Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analyzed in the first culture cycle of 96 clinical isolates from 4 populations with various levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator . Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing did not express , consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission.