Authors:Jean Chepngetich, Brenda Muriithi, Beatrice Gachie, Kevin Thiong'o, Mercy Jepkorir, Jeremiah Gathirwa, Francis Kimani, Peter Mwitari, Daniel Kiboi Abstract: Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we finally measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and the impact on drug responses require further investigations in malaria parasites. PubDate: 2023-03-07T08:49:40Z DOI: 10.12688/openresafrica.13436.2 Issue No:Vol. 5 (2023)
Authors:Beatrice Gachie, Jean Chepngetich, Brenda Muriithi, Kelvin Thiong’o, Jeremiah Gathirwa, Francis Kimani, Peter Mwitari, Gabriel Magoma, Daniel Kiboi Abstract: Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. The recent report on the emergence of artemisinin-resistant parasites portends an imminent failure of the partner drug in clearing the high residual parasite densities. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. We first verified the stability of the LM, PQ, and AQ-resistant parasites in the standard 4-Day Suppressive Test. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) in the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals differential expression in both LM and PQ-resistant parasites. Conversely, nfs1 expression remained unchanged in the AQ-resistant parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and differential expression of the nfs1 gene in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum. PubDate: 2023-03-07T08:45:26Z DOI: 10.12688/openresafrica.13457.2 Issue No:Vol. 5 (2023)
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