Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia

Abstract

Background: The malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles.

Methods: Blood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates.

Results: Though the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years.

Conclusions: Overall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.

Keywords: Erythrocyte; Expression; Inhibition; Invasion; Ligand genes; Malaria; P. falciparum.

Fig. 1

Prevalence and allelic frequencies of msp2. A Frequencies of 3D7 allelic family, B frequencies of FC27 allelic family, C prevalence of msp2 repeat polymorphism allelic families of Plasmodium falciparum in clinical isolates collected in 2015, 2016, and 2021 from Western Gambia. bp base pairs

Fig. 2

Relative gene expression and correlation between gene expression levels. A Hierarchical clustering heatmap of relative expression levels of six Plasmodium falciparum ligand genes (rows) between clinical isolates (columns). White to light red colour signifies lower expression while brown colour indicates higher expression. B Correlation between gene expression levels; numbers out of the brackets in square boxes represent the correlation coefficients while those in brackets represent the P values. Significant P values are indicated in red

Fig. 3

Invasion phenotypes of P. falciparum isolates across 3 years in The Gambia. NM neuraminidase, LT low trypsin, HT high trypsin, CHY_LT chymotrypsin and low trypsin, and CHY chymotrypsin. Each point represents an isolate while the vertical line of each box represents the median. P < 0.05 was considered statistically significant

Fig. 4

Comparison of erythrocyte invasion inhibition phenotypes of Plasmodium falciparum isolates across 3 years in The Gambia. A Neuraminidase, B low trypsin, C chymotrypsin, and D chymotrypsin and low trypsin. Each box shows the invasion efficiencies in enzyme-treated erythrocytes relative to untreated erythrocytes, and the horizontal black lines in each box represent the median. P values < 0.05 were considered statistically significant

Fig. 5

Correlation among enzyme inhibition, age, and parasitaemia. Numbers out of the brackets represent the correlation coefficients while those in brackets indicate the P-values with significant P-values highlighted in red. PCT parasitaemia