 2011). The prevalence of P. knowlesiis however, more apparent in

Sabah compared to the other Plasmodium species (Cox-Singh et al., 2008). Despite the decrease in

occurrence of malaria cases caused by P. falciparum and P. vivax in Sabah, the incidence of P.

knowlesi infections continues to increase (William et al., 2013). Barber et al. (2011) reported that the

highest proportion of malaria cases caused by P. knowlesi is located at the north-east region of

Sabah.This was further validated when P. knowlesiwas reported in all four districts of Sandakan,

Sabah, withthe highest incidence being reported in the Kinabatangan district (Goh et al., 2013).

Plasmodiumknowlesiwas also highly prevalent in the interior divisions of Sabah, where Lau et al.

(2011) reported P. knowlesias the most prevalent Plasmodium sp., accounting for more than half of

the positive malaria cases involved. The significance of P. knowlesiinfections in Malaysia is

emphasized on the fatality rates associated with it, which is higher or comparable to that of P.

falciparum (William et al., 2011).

2. Materials and Method

2.1 Study sites and sample collection

In this study, blood samples of human malaria patientswere collectedfrom Hospital Kudat,

Hospital Keningau and Hospital Queen Elizabeth in 2012 after written consent for sample collection

was obtained. This study was approved by Medical Research & Ethics Committee of the Ministry of

Health Malaysia.27 blood samples were collected from Kudat while 4 and 5 blood samples were

collected from Keningau and Kota Kinabalu respectively. The blood samples were spotted on the 3

MM chromatography paper. The volume of each blood spot is approximately 25 µl and the blood

spots were air dried before the chromatography paper was sealed in a plastic bag. The blood samples

collected were stored at room temperature prior to genomic DNA extraction.

2.2 Extraction of genomic DNA and Pkama1 gene amplification

The genomic DNA of Plasmodium parasites infecting human patients were extracted from

dried blood spots on chromatography paper using QIAamp DNA Mini Kit (QIAGEN, Inc., USA) and

stored at -20 ℃ before usage.

The extracted genomic DNA was subjected to Plasmodium genus-specific PCR in order to

determine the presence ofPlasmodium spp. in the patient’s blood samples. The PCR amplification

reactions were based on the small subunit ribosomal RNA (ssrRNA) gene of Plasmodium spp. as

previously described (Snounouet al., 1993; Imwonget al., 2009).

The patient samples with confirmed single P. Knowlesi i nfection were included in the

amplification ofPkama1 gene. Nested PCR primers were designed using Primer3 v. 0.4.0

(Untergasseret al., 2007)based on the Pkama1 gene of strain H (Accession no. XM_002259303).Nest

1 PCR primers PkAMA1-F1 (5’-CTG AAC TCG GTT GCT ACT AC-3’) and PkAMA1-R1 (5’-CAC ACC CAC

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AGT TGT TAC GA-3’)annealed at the flanking region of PkAMA-1, with an expected size of 1847 bp for

nest 1 PCR product. Nest 2 PCR primers PkAMA1-F2 (5’-CCG ATT AAT GAA GAG AGG GAG AA-3’) and

PkAMA1-R2 (5’-GCC CTG ACG AAT AAT ACG TTG CT-3’) alsoannealedat the flankingregion of PkAMA-

1, with an expected size of 1792 bp.

The nest 1 PCR amplification was carried out in a 20 µl reaction containing 1x PromegaGoTaq

buffer, 2 mM of MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 0.25 µM of each

primer, 1 UTaq DNA polymerase (PromegaCoorporation, USA) and 4 µl of genomic DNA. The PCR

conditions were as follows; initial denaturation at 94°C for 10 minutes, 40 cycles of denaturation at

94°Cfor 1 minute, annealing for 1 minute at 52°C,extension at 72°C for 1 minute and final extension

at 72°C for 10 minutes.

The nest 2 PCR amplification was carried out in 20 µl reactions containing 1x PromegaGoTaq

buffer, 1.6 mM of MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 0.25 µM of each

primer, 1 UTaq DNA polymerase (PromegaCoorporation, USA) and 2 µl of nest 1 PCR product as the

DNA template. The PCR conditions were as follows; initial denaturation at 94 °C for 10 minutes, 35

cycles of denaturation at 94°C for 1 minute, annealing for 1 minute at 54°C, extension at 72°C for 1

minute and a final extension at 72°C for 10 minutes. The nest 2 PCR products were analyzed by

electrophoresis on 1% agarose gel.

2.3. Nucleotide sequencing and sequence analysis

Gel purification of PCR products was carried outusing QIAquick Gel Extraction Kit (QIAGEN,

Inc., USA) prior tobidirectional sequencing. The forward and reverse sequencing of Pkama1 were

aligned and assembled into one single contig using the SeqMan module of the DNASTAR software.

Basic Local Alignment Search Tool (BLAST)program was then used to confirm that the sequencing

results were the desired Pkama1 gene. ThePkama1 sequences were then aligned and compared with

the strain H reference sequences(Accession no. XM_002259303 and AF298218) using the MEGA6

software (Tamura et al., 2013).The ratio of non-synonymous to synonymous substitutions (dN/dS)

was determined using the Nei and Gojobori’s method with the Jukes and Cantor correction (Nei and

Gojobori, 1986). Nucleotide diversity (Pi), haplotype diversity (Hd), Tajima’s D, Fu and Li’s D and F

tests were calculated for each of the three domains of Pkama1 as well as the whole Pkama1

sequences usingDNASP version 5.10.01 (Librado and Rozas, 2009).Haplotypes were classified based

on the pattern of amino acid variations observed among the AMA-1 of P. knowlesiisolates from

Sabah.

3. Results

3.1 Molecular characterization of Pkama1 gene

The complete Pkama1gene sequence is 1692 bp in length and PCR amplicons corresponding

to the 4-1662 bp region of the gene was successfully amplifiedand sequenced. ThePkama1

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sequences were deposited in Genbank database under the Accession no. KM061722-KM061757.A

total of 36 Pkama1 sequences were obtained, 27 from Kudat (KT), 4 from Keningau (KG) and 5 from

Kota Kinabalu (KK). The amplified nucleotide sequence encodes for amino acids 2-554 of the AMA-1

protein. 36 Pkama1 sequences were aligned with 2 reference sequence ofP. knowlesi ama1

(Accession no. XM_002259303 and AF298218).In comparison with reference sequence

XM_002259303, 37 point mutations that comprised of 25 synonymous and 12 non-synonymous

mutations were detected (Table 1). When compared to reference sequence AF298218, only 36 point

mutations were detected with 24 synonymous and 12 non-synonymous mutations.The reference

sequence AF298218 shared the same nucleotide base at the 1629 nucleotide position with the

isolates of this study.The Pkama1 sequences were subjected to BLAST against NCBI’s nucleotide

database (Wheeler et al., 2003), and most were found to be novel except for isolates KK100 and

KT010, which shared 100% identity with an isolate (Accession no. KP067836) from Betong, Sarawak

(Faber et al., 2015).

When amino acid sequences of PkAMA-1 were aligned and compared with reference

sequence 1 and 2, 12 amino acid variants were detected, all of which lies in the ectodomain region

(Table 2). Six of the variants were located in domain I of PkAMA-1, which three variants were each

found in domains II and III. Ten of the variants were dimorphic and one was trimorphic

(H481N/Q).Based on the polymorphic patterns in the amino acid sequence of PkAMA-1, 14

haplotypes were classified (Table 3). Haplotypes K1 and K3 were obtained from blood samples

collected in Keningau, haplotypes K2, K9, K10, K11, K12, K13 and K14 were exclusively from Kudat

while haplotypes K6 and K8 were found only from Kota Kinabalu. Haplotypes K4, K5 and K7 consisted

of samples from all the three regions, indicating similar patterns of polymorphism from the different

regions in Sabah.

3.2 Sequence analysis of Pkama1

The ratio of synonymous to non-synonymous mutations (dN/dS) of the 36 Pkama1 nucleotide

sequences was determined using the MEGA6 software (Table 4). Using the entire Pkama1 gene for

analysis, the dN/dS ratio obtained was 0.173, which gave an indication of purifying selection. Similar

dN/dS ratios were obtained for each of the domains when analyzed independently. Nucleotide

diversity (Pi), Tajima’s D and Fu & Li’s D and F values were also determined for the entire Pkama1

gene and each of its domains. The values obtained for nucleotide diversity was low (Table 4), which

was consistent with previous reports on Pkama1 (Faber et al., 2015; Fong et al., 2015). As for

Tajima’s D and Fu & Li’s D and F values, negative values were obtained for each neutrality test,

except for Fu & Li’s D on the domain III of Pkama1 (Table 4). Although negative values of these

neutrality tests points towards purifying selection, most of these values however, were statistically

insignificant (P > 0.05). The only exception was for the Fu & Li’s D and F values obtained for domain II

of Pkama1, which showed statistical significance (P < 0.05).

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4. Discussion

Among the 36 polymorphic sites identified in Pkama1 isolates from Sabah, 37 point

mutations were reported and they were mostly synonymous mutations (Table 1). At nucleotide

position 564, both synonymous and non-synonymous mutations were reported among the

Pkama1isolates. This indicates that two different single nucleotide polymorphisms (SNPs) occurred at

the same polymorphic site, one of which resulted in an amino acid change while the other had no

effect on the amino acid sequence of PkAMA-1. The nucleotide polymorphisms reported in Pkama1

for this study were found to be concentrated (45.95 %) in the domain I of Pkama1, while 24.32% and

13.52% of nucleotide polymorphisms were found in domain II and III respectively. This finding was

similar to previous reports onPfama1 (Mardaniet al., 2012) and Pvama1 (Putaporntipet al., 2009),

which indicates that domain I is the most polymorphic region in the ama1 gene of Plasmodium

parasites. This was expected, given that domain I is the largest domain in the Pkama1 gene, encoding

for almost half of the ectodomain region. Further validation on the mutations observed in the

Pkama1 isolates in this study is however, required as TaqDNA polymerase was used for PCR

amplification.

There were a total of 12 amino acid polymorphic sites identified within thePkAMA-1isolates

in this study, one of which was trimorphic. All of the amino acid variants detected in this study were

located in the ectodomain region of PkAMA-1 and this infers that this region of the PkAMA-1 antigen

could be one of the main targets of host immunity (Coley et al., 2006).This is because these

polymorphic sites may have been the consequence of selective pressures induced by the host’s

immunity. Out of the 12 point mutations detected in this study, 6 of them occurred only once among

the 36 P. knowlesi isolates in this study, 6 of which occurred in domain I of PkAMA-1. It was proposed

that novel point mutations are mostly expected to be highly deleterious, with only a small portion

being neutral (Kimura, 1983).

With reference to the amino acid variations, 14 haplotypes were identified (Table 3), most of

which were novel and have not been reported previously. Haplotype K7 has been reported by Faber

et al. (2015), where 100% identity was shared with a Pkama1 isolate (Accession no. KP067836) from

Betong, Sarawak. Haplotype K5 was the most prevalent haplotype (30.56 %) among the 36 P.

knowlesiisolates, where it comprised of isolates collected from all three sites of study in Kota

Kinabalu, Keningau and Kudat.Haplotypes K4 and K7 also consisted of isolates from more than one

region in Sabah, which further indicates that polymorphic patterns are not entirely restricted by

geographical barriers. The fact that haplotype K7 was also found in Betong, Sarawak further validates

this claim.In this study, eight PkAMA-1 haplotypes comprises of only a single isolate, each with their

own unique pattern of amino acid polymorphism. It was suggested that these haplotypes with only

single copies were either selectively advantageous or they may have occurred by recombination in

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other geographical areas with higher endemicity (Heidariet al., 2013).Unlike other Plasmodium spp.,

malaria caused by P. knowlesiis primarily a zoonosis with wild macaques as the reservoir hosts (Lee et

al., 2011). This could explain why the ama1 gene in P. knowlesiis less conserved as expected since the

parasite is not restricted to a single vertebrate host (Otto et al., 2014).This was similar to what was

observed in themerozoitesurface protein 1 (MSP-1) gene of P. knowlesi, which displayed

highvariability within the gene as well(Putaporntipet al., 2013). Nevertheless, there are certain genes

in P. knowlesithat do not display similar levels of genetic variability, such as the circumsporozoite

protein (csp) and MSP-3 genes, both of which lack in genetic diversity (Jeslynet al., 2011; De Silva et

al., 2017).

The dN/dS ratiosobtained (Table 4) gave an indication of negative or purifying selection

acting on thePkama1 gene. This is because the dN/dS ratio was less than 1 when the Pkama1 gene

was analyzed as a whole or separately as domains I, II and III. This observation was similar to what

was reported on domain I of Pkama1 isolates from Peninsular Malaysia (Fong et al., 2015). The

presence of negative selection on Pkama1 would imply that mutations detected in this gene are

generally deleterious and confer lower fitness to the parasite (Escalante et al., 2004). Purifying

selection has been reported in other antigens of P. knowlesi, such as MSP-9 (Chenetet al., 2013),

RAP-1 (Pacheco et al., 2010), MSP-8 and MSP-10 (Pacheco et al., 2012).The prevalence of purifying

selection infers that Pkama1 is in the process of screening for best-adapted variants, possibly in the

Borneo region (Loewe, 2008).The transmission of P. knowlesifrom its natural host, macaques, to

humans (Lee et al., 2011) could also explain why Pkama1 is undergoing adaptation. Lim et al. (2013)

have reported that P. knowlesihas shown increased proliferation in human hosts after undergoing

adaptation to increase its virulence through cellular niche expansion. Demographic processes such as

population increases could also be attributed to the polymorphic patterns observed among the

Pkama-1 isolates in this study. Evidence of population increases involving P. knowlesiin Southeast

Asia has been reported (Lee et al., 2011).In the event of population increase, the occurrence of

singleton sites increases, which subsequently lead to higher number of alleles with low frequency.

Although neutrality tests using Tajima’s D and Fu & Li’s D and F values also indicated

purifying selection on Pkama1, most of the values obtained were statistically insignificant (P > 0.05).

Fu & Li’s D and F values for domain II of Pkama1, on the other hand, were statistically significant (P <

0.05) with strong negative values that indicate purifying selection. The presence of purifying

selection on domain II of Pkama1 suggests that it may be under functional constraints. Furthermore,

nucleotide diversity wass also relatively low in Pkama1 as compared to Pfama1 and Pvama1, which

was similar to what was reported in Peninsular Malaysia (Fong et al., 2015) and Sarawak (Faber et al.,

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2015). The presence of purifying selection and low nucleotide diversity serve as good indications that

domain II ofPkama1can be further studied to serve as a target for vaccine development.

5. Conclusion

The nearly full length ama1 gene of 36 P. knowlesiisolates from Sabahwere successfully

amplified and sequenced in this study. Most of the polymorphisms were found in the region

encoding for the domain I of PkAMA-1. Preliminary findingsbased on dN/dS ratio implied that

purifying selection was prevalent in Pkama1 gene.The prevalence of selective pressures on specific

regions of Pkama1 gene was also elucidated in this study, where purifying selection was strongest in

domain II for Pkama1. This finding implies that most of the mutations observed in Pkama1 are

deleterious, conferring lower fitness to the parasite.The presence of purifying selection and low

nucleotide diversity indicated that domain II of Pkama1 can be further studied to serve as a target for

vaccine development.

6. Acknowledgement

We are grateful to all patients who donated their blood samples and the health workers at

the hospitals for theirassistance in sample collection for this study. Many thanks to all the colleagues

at Biotechnology Research Institute (BRI) that have provided assistance in the lab work involved for

the completion of the study. We would also like to thank our grant provider, Ministry of Education

under the grant of FRGS:FRG0322-SG/1-20/3, for funding this research.

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Received 16 October 2016, in revised form 16 January 2016; accepted 23 January 2017

Unedited version published online: 27 January 2017

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Appendix

Table 1: Polymorphic sites in the Pkama1 gene as compared to reference sequence 1 and 2 (Accession no. XM_002259303 and AF298218)

Nucleotide number

Domain I Domain II Domain III

2

4

6

6

7

2

1

1

4

1

7

4

1

9

9

2

7

7

3

2

1

3

5

7

4

9

2

5

6

4

5

9

4

6

3

0

6

4

8

6

8

4

7

1

7

7

1

9

7

3

2

7

3

5

7

3

7

7

4

4

8

3

0

8

4

6

8

5

8

8

7

3

8

8

2

8

8

6

1

0

4

7

1

0

5

0

1

0

7

5

1

2

9

6

1

3

5

0

1

3

9

3

1

4

4

1

1

4

4

3

1

6

2

9

S S S S S N N S S S SN S S S N S N S S N S N S S S S N S S N S S N N N S

Ref Seq 1 A A C A C G A C G G G A A C A A G A C A G G C T T A C T A A T C A C T T

Ref Seq 2 A A C A C G A C G G G A A C A A G A C A G G C T T A C T A A T C A C T C

KG004 . G T G . A . . . . . . G T . . . . T . . . T . . . . . . G . . . . . C

KG005 . . . G . . . . . . . . . . . . . . . . . . . . . . A . . G . . . . . C

KG006 G . T G . . . . . . . . . . . . . . . . . . . C . . A . . G . . . A . C

KG009 . G T . . . . . . . . . G T . . . . . . . . . . . . . . . G . . . A . C

KK015 . G . G . . . . . . T . . . . . . . . . A C . . . . A . . G . . T . G C

KK017 . G T . . . . . . . A . G . . . . G . . . . . . . . . . . G . . . A . C

KK018 . G T . . . . . A . A . . . . . . . . . A . . . . . . . . G . . . A . C

KK100 . G . G . . . . . . . . . . T G . . . . A . . . . . A . . G . . T . G C

KK101 . . . G . . . . . A A . . . T . . . . . . . . . . . A . . G . . . A . C

KT001 . . . G . . . . A . . . . T . . . . . . A . . . . . A . . G . . . A . C

KT002 . . . G . . . . . . . . G T T . . G . . . . . . . . . . . G . . . A . C

KT004 G . . G . . . . A . A . G . T . . . . . . . . . . . A . . G . . . . . C

KT007 . G T . . . . . A . . . G T . . . G . . . . . . . . A . . G . . . A . C

KT009 . . T G . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . C

KT010 . G . G . . . . . . . . . . T G . . . . A . . . . . A . . G . . T . G C

KT011 . . T G . . . . A . . . . . . . . . . . . . . . . . . . . G . . . A . C

KT012 . . T G . . . . A . . . . . T . . . . . . . . . . . . . . G . . . . . C

KT013 . G T . T . . . A . . . . . . . T . . G . . . . . . . . . G . . . A . C

13

Nucleotide number

Domain I Domain II Domain III

2

4

6

6

7

2

1

1

4

1

7

4

1

9

9

2

7

7

3

2

1

3

5

7

4

9

2

5

6

4

5

9

4

6

3

0

6

4

8

6

8

4

7

1

7

7

1

9

7

3

2

7

3

5

7

3

7

7

4

4

8

3

0

8

4

6

8

5

8

8

7

3

8

8

2

8

8

6

1

0

4

7

1

0

5

0

1

0

7

5

1

2

9

6

1

3

5

0

1

3

9

3

1

4

4

1

1

4

4

3

1

6

2

9

S S S S S N N S S S SN S S S N S N S S N S N S S S S N S S N S S N N N S

Ref Seq 1 A A C A C G A C G G G A A C A A G A C A G G C T T A C T A A T C A C T T

Ref Seq 2 A A C A C G A C G G G A A C A A G A C A G G C T T A C T A A T C A C T C

KT014 . . T G . . . . A . . . . . T . . . . . . . . . . . . . . G . . . . . C

KT019 . . . G . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A . C

KT020 . . . G . . . . . . A . . . T . . . . . A . . . . . . . . . . . . A . C

KT022 . . . G . . . . A . A . G . . . . G . . . . . . . . . . . G . . . A . C

KT023 . G T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . A . C

KT024 . . . G . . . . A . A . G . . . . G . . . . . . . . . . . G . . . A . C

KT026 . G T G . . . T . . A C . . T . . . . . . . . . . . . . T G . . . . . C

KT027 . G . G . . . T . . . . G . T . . . . . . . . . . . . . . G . . . A . C

KT040 . G T G . . . . . . . . . . . . . . . . . . . . . . . . T G . . . . . C

KT042 . G T . . . G . . . . . . . T . . . . . A . . . . G . . . G . . . A . C

KT043 . G T G . . . . . . A . . T T . . . . . . . . . . . . . . G . . . A . C

KT053 . . . G . . . . A . A . G . . . . G . . . . . . . . . . . G . T . A . C

KT057 . . . G . . . . . . . . . . T . . G . . . . . . . . . . T G . . . . . C

KT058 G . . G . . . . . . A . . . T . . . . . A . . . . . . . . G C . . . . C

KT065 . . . G . . . . . . A . . . . . . . . . . . . . . . . . . G . . . . . C

KT074 . . . G . . . . A . A . G . . . . G . . . . . . . . . . . G . T . A . C

KT077 . . . G . . . . . . . . . . T . . . . . A . . . . . . G . G . . . A . C

KT080 . . T G . . . . A . . . . T . . . . . . A . . . C . . . . G . . . A . C “S” indicates synonymous mutations; “N” indicates non-synonymous mutations ; “.” indicates identical nucleotide bases as compared to strain H reference sequence “S N” indicates that both synonymous and non-synonymous mutations; “KG” indicates blood samples collected from Keningau, Sabah; “KK” indicates blood samples collected from Kota Kinabalu; “KT”

14

Table 2: Amino acid polymorphisms in PkAMA-1 isolates from Sabah compared to reference sequence 1 and 2

Amino acid positions

Domain I Domain II Domain III

Haplotype

Frequency

(%)

A6

7T

K9

3E

K1

88

N

K2

28

N

S24

0I

K2

46

R

R2

77

T

R2

96

S

T35

9A

I46

5F

H4

81

N/Q

Reference sequence 1 (Accession no. XM_002259303)

A K K K S K R R T I H

Reference sequence 2 (Accession no. AF298218)

A K K K S K R R T I H

K1 2.78 T . . . . . . . A . .

K2 8.33 . . . . . . . . A . .

K3 2.78 . . . . . . . S A . .

K4 8.33 . . . . . . . S A . N

K5 30.56 . . . . . . . . A . N

K6 2.78 . . N . . . T S A F Q

K7 5.56 . . . N . . . S A F Q

K8 2.78 . . . N . . . S A . N

K9 11.11 . . . N . . . . A . N

K10 2.78 . . . N . . . S A . .

K11 13.89 . . . N . . . . A . .

K12 2.78 . . . . I R . . A . N

K13 2.78 . . . N . . . . . . N

K14 2.78 . E . N . . . . A . N

“.” indicates identical amino acid residues with reference sequence 1 and 2

15

Table 3:Haplotypes of P. knowlesiisolates from Sabah

Table 4: Sequence analysis and neutrality tests forPkama1 isolates from Sabah

Region Residues Nucleotide position

dN/dS Pi Hd Tajima’s D Fu&Li’s D Fu&Li’s F

Entire gene 1-554 1-1662 0.173 0.0042 0.994 -0.662* -1.637* -1.548*

Domain I 43-248 127-744 0.171 0.0059 0.960 -0.513* -1.642* -1.501*

Domain II 249-385 745-1155 0.185 0.0023 0.690 -1.704* -2.910 -2.968

Domain III 386-487 1156-1461 0.439 0.0032 0.621 -0.517* 0.218* -0.004*

“dN” indicates non-synonymous mutations; “dS” indicates synonymous mutations; “Hd” indicates haplotype

diversity; “Pi” indicates nucleotide diversity; “*” indicates values that are not statistically significant (P > 0.05)

Figure 1: Nest 2 ama1-specific PCR amplification for P. knowlesi with expected product size of

1792 bp in 1% agarose gel. Lane A: 1 kb DNA ladder (Promega Coorporation, USA); lane 1

represents the negative control. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 represent the successfully

amplified Pkama1 PCR amplicons for KK015, KK017, KK018, KG004, KT001, KT002, KT004,

KT007, KT009 and KT012 respectively.

Haplotype Isolate(s) of P. knowlesi

K1 KG004

K2 KT009, KT04o, KT065

K3 KG005

K4 KG006, KT001, KT007

K5 KG009, KK017, KK018, KT011, KT019, KT022, KT023, KT024, KT053, KT074, KT080

K6 KK015

K7 KK100, KT010

K8 KK101

K9 KT002, KT027, KT043, KT077

K10 KT004

K11 KT012, KT014, KT026, KT057, KT058

K12 KT013

K13 KT020

K14 KT042

16

Figure 2: Nest 2 ama1-specific PCR amplification for P. knowlesi with expected product size of

1792 bp in 1% agarose gel. Lane A: 1 kb DNA ladder (Promega Coorporation, USA); lane 1

represents the negative control. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 represent the

successfully amplified Pkama1 PCR amplicons for KG005, KG006, KG009, KT010, KT011,

KT013, KT014, KT019, KT020, KT022 and KT040 respectively.

17

Figure 3: Nest 2 ama1-specific PCR amplification for P. knowlesi with expected product size of 1792

bp in 1% agarose gel. Lane A and B: 1 kb DNA ladder (Promega Coorporation, USA); lane 1 represents the negative control. Lanes 2, 3, 4, 5, 6, 7, 8, 9 and 10 represent the successfully amplified Pkama1 PCR amplicons for KT042, KT043, KT053, KK100, KK101, KT023, KT024, KT026 and KT027 respectively.

18

Figure 4: Nest 2 ama1-specific PCR amplification for P. knowlesi with expected product size of 1792

bp in 1% agarose gel. Lane A: 1 kb DNA ladder (Promega Coorporation, USA); lane 1 represents the negative control. Lanes 2, 3, 4, 5, 6 and 7 represent the successfully amplified Pkama1 PCR amplicons for KT057, KT058, KT065, KT074, KT077 and KT080 respectively.

19