Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.

DNA is wrapped in a left-handed fashion around histone octasomes containing the centromeric histone H3 variant CENP-A. However, DNA topology studies have suggested that DNA is wrapped in a right-handed manner around the CENP-A nucleosome that occupies the yeast point centromere. Here, we determine the DNA linking number difference (ΔLk) stabilized by the yeast centromere and the contribution of the centromere determining elements (CDEI, CDEII, and CDEIII). We show that the intrinsic architecture of the yeast centromere stabilizes +0.6 units of ΔLk. This topology depends on the integrity of CDEII and CDEIII, but it is independent of cbf1 binding to CDEI and of the variable length of CDEII. These findings suggest that the interaction of the CBF3 complex with CDEIII and a distal CDEII segment configures a right-handed DNA loop that excludes CDEI. This loop is then occupied by a CENP-A histone complex, which does not have to be inherently right-handed.

BackgroundMalaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes.ResultsAnalysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates.ConclusionsAnalysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5257-x) contains supplementary material, which is available to authorized users.