The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection

Abstract

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

Keywords: circadian rhythm; gene expression; host–parasite interaction; intraerythrocytic development cycle; plasmodium vivax.

Fig. 1.

Experimental protocol and data collection. (A) Diagram of the clinical study sampling design. Each participant underwent a single blood draw that is used for screening to ensure uncomplicated monoinfection by P. vivax, and a second draw used for RNA sequencing. If the microscopy screening showed asexual parasitemia ≥0.1% and hematocrit ≥25%, and parasites were observed to be well synchronized in the early or late trophozoite phase, the participant was enrolled, and a second blood draw was taken and divided into 16 parts for subsequent RNA-Seq analysis. (B) P. vivax developmental stage percentages at the screening time for each participant. (C) Times of day at which each study participant was screened and enrolled and the times of day of the first timepoints in the RNA-Seq time series experiments. (D) For each participant, the times relative to the start time at which each of the 16 samples were aliquoted and frozen for later RNA sequencing. (E) Representative z-score heatmaps of genes which exhibit periodic expression patterns at a specified period (24 h for human genes and 48 h for P. vivax genes) in participants 02 and 08. For each participant and each organism, periodic genes are taken to be those whose maximum expression across the time series is at least 1 QN TPM and which exhibit at least the same degree of periodicity as the top 5% of genes when ranked by the JTK periodicity score. Genes are consistently ordered in each pair by participant 02’s peak expression time during the first period.

Fig. 2.

Comparisons of RNA-Seq time series data to earlier studies. (A) The average pairwise Spearman correlation between one of the P. vivax (Sal-1) isolates from the ex vivo time series RNA-Seq experiment reported in ref. , the other two samples reported in that study, and each of the 10 participants in this study. Correlations are the average over all timepoints shifted by the specified amount, with wrapping assuming a 48-h period. (B) z-score heatmaps of highly periodic (JTK P value ≤ 0.005) P. vivax (P01) genes in participant 08 that also appear in sample D1 from ref. . All heatmaps contain 1,138 genes and are ordered by the time of peak expression in participant 08. Analogous plots can be generated for the other participants using data and code repositories associated with this manuscript (SI Appendix, Table S3). (C) Box-and-whisker plots comparing the average percentage of expressed genes common to the three conditions and achieving the specified JTK P value thresholds. (D) Box-and-whisker plots comparing the average percent overlap among the rhythmic genes achieving the specified JTK P value thresholds. Distributions in C represent either the 14 participants in the in vivo study (21), under baseline or sleep deprivation conditions, or the 10 participants in this study. Distributions in D represent all participant pairs within each study. In C and D, boxes indicate distribution quartiles, and whiskers extend from minimum sample values to samples at most 1.5 times the interquartile ranges, with values outside this range plotted as outliers.

Fig. 3.

Human phase, P. vivax phase, and wall time circular correlations. (A) Cartoon illustrating the correspondences between elapsed time and the phases of a 48-h cycle and a 24-h cycle, indicating that the difference between the circadian cycle phase and twice the intraerythrocytic developmental cycle phase is expected to be constant over time. (B) The times of day on 24-h clocks of the first timepoints in the RNA-Seq time series (Top) and the histograms of gene phase differences estimated from the overlapping periodic human (Middle) and P. vivax (Bottom) genes for participants 02 and 08. The radii of the histogram slices have been scaled so the area of each slice is proportional to the number of data points it represents. (C) Scatter plots of inferred human and P. vivax phases. For P. vivax, all phase difference estimates were first multiplied by two and reduced modulo $2\pi$ before visualization. All phases are shifted to the interval $[-\pi ,\pi )$ radians, and the 0 phases were chosen to be the circular-mean human phase. The left and right edges and the top and bottom edges should be identified since the phase/angle $-\pi$ is equal to $\pi$ on a circle.