Genetic Variation and Invasion Phenotypes of Plasmodium Falciparum in the Gambia

Abstract

Plasmodium falciparum is the deadliest of all Plasmodium species causing malaria in humans. The interactions between P. falciparum merozoite ligands and erythrocyte receptors during erythrocyte invasion is a vital stage in the parasite’s life cycle, linked with the initiation of the clinical signs/symptoms of the disease. The availability of multiple alternative invasion pathways gives the parasite the luxury of switching between invasion pathways as an immune evasion mechanism and a means of escaping from control interventions probably due to polymorphisms in both ligand and receptor genes or changes in gene expression profiles. Additionally, the malaria disease has been suggested to have imposed evolutionary selection pressure on the human genome resulting in evolutionary changes in genes encoding both human receptors and parasite ligands. However, research on variations in parasite ligands and their corresponding receptors has received far less attention. Thus, the main aim of this study was to explore host-parasite genetic and transcriptomic correlates of P. falciparum erythrocyte invasion phenotypes, and malaria outcomes in The Gambia. Specifically, the genetic variations and transcriptional changes of P. falciparum invasion ligands in isolates with different invasion phenotypes were determined, sequence variations in parasite ligand genes and their corresponding erythrocyte receptors in the same population were also studied and lastly, the distribution of two key human glycophorin B receptor structural variants (denoted as Deletion 1 and 2) among malaria-infected individuals in The Gambia was described. Following informed consent documentation, 2 ml of whole blood samples were collected from all age groups (one to seventy years) with confirmed malaria (severe and mild) in four health facilities across three regions in The Gambia, notably Basse (Upper River Region, mesoendemic), Brikama and Fajikunda (Western region, hypoendemic), and Edward Frances Small Teaching Hospital (Greater Banjul region, hypoendemic). Leukocyte-depleted infected red blood cells (iRBCs) were used for erythrocyte invasion experiments. The iRBCs were also cultured to schizonts for RNA extraction and expression profiling of six invasion ligand genes: erythrocyte binding antigen (EBA) 175, EBA-181, reticulocyte homologue (Rh) 2b, Rh4, Rh5, and cytoadherence antigen 2 (Clag2), which are important players in the invasion machinery. To determine sequence variations in ligand and receptor genes, malaria-positive samples were used for targeted DNA sequencing of a broad range of 12 P. falciparum genes: EBA-175, EBA 181, EBA140, Clag2, Clag8, Rh4, Rh5, merozoite surface protein (MSP)1, MSP6, Duffy binding-like MSP (DBLMSP), erythrocyte binding-ligand 1 (EBL1), and surface-associated interspersed protein 4.2 (SURFIN4.2), and four human red cell receptors: glycophorin (GP) A, GPB, GPC, and complement receptor 1 (CR1), using the Oxford Nanopore GridIon platform. GPB deletion 1 and 2 were also genotyped from malaria-positive individuals using a multiplex PCR and restriction fragment length polymorphism-PCR approaches. Any sample with any or both of the deletions were further genotyped for sickle cell. R statistic packages were used for most analysis and the Shapiro-Wilk normality test was performed to determine if the distribution of the data was Gaussian, and variables that passed the test were subjected to parametric analysis; otherwise, non-parametric techniques were employed. Linkage disequilibrium was determined using the PLINK package and the Tajima’s D test was used to estimate departures from neutral evolution in the selected parsite and host genes. The Hardy Weinberg equation was used to determine the allele frequencies of GPB deletions while association analysis between the GPB deletions and severe malaria was undertaken using the Fisher’s exact test. A total of 90 clinical isolates were successfully cultured and phenotyped for the first part of the study. It was observed that P. falciparum clinical isolates in The Gambia predominantly use sialic-acid-independent pathways to invade the erythrocyte and the use of these pathways have increased from the year 2015 to 2022. Specifically, the data shows the increased expression of reticulocyte-binding homologue (Rh) protein family of parasite genes involved in sialic-acid independent pathways such as Rh5. Indeed, significantly positive correlations were observed between the expression of Rh2b, Rh4, and Rh5 known to be involved in the sialic-acid independent pathways. The P. falciparum isolates genotyped by nanopore sequencing showed moderate to high levels of within-host complexity of infection across sites. Interestingly, of the 12 genes sequenced, high inter-SNP linkage disequilibrium (LD) was observed only in the parasite DBLMSP and SURFIN 4.2 genes. LD was also strong for single nucleotide polymorphisms (SNPs) in the human GPB, GPC, and CR1 genes but not in GPA. Variants from these human loci (especially GPC) clustered individuals into two distinct sub-populations mostly shaped by two significant SNPs not previously described in The Gambia population. Moreover, Tajima’s D analysis identified the parasite DBLMSP and SURFIN 4.2, and human host CR1, GPC, and GPB genes to be under balancing selection. A case (severe malaria) – control (mild malaria) univariable logistic regression models analysis identified a SNP in SURFIN 4.2 and some SNPs in the CR1 gene to be significantly associated with malaria susceptibility. Additionaly, GPB deletions PCR genotyping showed high allele frequency of GPB deletion 1 (9.9 %) among malaria-positive individuals compared to deletion 2 (1.7 %). The Fulani and the Serehule ethnic groups had the highest frequency of GPB deletion 1 (14.8 %) and deletion 2 (2.5 %) respectively. Both deletions were more prevalent in Basse, where the Fulani ethnic group is dominant. However, both deletions were not associated with severe malaria propably due to small sample size of severe cases. Sickle cell genotyping among GPB deletion positive individulas suggested that the sickle cell and the GPB deletions may be co-evolving in population. Overall, this study has provided valuable insights into the parasite biology and the complex parasite’s life cycle, while also identifying SNPs in both the parasite and host that could serve as potential targets for drugs and vaccines development.