Evolutionary ecology of biological rhythms in malaria parasites

Biological rhythms are a ubiquitous feature of life and are assumed to allow organisms coordinate their activities with daily rhythms in the abiotic environment resulting from the rotation of the Earth every 24 hours. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are relatively well understood in contrast to the evolution and ecology of circadian rhythms. Circadian rhythms mediate interactions between organisms; from predators and prey, to mating behaviours between males and females, to hosts and parasites. The role of daily rhythms in infections is gaining traction because explaining the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites and hosts offers new avenues to treat infections. Here, I explore how periodicity in parasite traits is generated and why daily rhythms matter for parasite fitness. My work focuses on malaria (Plasmodium) parasites which exhibit developmental rhythms during replication in the mammalian host’s blood and during transmission to insect vectors. Rhythmic in-host parasite replication is responsible for eliciting inflammatory responses, severe anaemia, fuels transmission, and can confer 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. My papers integrate an evolutionary ecology approach with chronobiology and parasitology to investigate how host-parasite-vector interactions shape the evolution of rhythmicity in parasites traits. I have used a rodent malaria parasite model system (Plasmodium chabaudi) for my experiments, capitalising on the tractability of this model for the human malaria, P. falciparum. P. chabaudi exhibits a 24-hour rhythm in replication, facilitates ecologically realistic studies because experiments can be carried out in vivo (compared to the in vitro limitations on studying human parasites), and perturbations to the timing of the in-host and in-vector environments are straightforward. My findings include: 1) Perturbing the timing of parasite rhythms with respect to the timing of host rhythms (analogous to giving the parasites “jet lag”), results in a fitness cost to the parasites, evident by a 50% reduction in both asexually replicating and transmission stage parasites. 2) The consequences of temporal mismatch to the host manifest very early in the infection (within 48 hours, i.e. the first 1-2 cycles of replication) and are dependent on the parasite stage by which infections are initiated (0-12 hour old parasites suffer a cost, whereas 12-24 hour parasites benefit). 3) The timing of the parasite replication cycle is independent of the canonical ‘core’ host clock (i.e. transcription translation feedback loop) and instead depends on the timing of feeding-fasting rhythms of the host. 4) If perturbed, the timing of the parasite’s rhythm reschedules to regain synchrony with the timing of the host’s rhythm within 7 replication cycles. Specifically, parasites achieve this by speeding up the replication rhythm by 2-3 hours per cycle, and the rate of rescheduling is independent of parasite density. 5) Naturally asynchronous Plasmodium species are ‘resistant’ to conditions that lead to alignment with host rhythms in synchronously replicating species. This suggests that unknown ecological differences between these parasite species selects for vastly different schedules of within-host replication rather than some species being constrained to replicate asynchronously. 6) In addition to the timing of parasite rhythms impacting directly upon within-host dynamics, timing also matters – albeit indirectly - for transmission, via impacts on the population dynamics of the vector. For example, receiving a blood meal in the morning makes mosquitoes more likely to lay eggs, lay slightly sooner and have a larger clutch size than those feeding at night. Yet, whilst mosquitoes infected with malaria die sooner, the effects of taking a blood meal at different times of day do not impact transmission of an asynchronously replicating malaria parasite. It is beneficial for parasites to be in synchronization with their host’s feeding-fasting rhythms and plasticity in the IDC duration facilitates this synchrony by enabling parasites to make small daily changes to their IDC schedule when necessary. Understanding the extent of, and limits on, plasticity in the IDC schedule is important as it may reveal targets for novel interventions, such as drugs to disrupt IDC regulation and preventing IDC dormancy conferring tolerance to existing drugs. More generally, our results provide a demonstration of the adaptive value of biological rhythms and the utility of using an evolutionary framework to understand parasite traits

Mistimed malaria parasites re‐synchronise with host feeding‐fasting rhythms by shortening the duration of intra‐erythrocytic development

AIMS: Malaria parasites exhibit daily rhythms in the intra‐erythrocytic development cycle (IDC) that underpins asexual replication in the blood. The IDC schedule is aligned with the timing of host feeding‐fasting rhythms. When the IDC schedule is perturbed to become mismatched to host rhythms, it readily reschedules but it is not known how. METHODS: We intensively follow four groups of infections that have different temporal alignments between host rhythms and the IDC schedule for 10 days, before and after the peak in asexual densities. We compare how the duration, synchrony and timing of the IDC differs between parasites in control infections and those forced to reschedule by 12 hours and ask whether the density of parasites affects the rescheduling process. RESULTS AND CONCLUSIONS: Our experiments reveal parasites shorten the IDC duration by 2–3 hours to become realigned to host feeding‐fasting rhythms with 5–6 days, in a density‐independent manner. Furthermore, parasites are able to reschedule without significant fitness costs for them or their hosts. Understanding the extent of, and limits on, plasticity in the IDC schedule may reveal targets for novel interventions, such as drugs to disrupt IDC regulation and preventing IDC dormancy conferring tolerance to existing drugs

Host circadian rhythms are disrupted during malaria infection in parasite genotype-specific manners

Infection can dramatically alter behavioural and physiological traits as hosts become sick and subsequently return to health. Such “sickness behaviours” include disrupted circadian rhythms in both locomotor activity and body temperature. Host sickness behaviours vary in pathogen species-specific manners but the influence of pathogen intraspecific variation is rarely studied. We examine how infection with the murine malaria parasite, Plasmodium chabaudi, shapes sickness in terms of parasite genotype-specific effects on host circadian rhythms. We reveal that circadian rhythms in host locomotor activity patterns and body temperature become differentially disrupted and in parasite genotype-specific manners. Locomotor activity and body temperature in combination provide more sensitive measures of health than commonly used virulence metrics for malaria (e.g. anaemia). Moreover, patterns of host disruption cannot be explained simply by variation in replication rate across parasite genotypes or the severity of anaemia each parasite genotype causes. It is well known that disruption to circadian rhythms is associated with non-infectious diseases, including cancer, type 2 diabetes, and obesity. Our results reveal that disruption of host circadian rhythms is a genetically variable virulence trait of pathogens with implications for host health and disease tolerance

Automated detection and staging of malaria parasites from cytological smears using convolutional neural networks

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis

Timing of host feeding drives rhythms in parasite replication

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the hosts' peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the hosts' peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience

Single-cell multi-omics analysis of the immune response in COVID-19

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255Funder: University College London, Birkbeck MRC Doctoral Training ProgrammeFunder: The Jikei University School of MedicineFunder: Action Medical Research (GN2779)Funder: NIHR Clinical Lectureship (CL-2017-01-004)Funder: NIHR (ACF-2018-01-004) and the BMA FoundationFunder: Chan Zuckerberg Initiative (grant 2017-174169) and from Wellcome (WT211276/Z/18/Z and Sanger core grant WT206194)Funder: UKRI Innovation/Rutherford Fund Fellowship allocated by the MRC and the UK Regenerative Medicine Platform (MR/5005579/1 to M.Z.N.). M.Z.N. and K.B.M. have been funded by the Rosetrees Trust (M944)Funder: Barbour FoundationFunder: ERC Consolidator and EU MRG-Grammar awardsFunder: Versus Arthritis Cure Challenge Research Grant (21777), and an NIHR Research Professorship (RP-2017-08-ST2-002)Funder: European Molecular Biology Laboratory (EMBL)Abstract: Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy