The k13 gene of P. ovale is in the 404824-407001 rt region of chromosome 12, with a coding region in full length of 2178 bp. Its encoded kelch protein has a skeletal region near the N-terminus, and a propeller domain near the C-terminus consisting of about 290 amino acids from 440th aa-725th aa [26]. Studies have shown that amino acid substitutions in the propeller domain of the kelch protein in P. falciparum are genetically related to artemisinin resistance [25, 27]. Moreover, there are very few bases with more than two substitution loci in the entire coding region, which demonstrates [25, 28, 29] high conservation. Therefore, k13 gene can be used as a stable molecular marker to predict the artemisinin resistance in P. falciparum [30-32].
In this study, the polymorphism of the entire propeller domain and a fraction of the upstream skeletal domain in k13 gene of the P. ovale isolates imported into Yunnan Province from Myanmar and some African countries were analysed. Of the 15 CDS sequences analysed, base substitutions were found at 38 loci, such as c.711 ~ c.2118 (Table 2), showed the inter-type dimorphism of curtisi subtype and wallikeri subtype, as well as the complete intra-type monomorphism (Fig.2). The finding of such stable monomorphism and dimorphism characteristics at each locus is consistent with the results of polymorphism analysis conducted by Sutherland et al. [12], Fuehrer et al. [33], Chavatte et al. [7] on reticulocyte-binding protein 2 gene (rbp2), and glyceraldehyde-3-phosphatase (g3p) gene. All the above-mentioned research found the dimorphism of different genes in P. ovale, such as at 22 loci in rbp2 gene with approximately a 793 bp fragment and at 19 loci in g3p gene with a 662 bp fragment between curtisi subtype and wallikeri subtype sequences. Moreover, the loci showed highly monomorphic within curtisi subtype and wallikeri subtype sequences. While the P. ovale tryptophan-rich antigen gene (Potra) in wallikeri subtype had a 54-bp deletion compared to it in the curtisi subtype [12]. Fuehrer et al. [33] also found there were different dominant short peptide chain repeat in circumsporozoite surface protein gene (csp) between curtisi subtype and wallikeri subtype. For the csp gene of wallikeri subtype, the "DPPAPVPQG" short peptide chain was more frequent, while for curtisi subtype was the "NPPAPQGEG" short peptide chain. It seems that the polymorphism of csp gene could be used to establish the genotyping method for distinguishing two subtypes, but Fueher et al. [33] believed it was only applicable to determination of P. ovale evolutionary relationships. In the current research, the authors discovered the dimorphism at 38 base loci in k13 gene propeller domain existed between two subtypes and the CDSs of curtisi subtype (n=11) and wallikeri subtype (n=4) could be separated in the Neighbour-Joining evolutionary tree. However, on account of the failure to find the predisposing structural features like csp gene in CDSs or peptide chain of k13 gene propeller domain. Therefore, it is difficult to establish a suitable discriminating method between curtisi subtype and wallikeri subtype based on the polymorphism of k13 gene, propeller domain, and further analysis on the backbone domain of k13 gene might be helpful to find more evidence.
Consequently, these findings about k13 gene in this article only emphasize that k13 gene polymorphism in P. ovale is like the differentiation of other members in the genome, resulting in the distinction between curtisi subtype and wallikeri subtype. However, it is noted that the degree of k13 gene differentiation is weaker than circumsporozoite protein / thromspondin-related anonymous protein (ctrp), circumsporozoite protein (csp) and merozoite surface protein 1 gene (msp1), which were reported by Saralamba et al. [34]. The Pi value of these three genes was predicted to be between 0.12 and 0.11, which is greater than 0.0095 in this study.
Evidence indicated that P. ovale originated from Southeast Asian countries is mostly curtisi subtype, while Africa showed a sympatric distribution of P. o. curtisi and P. o. wallikeri [7, 12, 35, 36, 37], and the mutation type is mainly restrained in Western Africa [20]. In this study, the distribution pattern of similar P. ovale subspecies was almost restored. The sequences of k13 propeller domain in five Myanmar isolates were all identified as curtisi subtype, while the ten African isolates included six curtisi subtype, three wallikeri subtype and one mutation type (Table 1). This result serves as a constant reminder that the population structure of P. ovale isolates imported into Yunnan province maybe are more complicated than those of the original population [34, 38]. Therefore, greater discretion and accuracy are needed in the diagnosis and antimalarial treatment of these P. ovale infections. The current study is the first to ascertain that the infected isolates in malaria cases officially reported in Yunnan Province include the two sub-species of P. ovale curtisi and wallikeri and further providing a favourable basis for the control of ovale malaria epidemic in Myanmar [39]. In addition, although amino acid substitution variation in the skeleton region of kelch protein was detected in only one sample, the same amino acid variation has also detected and demonstrated by Jin’s study on the samples from Hangzhou city, China (personal communication), which increase credence of the result. However, double DNA sequencing process could further help rule out sequencing errors.
Although this study was not dedicated to exploring the genetic correlation between k13 gene mutations and artemisinin resistance in P. ovale, the spatial structure prediction on the peptide chain near the C-terminus from 224th aa to 725th aa in k13 gene found that curtisi subtype peptide chains and wallikeri subtype peptide chains share almost analogous monomeric crystal structures (Fig 3A, B). Moreover, with one amino acid variation in the skeleton region, yet the homology model has dramatically changed into a dimeric structure (Fig. 3C). The finding is completely different from that of Choowongkomon et al. [37] in terms of the spatial structure prediction of dihydrofolate reductase (dhfr) gene in P. ovale. Their results showed the identities of dhfr peptide chain in P. ovale were merely 67.4%, 64.7% and 75.4% in comparison with P. vivax, P. falciparum, and P. malariae, respectively. However, the crystal structures of the four dhfr peptide chains are similar regarding subunit composition and the tendency of overall folding. All display monomeric and α-helix structure, which are folded on the surface of the homology model [36]. This pattern might be related to the different proportions and intensities of α-helix and β-helix structures in the two peptide chains of k13 gene and dhfr gene. In the current study, β-helix structures accounted for 75.1% (377 aa / 505 aa) in the k13 peptide chain, and were mainly located in the C-terminus of the peptide chain to fold into a "propeller" shape. In addition, Bayih et al. [40] had proposed the substitution from basic-to-aliphatic residue at the kelch 13 propeller domain, especially β-helix structures region, may impact the protein function. However, further studies should be carried out to investigate whether the predicted structural change in skeletal region of the kelch protein in P. ovale, just like the mutation of the propeller domain, is related to the artemisinin-resistant phenotype [25, 27].
In this study, the understanding that there are numerous dimorphisms in the genome of P. o. curtisi and P. o. wallikeri was broadened. By using the multi-loci dimorphism of the k13 gene, it might be possible to establish a stable and accurate genotyping method for distinguishing different subtypes of P. ovale. Nevertheless, this study is not without limitations. Firstly, given the difficulty to accurately calculate the parasitaemia of P. ovale in some blood slides, it is impracticable to explore the correlation between the density of the parasites and the copy number of k13 gene. Secondly, the polymorphic analysis of the full sequence of the k13 gene has not yet been performed, and the incomplete identification of the dimeric loci in the skeleton region of kelch protein and the DNA sequence of P. o. curtisi and P. o. wallikeri might not be conducive to assess of the degree of k13 gene differentiation accurately. Thirdly, this protocol is based on nested PCR and DNA sequencing, which is labour- and cost-intensive due to the second PCR reaction, and also increase risk of contamination due to PCR product transfer from the initial reaction to the second, while one-step real time PCR assay to discriminate P. ovale subspecies using specific primers and hydrolysis probes targeting rbp2 gene has been reported and applied in West Kenya [41]. Lastly, given 16.7% (3/18) of the samples fail to be detected by this protocol, low parasitaemia of these samples (not counted due to poor quality of slides) might be one cause, or potentially due to multiple reference sequences were not included as template during the primer design stage, which could limit the sensitivity of the experiment. Local wild type sequences could be used as reference to design the primers to increase the sensitivity of this protocol.