Periodic Parasites and Daily Host Rhythms

Abstract

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.

Keywords: Plasmodium; circadian clock; circadian rhythm; entrainment; fitness; host-parasite interactions; inflammatory response; intra-erythrocytic development cycle; metabolism; nutrient sensing; periodicity; synchronicity.

Figure 1.. Periodicity in malaria parasites

a) Following transmission from a mosquito, malaria parasites replicate in the liver before invading red blood cells and undergo successive cycles of asexual replication (the intra-erythrocytic development cycle, IDC). The IDC begins when a merozoite invades an RBC, which then becomes a ring stage parasite (purple), before developing through several developmental stages of the trophozoite stage (blue, orange, pink), and finally becoming a mature schizont (teal) which ruptures to release merozoites to initiate the next IDC. Each stage within the IDC has different roles and requirements from the host. The ring stage remodels the RBC and has no specific requirements, trophozoites undergo DNA replication and need amino acids, glucose, purines, folates and lysophosphatidylcholine, while schizonts complete cell division and require phospholipids for membrane production. b) Throughout the rodent host’s circadian cycle (white bar = day, grey bar = night), each IDC stage develops in sequence. Later stages (mid trophozoites to schizonts) sequester out of circulation in capillaries and organs and have very low abundance (and therefore dampened rhythms) in circulation. However, zooming in, for example, on schizonts (inset) reveals rhythmicity, with their peak occurring just before their ring stage progeny appear. Rhythms shown are model fits from data in Prior et al., (unpublished). Zeitgeber Time refers to the number of hours since lights on/dawn.

Figure 2.. Chronobiology concepts

a) Core molecular components of the mammalian TTFL (Transcription-Translation Feedback Loop) clock consist of the proteins CLOCK (CLK), BMAL1, PERIOD1 and PERIOD2 (PER) and CRYPTOCHROME1 and CRYPTOCHROME2 (CRY). These components, with the help of other transcription factors and proteins, cycle through transcription, translation and degradation cycles with a period of ~24h, instructing downstream target genes to orchestrate rhythms in physiology and behavior (Reppert and Weaver, 2002). Figure modified from Rijo-Ferreira et al., (2017a). b) Circadian clocks have a built-in ability to anticipate daily environmental changes. The activities of circadian clocks can be assessed by directly measuring clock-controlled gene expression (either clock genes or those downstream), commonly done using a luminescent reporter (Yamazaki et al., 2000). Circadian rhythms are characterized by their “period” (of around 24 hours), “phase” (time-of-day the rhythm reaches a point of biological relevance), and “amplitude” (difference between peak and trough values). These features persist due to clock-control even in the absence of environmental rhythms (“free running”); here the subject is kept in constant darkness and the light and dark grey bars illustrate “subjective” day and night. c) Circadian clocks are also “temperature compensated” and so, maintain the same period across a gradient of environmental temperatures (Pittendrigh, 1960). d) Circadian clocks usually operate in rhythmic environments and all clocks require a “Zeitgeber” or time cue to “entrain”. When cultured without external stimuli for many days, clocks retain individual oscillations but usually become desynchronized from each other. Synchronization is achieved by entrainment. The features illustrated (b-d) ensure that clock oscillators balance being robust to perturbation whilst being flexible enough to keep up with (for example) changing photoperiod across seasons.

Figure 3.. Evolutionary explanations for the IDC schedule

Understanding the evolution of the IDC schedule requires explaining why both synchrony and timing occur. Selection could favor both traits, one trait, or neither of the traits, as illustrated by these scenarios. a) Synchrony is beneficial: Synchronous development enhances fitness, but timing does not. Timing may vary or be used as the mechanism to achieve synchrony. b) Timing is beneficial: An IDC transition at a particular time of day enhances fitness, so synchrony occurs as a by-product of selection for timing. For example, timing may be beneficial to parasites simply because it enables them to maximally exploit a rhythmic resource inside the host. In the scenarios in which one trait is favored, the other could be costly (i.e., a constraint) but the benefits of the useful trait outweigh these costs. The alternative is that both (c) or neither (d) trait are beneficial to parasites. c) Both traits are beneficial: Fitness is enhanced by each trait for different reasons so both traits are independently favored by natural selection. For example, synchrony may provide safety in numbers against a harmful host factor, and if this harmful factor is rhythmic and IDC stages vary in their vulnerability to it, then timing is also beneficial. d) Neither are beneficial: Synchrony and timing could be an unavoidable by-product of a different trait that confers sufficient fitness benefits to offset the costs of synchrony and timing. Alternatively, parasites may have no control of the IDC schedule and synchrony and timing are forced upon the parasite by host rhythms (regardless of whether this is beneficial to the host or not).

Figure 4.. What sets the timing of the IDC?

a) In P. chabaudi, the timing of the IDC can be driven by the feeding rhythm of the host. Mammalian hosts have a clock in the suprachaismatic nucleus (SCN) and peripheral clocks in other organs and tissues. The SCN clock uses light as its primary time cue and the timing of peripheral clocks is most sensitive to cues from food/metabolism. The cartoon illustrates the results of Prior et al., (2018) and Hirako et al., (2018) in which schizogony peaks during the period in which hosts are provided with food, irrespective of the timing of the hosts SCN clock. The precise mechanism by which parasite rhythms result from the timing of food intake is unclear, as indicated by the question marks (?). b) The generation of synchrony and timing of the parasite IDC is still not fully understood. Parasites develop through the IDC, from being relatively metabolically inactive to undergoing DNA replication and cell division, as hosts move from the rest to the active phase of their daily rhythm. Host activity corresponds with food intake, which elevates blood glucose concentration. As infections progress, pathogen-associated molecular patterns (PAMPs) activate immune cells via TNF-α which alters their energy metabolism. This, in concert with the production of inflammatory cytokines stimulating glucose uptake from the blood (by for example, the liver), exacerbate the host’s daily rhythm in blood glucose. The resulting daily window of hypoglycaemia corresponds with metabolically inactive stages in the IDC, and hyperglycaemia corresponds with IDC completion, generating both synchrony and timing of the IDC (Hirako et al., 2018, Prior et al., 2018, Reece and Prior, 2018).

Figure 5.. Who controls the IDC schedule?

a) To what extent the IDC schedule is organized by the parasite versus forced by the host is unclear. The possible scenarios (i – iv) are illustrated by considering how P. chabaudi parasites (dashed line) whose IDC is mismatched to the host feeding rhythm become rescheduled to follow the same pattern as control (i.e., matched; grey lines) infections. If parasites control the IDC schedule, they can (i) slow or (ii) speed up the IDC (within constraints set by the minimum and maximum possible duration of each IDC stage), or (iii) become quiescent until an IDC stage encounters the correct time-of-day to develop. Alternatively, (iv) if the parasites are passive to being scheduled solely by the host, mismatched parasites will be forced to stop at some point in the IDC. Depending on how long parasites can remain quiescent and the degree of stress imposed on parasites by being forced to stop developing, scenario (iv) is hard to differentiate from scenario (iii). But, if parasites whose development is stopped die, then examining densities during rescheduling (v) can differentiate the scenarios. (v) Assuming burst size, invasion rate etc. do not differ between the scenarios, and all infections replicate at the same rate once rescheduled (i.e., slopes are parallel), comparing densities during (solid lines) and after rescheduling (dashed lines), differentiates the scenarios. Observations that the IDC remains mismatched for a few cycles suggests scenarios iii and iv are unlikely. b) If the P. chabaudi IDC is mismatched to the host rhythm, the IDC returns to synchrony with the host rhythm within 5-7 cycles. When infections are initiated using ring stages transferred from donor hosts to recipient hosts on an opposite light schedule (O’Donnell et al., 2011), the recipient host rhythm (dashed green line) remains unaltered but the parasite IDC (purple line) becomes rescheduled to the recipient host rhythm, whilst remaining synchronous (Prior et al., 2018). Dark bars indicate night and light bars indicate day.