Molecular Investigation of Genetic Signatures of Selection in Plasmodium falciparum Actin-Binding Protein Coronin, Cysteine Desulfurase, and Plasmepsin 2 Gene in Mbita Field Isolates, Western Kenya

Background: Plasmodium falciparum (Pf) resistance to antimalarial drugs is a major impediment to malaria control. The Pf.Kelch 13 (PfK13) gene has been largely reported to be associated with artemisinin resistance. However, recent studies have shown artemisinin resistance without Kech13 mutations suggesting the implication of others genes in artemisinin resistance. In this current study, we focused on mutations in Pf.actin-binding protein coronin, Pf.cysteine desulfurase and Pf.plasmepsin 2 gene, three putative candidates recently were reported to be involved in artemisinin, lumefantrine and piperaquine resistance respectively. Method: Archived blood samples previously collected from asymptomatic school children from December 2016 to October 2018 were used in this reaching 11.1%. Lumefantrine selection marker K65, in codon (K65Q) was observed at 14.2% in Pfcysteine desulfurase, and the mutant’ allele 65Q gradually increased frequency from 28.5% in 2016/17 to 57.1% in 2018. Pf.plasmepsin 2 was the less polymorphic gene. Several other polymorphism codons and single nucleotide variants were detected. Conclusion: The findings indicate the presence of mutations associated with reduced artemisinin susceptibility and lumefantrine selection marker. Therefore, the results call for continuous monitoring of molecular makers in Mbita parasites.

first-line drugs (chloroquine, and sulfadoxine-pyrimethamine) resistance started and subsequently spread to Africa [16]. Drug resistance remains a major impediment to malaria eradication. To this end, the recent prevalence of resistance to artemisinin and its partner drugs in Africa is highly alarming. Mutations (P553L, M476I, P574L, and R561H) have been detected in Africa, with an increased prevalence of R561H, found in 12.8% in Rwanda, associated with late parasite clearance [17]. Recently, in Uganda, evidence of P.falciparum resistance to artemisinin has been reported in patients with K13 mutations (C469Y and A675V), which were confirmed to be associated with late parasite clearance after evaluating parasite susceptibility by ring-stage survival assay and genotyping [17] Continuous monitoring of point mutations and temporal trends of mutant frequency in genes associated with P. falciparum resistance to artemisinin and its partner drugs is crucial, especially the identification of single nucleotide polymorphism (SNP) markers associated with treatment failure. Mutation in P.falciparum kelch13 gene located on chromosome 13 is associated with artemisinin resistance in Cambodia. kelch13 gene point mutations (C580Y, R561H, P553L, R539T, I543T, F446I, N458Y, Y493H and M476I) are providing insight into artemisinin resistance in South East Asia [18] [19] [20] among which five (C580Y, Y493H, R539T, M476I, I543T) have been validated to confer artemisinin resistance [21] [22] [23]. However, recently artemisinin resistance mutations were observed in parasites at other loci, notably a mutation in codon I356T of chloroquine resistance transporter gene (crt), codon V127M in apicoplast ribosomal protein S10 precursor gene (arps10), and codon D193Y in ferredoxin (fd) gene, suggesting that, in addition to the Kelch13 gene, other genes may be involved in artemisinin and its partner drugs resistance [24]. This was supported by a recent study in which parasites lacking kelch13 mutations exhibited an increased ring-stage survival phenotype [11] [25]. Plasmodium falciparum actin-binding protein coronin is a WD40-propeller domain protein family sharing the β-propeller motif with Kelch13 protein [26]. Interestingly, mutations (R100K, E107V and G50E) in the P.fcoronin gene (PF3D7_1251200) on chromosome 12, were reported to confer reduced artemisinin susceptibility after a CRISPR/Cas9-based mutagenesis study [26], making the Pf.coronin gene, a potential target for tracking artemisinin resistance. Although much attention has been focused on Kelch13 gene, it is of importance to monitor artemisinin-resistant signatures in other putative genes. On the other hand, it is important to also consider artemisinin partner drugs resistance, mainly lumefantrine and piperaquine since Artemether-lumefantrine (AL) and Dihydroartemisinin-piperaquine (DP) are most commonly ACTs used in Africa [27] [28]. Lumefantrine resistance has been associated with Plasmodium falciparum multidrug resistance 1 (Pfmdr1) gene and the increase of its copy number) [16] [29], and the combination (AL) shown selection for polymorphisms in the 1 (mdr1) (N86/184F/D1246-NFD haplotype and chloroquine resistance transporter (crt) (K76) [16] [30]. Furthermore, the cysteine desulfurase gene (Pfnfs) (PF3D7_0727200), a novel putative gene for resistance to lumefantrine, located on chromosome 7 has recently shown significant temporal Open Journal of Genetics trend polymorphism in Kenya [16]. Mutation in Pfnfs in codon K65Q has been reported to have a significant trend pre and post-ACT introduction with a decline in the K65 wild-type allele while the mutant allele (65Q) increased in the Kilifi population since the introduction of artemether-lumefantrine treatment [16]. This calls for further investigation. The same codon, (K65Q) in Pfnfs has also been found to have strong temporal differentiation 7years after the introduction of ACTs in Gambian isolates, with significantly higher IC50 (inhibitory concentration) in the wild type allele K65 [31]. Parasites with artemisinin resistance may develop resistance to piperaquine, compromising the efficacy of dihydroartemisinin-piperaquine as well as all other combinations [10]. Piperaquine resistance is now reported to be spreading in Cambodia [32], and DHA-PPQ treatment failure has been reported in Ethiopia and in Cameroon [33] Increased copy number of P. falciparum plasmepsin 2 (Pfpm2) gene (PF3D7_ 1408000), located on chromosome 14, and Pfmdr1 copy number have been associated with resistance to piperaquine and lumefantrine respectively [16] [34] [35] [36]. The reported signature of PPQ resistance also includes SNPs in Pfcrt [25] [34]. Perpiraquine resistance call for continuous surveillance. Although no evidence of ACT resistance is yet been reported in Kenya, is crucial to continuously investigate artemisinin and its partner drugs resistance markers in the Kenyan population as artemether-lumefantrine (Coartem TM ) has been adopted in 2004 [7] and dihydroartemisinin-piperaquine (Artekin R ) since 2009 [37] as first-line and second-line drugs in the treatment of uncomplicated malaria respectively. Investigation of genetic mutations associated with antimalarial resistance provides molecular markers for monitoring resistance. To this end, the purpose of this study was to investigate mutations associated with artemisinin, lumefantrine and piperaquine resistance in Plasmodium falciparum actinbinding protein coronin gene, cysteine desulfurase gene and plasmepsin2 gene respectively in Mbita Sub-county isolates, a region of high infection of malaria where the three putative genes have yet been studied. The use of such information is of paramount importance in the management of antimalarial drugs policy and the adoption of strategies to track the emergence and spread of resistance.

Ethical Approval
The study was performed under the granted approval for the original study (KEMRI/RES/7/3/1) provided by the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU). All experiments were conducted consistently with good laboratory practice regulations and relevant guidelines.

Study Site
The study was conducted in Mbita sub-county located on the shores of Lake Victoria in Homa Bay County, western Kenya [6]. The district is bordered by Open Journal of Genetics Lake Victoria to the north, west and south and situated between longitudes 34˚04' and 34˚24' East and latitudes 0˚21' and 0˚32' South [38]. It is located about 400 km west of Nairobi and has an area of 163.28 km [6]. Mbita subcounty has a population of approximately 115,896 [6]. The area experiences two rainy seasons annually from March to June and October to November [39], with an average annual rainfall estimated at 1300 mm and daily temperatures ranging between 26˚C to 34˚C [40]. Malaria transmission is intense among the local population with P. falciparum prevalence ≥ 40% [41] [42] [43]. The high infection rate of malaria among the residents makes the disease a burden for health care in the region.

Sample Collection
The study used archived samples previously collected over two years from December 2016 to October 2018 in the context of a study evaluating symbiotic microbes and mosquito vector ability using membrane feeding assay in the bid to characterize candidates that potentially block malaria transmission. The samples were collected from asymptomatic schools children (male and female) of various ages ranging from 5 to 15 years. Eligibility criteria for study participants in-

Genomic DNA Extraction, PCR, and Sanger Sequencing
DNA was extracted from 85 frozen blood samples using ISOLATE II Genomic DNA kit (Bioline, Meridian Bioscience, UK) as per the manufacturer's protocol.
Amplicons were obtained from the three target genes (Pfcoronin, Pfnsf and Pfpm2) using gene-specific primers targeting the regions of interest. All primers used were designed in this study (Table 1)

Data Analysis
Sequencing results were processed and analyzed in Geneious 10.2.3 software (Biomatters, New Zealand). The sequences were trimmed, edited, and BLAST (Basic Local Alignment Search Tool) alignment was performed in NCBI (National Center for Biotechnology Information), to check the per cent identity of the sequences vis à vis the corresponding reference genes. We used Plasmodium falciparum 3D7 strain as the reference genome. The sequences were subsequently assembled in Geneious, and aligned using ClustalW alignment. Alignment output was mapped to the corresponding reference genes for variants calling. The Geneious software automatically assigns a P-value to the variant, if the same variant is commonly observed (SNP that are observed in more than one isolate ( Table 2, Table 3). The rare single point mutations (Tables 2-4) were recovered without a P-value assigned based on, if the base quality was ≥20 (the Phred quality score), and if the point mutation does not contain mixed peaks. Open Journal of Genetics All variants that had mixed peaks were removed from the data. To reduce noise in SNP calling, we set up SNPs calling parameters such that only highly significant SNPs (P < 10 −6 ) were filtered out. Statistical analyses were carried out using

SNPs Markers Detected in Pfnfs Gene
A total of 24 polymorphism codons were recovered within the target region of interest ( Table 3). The polymorphism codon K65Q which is lumefantrine selection marker [16] was identified. The strongest signal reported was the mutant's allele 65Q (P = 2.5 × 10 −76 ) and the allele was recovered to increase frequencies over the time points from 28.5% in 2016/17 to 57.1% in 2018 while the wild type K65 which is responsible for lumefantrine selection [16], was detected at 14. A transversion was found to occur in codons S120I and E130D with frequencies that ranged from 7.8% to 15.3% for 130D (P = 3 × 10 −9 ) from 2016/17 to 2018 respectively. The allele 120I (P = 1 × 10 −10 ) had stable frequency (23%) over time. Genetic drift is a non-directional change in allele frequency that occurs by chance between generations by decreasing or increasing a certain allele's frequency, but it does not result from the accommodation of individuals to the environment since the affected allele may be beneficial or harmful allele [44].  Table 3). The frequency of wild type and mutants' alleles in the Pfcysteine desulfurase gene is shown in (Figure S2).

Discussion
Pf.coronin is one of the members of the WD40-propeller residues protein family Adoption of resistance fitness mostly results from the resistant mutations due to drug pressure, for instance, mutation C580Y has been reported to be associated with artemisinin resistance in Cambodia [21]. In Kenya, malaria parasites are  some cases, however, the frequency of drug selection alleles increases under drug pressure over time and decreases after withdrawal or discontinuation of the drug responsible for the pressure [48], therefore, the temporal increase in the frequency of artemisinin selection allele 100k may be attributed to the continuous use of artemether-lumefantrine and dihydroartemisinin-piperaquine regimen.
We also noticed an increase in the frequencies of mutants 96N, 95K, 59K, 57K, and 47F suggesting that these alleles may be under ACT drug pressure. Importantly, care must be taken in the surveillance of alleles 100k, and 50E, two of the alleles recovered by Damas et al. [26] to confer reduced artemisinin susceptibility. No variants were recovered in codon I68K, Q61P, and G50E, in 2017 time point, while mutants were observed in 2018, indicating that, pressure in these alleles has started in 2018. For further analysis, we used InterPro database, SMART tool, and NCBI CD-Search, and we found in a conserved domain (WD40 repeats), five successive predicted WD40-repeats motifs covering a region of 199aa, in which the following mutations (E95K, D96N, R100K, S149S, K71K, S75S, S81S, K125K, K159K, E158E, A161P, K168K, G179G, and S183G) were found to occur in WD40 motifs ( Figure S4). Interestingly, these findings correlate with the results of the selection mutations reported by Dams, et al., [26] which were also discovered in WD40 motifs. G50E, N60N, E59K, R57K, Q61P, E156K, and I68K were localized outside WD repeats motifs, but took part of the WD40 domain. Only V47F was found outside the domain. Interestingly, WD40 repeats (repetitive tandem residues, containing tryptophan (W)-aspartic acid (D) at the C-terminus while having glycine-(G) and histidine-(H) at the N terminus), are conserved domains that contain a β-propeller structure providing a scaffold for protein-protein and protein-DNA interaction and are known to involve in multiples functions viz, signal transduction, regulation of transcription Open Journal of Genetics [49] [50], and also involved in drugs and chemical inhibitors pathways [51].
Mutations in these motifs are of crucial importance as they may impair drugs effect pathways . Variants, 125K, 75S, 71K, 168K, 81S, 60N, 149S, 179G, 150E, and 159K, are synonymous mutants. Although synonymous mutations are thought to be less harmful, they can affect the stability of the stem-loops, helices [52], therefore, proteomic analyses will be helpful to elucidate their harmless. Lumefantrine selection marker, K65 was previously reported in Kilifi isolates together with its neighbour's mutants 62N, and 67G [16]. Mutants, 65Q, 62N, and 67G in addition to 120I and 130D were identified to increase frequencies in Kilifi (65Q (80%), 62N (20%), 67G (80% -80%), 120I (32%), 130D (22%), and 188K (5.9%)) in 2017/18 period [16]. Consistently with our findings, an increase in the frequency of these alleles is observed in the present study although slightly lower in mutants 65Q (57.1%), 67G (57.1% -50%), 120I (23%), 130D (15.3%), compared to the findings in Kilifi field. The author also reported a decline in the frequency of wild type K65 (from 38% to 20%) in Kilifi isolates [16], this supports our results, as we found K65 at low frequency (14.2%) in Mbita isolates. Conversely, mutant 62N (50%), and 188K (45.4%) frequencies are higher than found in Coastal Kenya. This irregularity may be due to the differences in drug pressure in both settings. The increase in frequencies observed in mutant's alleles may be a result of Arthemetr-lumefantrine drug pressure as observed in Pfcoronin mutants. Conserved domain search using NCBI-CDD and InterPro revealed a conserved sequence (Aminotran_5 domain) in Pfnfs protein, composed of 367aa ( Figure S5), localized within ATT-I region, a region of 387aa. Excluding the mutations (R146K, S62N, K65Q, E67G, S120I, E130D), all the rest of the variants codons were found in the Aminotran_5 domain. 120I was found in a predicted disordered region made up of 23aa, the function of which includes protein modification, molecular assembly, molecular recognition, and serve as entropic chains [53].
By using SMART database, the predicted disorder region was found to span a low complexity motif of 20aa, and another low complexity motif was predicted downstream (14aa), in which occurred mutation N203K. These motifs play adhesive and structural roles, they transduce molecular movement, interact with phospholipid bilayers, modulate protein translation and act as frame-shift checkpoints [54]. From 2016/17 to 2018, the mutant 150N, 155P, 160K, 163N, 195Q, and 164K increased frequencies (9.1%), while 194A gained (18.2%). These alleles in addition to 238k, and 146K which decreased in frequencies (20%) and (9.1%) and also (179K, 271N, 148F, 203K, 306N) were not previously reported. Therefore we could not confirm their selection, though, the evolution their frequencies need to be monitored. 279H, 305L, were not observed in the 2018 time point indicating the restoration of the wild-types. All these mutants were localized in protein conserved features except the ones aforementioned. Together, knowing the functions of Pf.cysteine desulfurase IscS (Iron-sulfur cluster System), mutants reported in this study may have an impact on parasites and this may result in acquiring selective fitness. Cysteine desulfurase IscS is a member of H. Diarra et al. Open Journal of Genetics aspartate aminotransferase superfamily fold type I (AAT_I) and belongs to the class-V pyridoxal-phosphate-dependent aminotransferase family. The protein IscS is involved in the biosynthesis and delivery of iron-sulfur clusters to diverse metabolic pathways, such as Fe-S complex synthesis [31] [55]. IscS pathway is crucial for erythrocytic stage parasite development and is an inherent drug target [56]. Iron homeostasis is capital for erythrocytic stage parasite growth and is involved in quinolones drug mechanisms [31], lumefantrine drug family. In apicomplexans, proteins containing Fe-S cluster are modulated during parasites' resistance, stress conditions and cell development [31] [56] [57]. They occupied important roles in gene expression and are also antimalarial drugs targets [58].
The remarkable observation we made in Pfpm2 point mutations is that all the variants discovered were transversion mutations, among which five mutants (188T, 260L, 261H, 309L, and 322E) were localized in an active site (aspartic-peptidase active site), predicted within peptidase A1 domain, a region makes up of 308aa, (Figure S6), found in the amino acid sequence of Pfpm2 protein.
Only mutation H79N was localized outside the peptidase A1 domain. Aspartic-peptidase active site is involved in catalysis, and binding of substrates for cleavage [59] [60]. Transversions mutations largely impact gene expression and impair the amino acid sequence of proteins [61]. Pfpm 2 is a digestive vacuole enzyme of aspartic proteases family A1 and is a potential antimalarial drug target [62] [63] [64], expressed in the erythrocytic parasite stage as integral membrane protein. The enzyme is implicated in the digestion and host haemoglobin degradation, processing of cytoskeletal protein, host cell remodelling, and effector export [64] [65] and the amplification of its copy number has been to be associated with piperaquine resistance. Mutations found in the active site may alter the binding affinity of substrates, protein structure, functions, therefore may impart to parasites reduced sensitivity fitness to the drugs. Taking together, mutations found in the three genes are localized in critical regions of proteins in which they were identified, and the trend analysis was found to be significant in both Pfcoronin and Pfcysteine markers' data with a positive correlation between time points. This gives importance to our findings, and the slight increase in mutant frequencies over time may be the result of ACT drugs pressure used in the study area. We detected several SNPs at increased frequencies that were not previously reported, this call for further study to lighten their role as selection markers or resistant point mutations.

Conclusion
The present study has identified two point mutations in Pfcoronin previously reported to be associated with reduced artemisinin susceptibility and also a marker of selection for lumefantrine in Pfcysteine desulfurase. The results call attention to continuous surveillance of P.falciparum molecular markers associated with drug resistance in the study setting known for high transmission of malaria. To our knowledge, artemether-lumefantrine, as well as dihydroartemisinin-piperaquine, Open Journal of Genetics are still efficacious in western Kenya, and no clinical failures have yet been reported. Despite this, parasite's genetic monitoring will be important in informing about prospective resistance. We have also identified several other SNPs markers that were first reported in this study, but more remains to be known about their impacts. Close study of these mutants and the corresponding genes through gene expression analysis, linkage disequilibrium and the mechanism by which the three genes might confer resistance will be useful in tracking resis-