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Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 hours (depending on the species), suggesting a circadian basis to the asexual cell cycle, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches using rodent and human malaria parasites, we reveal that: (i)...
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... in lights on/off). In one group, the light regime was maintained as for the donor mouse (i.e. host rhythm matched, lights on: 7.30 (ZT0/24 (Zeitgeber Time: hours after lights on)) and lights off: 19.30(ZT12)). In the other group, the light-dark cycle was reversed (host rhythm mismatched, lights on: 19.30 (ZT0/24), lights off: 7.30 (ZT12)) (Fig. 1a). The effect of mismatch to host rhythms on the parasite was assessed by analyzing the parasite transcriptome every 3 h for 30 h (n=4 mice/group/time ...Context 2
... mismatch to host circadian rhythms, the IDC of P. chabaudi becomes rescheduled within approximately seven cycles to match the host's rhythms. By the time of sampling (days 4-5 post infection, PI), the parasites were six hours mismatched to the host's rhythm ( Supplementary Fig. 1a). Specifically, in mismatched parasites, merozoite egress from RBCs following schizogony peaked six hours after matched parasites (ZT . ...Context 3
... bioRxiv preprint first posted online Oct. 2, 2019; 0/24 and ZT 18 respectively; inferred from ring stage rhythms). Parasites in both the matched and mismatched infections remained synchronous throughout the sampling period ( Supplementary Fig. 1a). ...Context 4
... to as "circadian") if both algorithms independently detected a ~24 h periodicity in their transcription with a threshold of p < 0.05. Of a total of 5,343 genes in P. chabaudi (5,158 detected and considered for analysis), 3,057 (58%) in matched parasites, and 1,824 (34%) in mismatched parasites, exhibited circadian rhythms in transcription (Fig. 1b, Supplementary Data 1). A permutation test was performed to empirically determine the false discovery rate (FDR) in detecting circadian transcripts. The original time points were shuffled randomly for 1,000 iterations and circadian transcripts were identified each time by both the algorithms. The permutation test identified that the number of circadian ...Context 5
... original time points were shuffled randomly for 1,000 iterations and circadian transcripts were identified each time by both the algorithms. The permutation test identified that the number of circadian transcripts in both matched and mismatched parasites detected in the original sampling order by both the programs was significantly higher (FDR < 0.05, Supplementary Fig. 1b) than when the sampling order was randomly permuted indicating that the circadian genes identified were transcribed in a rhythmic manner above background noise. ...Context 6
... < 0.05) in matched parasites that exhibit an arrhythmic transcription profile in mismatched parasites (p > 0.05); 2) 1,292 genes with transcription profiles with 24 h rhythm in both matched and mismatched parasites; and 3) 532 genes whose transcription profiles were arrhythmic in matched parasites and rhythmic in mismatched parasites (p < 0.05, Fig. 1b and 1c, Supplementary Fig. 1c). Hierarchical . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in ...Context 7
... doi: bioRxiv preprint first posted online Oct. 2, 2019; clustering analysis identified biological replicates to be tightly clustered ( Supplementary Fig. 1d). Comparison of the 11 time points using principal component analysis identified the first component in both the conditions with a cyclic pattern that accounted for > 85 % of total variance (Supplementary Fig. 1e), which supports the hypothesis that the large number of transcripts detected as circadian are truly transcribed at a 24 h periodicity. ...Context 8
... of 1,292 genes that were rhythmically transcribed in both matched and mismatched parasites, we found 685 genes (53%) that had a delayed phase of transcription with a delay of about 6 h (± 1.5 h) in mismatched compared to matched parasites ( Supplementary Fig. 1f) which complements our phenotypic observation of the phase difference between mismatched and matched parasite IDCs (Supplementary Fig. 1a). ...Context 9
... of 1,292 genes that were rhythmically transcribed in both matched and mismatched parasites, we found 685 genes (53%) that had a delayed phase of transcription with a delay of about 6 h (± 1.5 h) in mismatched compared to matched parasites ( Supplementary Fig. 1f) which complements our phenotypic observation of the phase difference between mismatched and matched parasite IDCs (Supplementary Fig. 1a). ...Context 10
... and so might also be IDC associated genes affected by a loss of scheduling forces and / or genes undergoing readjustment of their phase of transcription to realign the IDC with the host rhythm. The amplitude of rhythmically transcribed genes is significantly higher (p < 0.0001, unpaired student t test) in matched compared to mismatched parasites (Fig. 1d), suggesting a loss of synchronicity in the transcription of mismatched parasites that is not severe enough to impact on IDC synchronicity as measured by stage proportions (Supplementary Fig. 1a). Such a dampening of rhythms is a typical consequence of misalignment of a circadian clock with its time-of-day cue 22 ...Context 11
... other 607 genes display broad differences in their phase of transcription between matched and mismatched parasites, and so might also be IDC associated genes affected by a loss of scheduling forces and / or genes undergoing readjustment of their phase of transcription to realign the IDC with the host rhythm. The amplitude of rhythmically transcribed genes is significantly higher (p < 0.0001, unpaired student t test) in matched compared to mismatched parasites (Fig. 1d), suggesting a loss of synchronicity in the transcription of mismatched parasites that is not severe enough to impact on IDC synchronicity as measured by stage proportions (Supplementary Fig. 1a). Such a dampening of rhythms is a typical consequence of misalignment of a circadian clock with its time-of-day cue 22 . ...Context 12
... times of the day (ZT 8 and ZT 20, Whilst these genes have a periodicity of transcription extremely close to 24 h in matched parasites (median periodicity =23.89h), 55% of these transcripts had shorter periodicities in mismatched parasites (between 20h to 24h), which in turn reduced the overall periodicity by ~ 1 hour (median periodicity 22.85h) (Fig. 1f). Period estimates from genes that lost rhythms in transcription profiles are used here to illustrate an overall trend, rather than provide information on individual genes. It is possible that the shorter periods for mismatched parasites correlates with a shorter IDC, thus explaining how mismatched parasites become rescheduled by ...Context 13
... a member of a seven-transmembrane receptor family, was the top ranked receptor in the P. falciparum circadian gene list (ranked 21 out of all 495 genes sorted based on q-values) (Fig. 3c, Supplementary Data 3). Its orthologue in P. chabaudi (PCHAS_1433600) was also circadian in transcription in both matched and mismatched parasites (Fig. 3c, Supplementary Data 1). In P. falciparum, SR10 expression peaked at 8 h and 32 h post invasion, which corresponds to ring and late trophozoite stages of the IDC. In P. chabaudi, expression peaked at ZT14, corresponding to the late trophozoite ...Context 14
... that lose rhythmicity are involved in diverse biological processes including glycolytic process, DNA replication, translation, ubiquitin/proteasome pathway and redox metabolism (Supplementary Data 2). This is not simply a consequence of mismatched parasites becoming desynchronised as they maintain synchrony during rescheduling (Supplementary Fig. 1a). Instead, disruption to these processes could be a result of stresses resulting from the IDC being misaligned to the host. ...Context 15
... (entrains) to a phase-shift in its time-cue (Zeitgeber). Such misalignment can manifest as a dampening (reduction in amplitude) of rhythmicity of transcription of genes driven by the clock 22,59 . In concordance with this, we observed a reduction in the mean amplitude of transcripts of genes that remain rhythmic in matched, mismatched (Fig. 1d), and in wild type and sr10KO parasites (Fig. ...Context 16
... in lights on/off). In one group, the light regime was maintained as for the donor mouse (i.e. host rhythm matched, lights on: 7.30 (ZT0/24 (Zeitgeber Time: hours after lights on)) and lights off: 19.30(ZT12)). In the other group, the light-dark cycle was reversed (host rhythm mismatched, lights on: 19.30 (ZT0/24), lights off: 7.30 (ZT12)) (Fig. 1a). The effect of mismatch to host rhythms on the parasite was assessed by analyzing the parasite transcriptome every 3 h for 30 h (n=4 mice/group/time ...Context 17
... mismatch to host circadian rhythms, the IDC of P. chabaudi becomes rescheduled within approximately seven cycles to match the host's rhythms. By the time of sampling (days 4-5 post infection, PI), the parasites were six hours mismatched to the host's rhythm ( Supplementary Fig. 1a). Specifically, in mismatched parasites, merozoite egress from RBCs following schizogony peaked six hours after matched parasites (ZT . ...Context 18
... bioRxiv preprint first posted online Oct. 2, 2019; 0/24 and ZT 18 respectively; inferred from ring stage rhythms). Parasites in both the matched and mismatched infections remained synchronous throughout the sampling period ( Supplementary Fig. 1a). ...Context 19
... to as "circadian") if both algorithms independently detected a ~24 h periodicity in their transcription with a threshold of p < 0.05. Of a total of 5,343 genes in P. chabaudi (5,158 detected and considered for analysis), 3,057 (58%) in matched parasites, and 1,824 (34%) in mismatched parasites, exhibited circadian rhythms in transcription (Fig. 1b, Supplementary Data 1). A permutation test was performed to empirically determine the false discovery rate (FDR) in detecting circadian transcripts. The original time points were shuffled randomly for 1,000 iterations and circadian transcripts were identified each time by both the algorithms. The permutation test identified that the number of circadian ...Context 20
... original time points were shuffled randomly for 1,000 iterations and circadian transcripts were identified each time by both the algorithms. The permutation test identified that the number of circadian transcripts in both matched and mismatched parasites detected in the original sampling order by both the programs was significantly higher (FDR < 0.05, Supplementary Fig. 1b) than when the sampling order was randomly permuted indicating that the circadian genes identified were transcribed in a rhythmic manner above background noise. ...Context 21
... < 0.05) in matched parasites that exhibit an arrhythmic transcription profile in mismatched parasites (p > 0.05); 2) 1,292 genes with transcription profiles with 24 h rhythm in both matched and mismatched parasites; and 3) 532 genes whose transcription profiles were arrhythmic in matched parasites and rhythmic in mismatched parasites (p < 0.05, Fig. 1b and 1c, Supplementary Fig. 1c). Hierarchical . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in ...Context 22
... doi: bioRxiv preprint first posted online Oct. 2, 2019; clustering analysis identified biological replicates to be tightly clustered ( Supplementary Fig. 1d). Comparison of the 11 time points using principal component analysis identified the first component in both the conditions with a cyclic pattern that accounted for > 85 % of total variance (Supplementary Fig. 1e), which supports the hypothesis that the large number of transcripts detected as circadian are truly transcribed at a 24 h periodicity. ...Context 23
... of 1,292 genes that were rhythmically transcribed in both matched and mismatched parasites, we found 685 genes (53%) that had a delayed phase of transcription with a delay of about 6 h (± 1.5 h) in mismatched compared to matched parasites ( Supplementary Fig. 1f) which complements our phenotypic observation of the phase difference between mismatched and matched parasite IDCs (Supplementary Fig. 1a). ...Context 24
... of 1,292 genes that were rhythmically transcribed in both matched and mismatched parasites, we found 685 genes (53%) that had a delayed phase of transcription with a delay of about 6 h (± 1.5 h) in mismatched compared to matched parasites ( Supplementary Fig. 1f) which complements our phenotypic observation of the phase difference between mismatched and matched parasite IDCs (Supplementary Fig. 1a). ...Context 25
... and so might also be IDC associated genes affected by a loss of scheduling forces and / or genes undergoing readjustment of their phase of transcription to realign the IDC with the host rhythm. The amplitude of rhythmically transcribed genes is significantly higher (p < 0.0001, unpaired student t test) in matched compared to mismatched parasites (Fig. 1d), suggesting a loss of synchronicity in the transcription of mismatched parasites that is not severe enough to impact on IDC synchronicity as measured by stage proportions (Supplementary Fig. 1a). Such a dampening of rhythms is a typical consequence of misalignment of a circadian clock with its time-of-day cue 22 ...Context 26
... other 607 genes display broad differences in their phase of transcription between matched and mismatched parasites, and so might also be IDC associated genes affected by a loss of scheduling forces and / or genes undergoing readjustment of their phase of transcription to realign the IDC with the host rhythm. The amplitude of rhythmically transcribed genes is significantly higher (p < 0.0001, unpaired student t test) in matched compared to mismatched parasites (Fig. 1d), suggesting a loss of synchronicity in the transcription of mismatched parasites that is not severe enough to impact on IDC synchronicity as measured by stage proportions (Supplementary Fig. 1a). Such a dampening of rhythms is a typical consequence of misalignment of a circadian clock with its time-of-day cue 22 . ...Context 27
... times of the day (ZT 8 and ZT 20, Whilst these genes have a periodicity of transcription extremely close to 24 h in matched parasites (median periodicity =23.89h), 55% of these transcripts had shorter periodicities in mismatched parasites (between 20h to 24h), which in turn reduced the overall periodicity by ~ 1 hour (median periodicity 22.85h) (Fig. 1f). Period estimates from genes that lost rhythms in transcription profiles are used here to illustrate an overall trend, rather than provide information on individual genes. It is possible that the shorter periods for mismatched parasites correlates with a shorter IDC, thus explaining how mismatched parasites become rescheduled by ...Context 28
... a member of a seven-transmembrane receptor family, was the top ranked receptor in the P. falciparum circadian gene list (ranked 21 out of all 495 genes sorted based on q-values) (Fig. 3c, Supplementary Data 3). Its orthologue in P. chabaudi (PCHAS_1433600) was also circadian in transcription in both matched and mismatched parasites (Fig. 3c, Supplementary Data 1). In P. falciparum, SR10 expression peaked at 8 h and 32 h post invasion, which corresponds to ring and late trophozoite stages of the IDC. In P. chabaudi, expression peaked at ZT14, corresponding to the late trophozoite ...Context 29
... that lose rhythmicity are involved in diverse biological processes including glycolytic process, DNA replication, translation, ubiquitin/proteasome pathway and redox metabolism (Supplementary Data 2). This is not simply a consequence of mismatched parasites becoming desynchronised as they maintain synchrony during rescheduling (Supplementary Fig. 1a). Instead, disruption to these processes could be a result of stresses resulting from the IDC being misaligned to the host. ...Context 30
... (entrains) to a phase-shift in its time-cue (Zeitgeber). Such misalignment can manifest as a dampening (reduction in amplitude) of rhythmicity of transcription of genes driven by the clock 22,59 . In concordance with this, we observed a reduction in the mean amplitude of transcripts of genes that remain rhythmic in matched, mismatched (Fig. 1d), and in wild type and sr10KO parasites (Fig. ...Similar publications
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Citations
... of parasites in vivo are inevitably confounded by synchronous development throughout the IDC of 24 h. Using P. falciparum would overcome some of these obstacles because its IDC duration is 48 h and it can be cultivated in vitro (Subudhi et al., 2019). Thus, experiments in which constant (''free-running'') conditions are generated by either not replenishing or continuously replenishing media could use P. falciparum to test for 24 h rhythms in gene expression and protein production, as well as temperature compensation. ...
Biological rhythms appear to be an elegant solution to the challenge of coordinating activities with the consequences of the Earth's daily and seasonal rotation. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are well understood. In contrast, the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites remain mysterious. Here, we explore how periodicity in parasite traits is generated and why daily rhythms matter for parasite fitness. We focus on malaria (Plasmodium) parasites which exhibit developmental rhythms during replication in the mammalian host's blood and in transmission to vectors. Rhythmic in-host parasite replication is responsible for eliciting inflammatory responses, the severity of disease symptoms, and fueling transmission, as well as conferring tolerance to anti-parasite drugs. Thus, understanding both how and why the timing and synchrony of parasites are connected to the daily rhythms of hosts and vectors may make treatment more effective and less toxic to hosts.
... A recent report has addressed a similar question in Plasmodium (Subudhi et al., 2019). Mice infected with P. chabaudi (which displays a 24 h development cycle in RBCs) were housed under a light-dark cycle, and blood sampled over 30 h was used for RNA sequencing: over 5,000 P. chabaudi transcripts displayed ∼24 h rhythms. ...
... A followup study showed that this mismatch effect does not depend on either the developmental stage of the parasite used for infection or on the route of infection, and has an impact at early infection stages (O'Donnell et al., 2013). A misalignment of the P. chabaudi and host rhythms also impacted rhythmic parasite genes, and led to a reduced development cycle period (Subudhi et al., 2019). ...
Parasites have evolved various mechanisms to favor infection of their hosts and enhance the success of the infection. In this respect, time-of-day effects were found during the course of parasitic infections, which can be caused or controlled by circadian rhythms in the physiology of their vertebrate hosts. These include circadian clock-controlled rhythms in metabolism and in immune responses. Conversely, parasites can also modulate their hosts' behavioral and cellular rhythms. Lastly, parasites themselves were in some cases shown to possess their own circadian clock mechanisms, which can influence their capacity to infect their hosts. A better knowledge of the circadian regulation of host-parasite interactions will help in designing new preventive and therapeutic strategies for parasitic diseases.
