Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma.

Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt's lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells. PAPERCLIP.

Cellular iron governs the host response to malaria.

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor (TfrcY20H/Y20H), which limits the ability of cells to internalise iron from plasma. At homeostasis TfrcY20H/Y20H mice appear healthy and are not anaemic. However, TfrcY20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that TfrcY20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected TfrcY20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4+ T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, TfrcY20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

CD4 T Cell-Dependent and -Independent Roles for IFN-γ in Blood-Stage Malaria.

Production of IFN-γ by CD4 T cells is widely theorized to control Plasmodium parasite burden during blood-stage malaria. Surprisingly, the specific and crucial mechanisms through which this highly pleiotropic cytokine acts to confer protection against malarial disease remain largely untested in vivo. Here we used a CD4 T cell-restricted Cre-Lox IFN-γ excision mouse model to test whether and how CD4 T cell-derived IFN-γ controls blood-stage malaria. Although complete absence of IFN-γ compromised control of the acute and the chronic, recrudescent blood-stage infections with P. c. chabaudi, we identified a specific, albeit modest, role for CD4 T cell-derived IFN-γ in limiting parasite burden only during the chronic stages of P. c. chabaudi malaria. CD4 T cell IFN-γ promoted IgG Ab class switching to the IgG2c isotype during P. c. chabaudi malaria in C57BL/6 mice. Unexpectedly, our data do not support gross defects in phagocytic activity in IFN-γ-deficient hosts infected with blood-stage malaria. Together, our data confirm CD4 T cell-dependent roles for IFN-γ but suggest CD4 T cell-independent roles for IFN-γ in immune responses to blood-stage malaria.

Type I interferon production elicits differential CD4+ T-cell responses in mice infected with Plasmodium berghei ANKA and P. chabaudi.

Plasmodium parasites that infect humans are highly polymorphic, and induce various infections ranging from an asymptomatic state to life-threatening diseases. However, how the differences between the parasites affect host immune responses during blood-stage infection remains largely unknown. We investigated the CD4+ T-cell immune responses in mice infected with P. berghei ANKA (PbA) or P. chabaudi chabaudi AS (Pcc) using PbT-II cells, which recognize a common epitope of these parasites. In the acute phase of infection, CD4+ T-cell responses in PbA-infected mice showed a lower involvement of Th1 cells and a lower proportion of Ly6Clo effector CD4+ T cells than those in Pcc-infected mice. Transcriptome analysis of PbT-II cells indicated that type I interferon (IFN)-regulated genes were expressed at higher levels in both Th1- and Tfh-type PbT-II cells from PbA-infected mice than those from Pcc-infected mice. Moreover, IFN-α levels were considerably higher in PbA-infected mice than in Pcc-infected mice. Inhibition of type I IFN signaling increased PbT-II and partially reversed the Th1 over Tfh bias of the PbT-II cells in both PbA- and Pcc-infected mice. In the memory phase, PbT-II cells in PbA-primed mice maintained higher numbers and exhibited a better recall response to the antigen. However, recall responses were not significantly different between the infection groups after re-challenge with PbA, suggesting the effect of the inflammatory environment by the infection. These observations suggest that the differences in Plasmodium-specific CD4+ T-cell responses between PbA- and Pcc-infected mice were associated with the difference in type I IFN production during the early phase of the infection.

Structure of the Plasmodium-interspersed repeat proteins of the malaria parasite.

The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.

ICOS signaling promotes a secondary humoral response after re-challenge with Plasmodium chabaudi chabaudi AS.

The co-stimulatory molecule ICOS is associated with the induction and regulation of T helper cell responses, including the differentiation of follicular helper T (Tfh) cells and the formation and maintenance of memory T cells. However, the role of ICOS signaling in secondary immune responses is largely unexplored. Here we show that memory T cell formation and maintenance are influenced by persistent infection with P. chabaudi chabaudi AS infection, as memory T cell numbers decline in wild-type and Icos-/- mice after drug-clearance. Following drug-clearance Icos-/- mice display a relapsing parasitemia that occurs more frequently and with higher peaks compared to wild-type mice after re-challenge. The secondary immune response in Icos-/- mice is characterized by significant impairment in the expansion of effector cells with a Tfh-like phenotype, which is associated with a diminished and delayed parasite-specific Ab response and the absence of germinal centers. Similarly, the administration of an anti-ICOSL antagonizing antibody to wild-type mice before and after reinfection with P. c. chabaudi AS leads to an early defect in Tfh cell expansion and parasite-specific antibody production, confirming a need for ICOS-ICOSL interactions to promote memory B cell responses. Furthermore, adoptive transfer of central memory T (TCM) cells from wild-type and Icos-/- mice into tcrb-/- mice to directly evaluate the ability of TCM cells to give rise to Tfh cells revealed that TCM cells from wild-type mice acquire a mixed Th1- and Tfh-like phenotype after P. c. chabaudi AS infection. While TCM cells from Icos-/- mice expand and display markers of activation to a similar degree as their WT counterparts, they displayed a reduced capacity to upregulate markers indicative of a Tfh cell phenotype, resulting in a diminished humoral response. Together these findings verify that ICOS signaling in memory T cells plays an integral role in promoting T cell effector responses during secondary infection with P. c. chabaudi AS.

Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria.

Although the relationship between hematopoietic stem cells and progenitor populations has been investigated extensively under steady-state conditions, the dynamic response of the hematopoietic compartment during acute infection is largely unknown. Here we show that after infection of mice with Plasmodium chabaudi, a c-Kit(hi) progenitor subset positive for interleukin 7 receptor-alpha (IL-7Ralpha) emerged that had both lymphoid and myeloid potential in vitro. After being transferred into uninfected alymphoid or malaria-infected hosts, IL-7Ralpha(+)c-Kit(hi) progenitors generated mainly myeloid cells that contributed to the clearance of infected erythrocytes in infected hosts. The generation of these infection-induced progenitors was critically dependent on interferon-gamma (IFN-gamma) signaling in hematopoietic progenitors. Thus, IFN-gamma is a key modulator of hematopoiesis and innate and adaptive immunity during acute malaria infection.

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

Mistimed malaria parasites re-synchronize 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.

Model of severe malaria in young mice suggests unique response of CD4 T cells.

Severe malaria occurs most in young children but is poorly understood due to the absence of a developmentally-equivalent rodent model to study the pathogenesis of the disease. Though functional and quantitative deficiencies in innate response and a biased T helper 1 (Th1) response are reported in newborn pups, there is little information available about this intermediate stage of the adaptive immune system in murine neonates. To fill this gap in knowledge, we have developed a mouse model of severe malaria in young mice using 15-day old mice (pups) infected with Plasmodium chabaudi. We observe similar parasite growth pattern in pups and adults, with a 60% mortality and a decrease in the growth rate of the surviving young mice. Using a battery of behavioral assays, we observed neurological symptoms in pups that do not occur in infected wildtype adults. CD4+ T cells were activated and differentiated to an effector T cell (Teff) phenotype in both adult and pups. However, there were relatively fewer and less terminally differentiated pup CD4+ Teff than adult Teff. Interestingly, despite less activation, the pup Teff expressed higher T-bet than adults' cells. These data suggest that Th1 cells are functional in pups during Plasmodium infection but develop slowly.

CD4+ICOS+Foxp3+: a sub-population of regulatory T cells contribute to malaria pathogenesis.

BACKGROUND: Regulatory T cells are known to play a key role to counter balance the protective immune response and immune mediated pathology. However, the role of naturally occurring regulatory cells CD4+CD25+Foxp3+ in malaria infection during the disease pathogenesis is controversial. Beside this, ICOS molecule has been shown to be involved in the development and function of regulatory T cell enhance IL-10 production. Therefore, possible involvement of the ICOS dependent regulatory CD4+ICOS+Foxp3+ T cells in resistance/susceptibility during malaria parasite is explored in this study. METHODS: 5 × 105 red blood cells infected with non-lethal and lethal parasites were inoculated in female Balb/c mice by intra-peritoneal injection. Infected or uninfected mice were sacrificed at early (3rd day post infection) and later stage (10th day post infection) of infection. Harvested cells were analysed by using flow cytometer and serum cytokine by Bioplex assay. RESULTS: Thin blood films show that percentages of parasitaemia increases with disease progression in infections with the lethal malaria parasite and mice eventually die by day 14th post-infection. Whereas in case of non-lethal malaria parasite, parasitaemia goes down by 7th day post infection and gets cleared within 13th day. The number of CD4+ ICOS+ T cells increases in lethal infection with disease progression. Surprisingly, in non-lethal parasite, ICOS expression decreases after day 7th post infection as parasitaemia goes down. The frequency of CD4+ICOS+FoxP3+ Tregs was significantly higher in lethal parasitic infection as compared to the non-lethal parasite. The level of IL-12 cytokine was remarkably higher in non-lethal infection compared to the lethal infection. In contrast, the level of IL-10 cytokines was higher in lethal parasite infection compared to the non-lethal parasite. CONCLUSION: Taken together, these data suggest that lethal parasite induce immunosuppressive environment, protecting from host immune responses and help the parasite to survive whereas non-lethal parasite leads to low frequencies of Treg cells seldom impede immune response that allow the parasite to get self-resolved.

Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ß2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ß2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ß2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

The contribution of host cell-directed vs. parasite-directed immunity to the disease and dynamics of malaria infections.

Hosts defend themselves against pathogens by mounting an immune response. Fully understanding the immune response as a driver of host disease and pathogen evolution requires a quantitative account of its impact on parasite population dynamics. Here, we use a data-driven modeling approach to quantify the birth and death processes underlying the dynamics of infections of the rodent malaria parasite, Plasmodium chabaudi, and the red blood cells (RBCs) it targets. We decompose the immune response into 3 components, each with a distinct effect on parasite and RBC vital rates, and quantify the relative contribution of each component to host disease and parasite density. Our analysis suggests that these components are deployed in a coordinated fashion to realize distinct resource-directed defense strategies that complement the killing of parasitized cells. Early in the infection, the host deploys a strategy reminiscent of siege and scorched-earth tactics, in which it both destroys RBCs and restricts their supply. Late in the infection, a "juvenilization" strategy, in which turnover of RBCs is accelerated, allows the host to recover from anemia while holding parasite proliferation at bay. By quantifying the impact of immunity on both parasite fitness and host disease, we reveal that phenomena often interpreted as immunopathology may in fact be beneficial to the host. Finally, we show that, across mice, the components of the host response are consistently related to each other, even when infections take qualitatively different trajectories. This suggests the existence of simple rules that govern the immune system's deployment.

Plasmodium-specific antibodies block in vivo parasite growth without clearing infected red blood cells.

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC.

Uncovering drivers of dose-dependence and individual variation in malaria infection outcomes.

To understand why some hosts get sicker than others from the same type of infection, it is essential to explain how key processes, such as host responses to infection and parasite growth, are influenced by various biotic and abiotic factors. In many disease systems, the initial infection dose impacts host morbidity and mortality. To explore drivers of dose-dependence and individual variation in infection outcomes, we devised a mathematical model of malaria infection that allowed host and parasite traits to be linear functions (reaction norms) of the initial dose. We fitted the model, using a hierarchical Bayesian approach, to experimental time-series data of acute Plasmodium chabaudi infection across doses spanning seven orders of magnitude. We found evidence for both dose-dependent facilitation and debilitation of host responses. Most importantly, increasing dose reduced the strength of activation of indiscriminate host clearance of red blood cells while increasing the half-life of that response, leading to the maximal response at an intermediate dose. We also explored the causes of diverse infection outcomes across replicate mice receiving the same dose. Besides random noise in the injected dose, we found variation in peak parasite load was due to unobserved individual variation in host responses to clear infected cells. Individual variation in anaemia was likely driven by random variation in parasite burst size, which is linked to the rate of host cells lost to malaria infection. General host vigour in the absence of infection was also correlated with host health during malaria infection. Our work demonstrates that the reaction norm approach provides a useful quantitative framework for examining the impact of a continuous external factor on within-host infection processes.

The plasma membrane calcium ATPase 4 does not influence parasite levels but partially promotes experimental cerebral malaria during murine blood stage malaria.

BACKGROUND: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. METHODS: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. RESULTS: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. CONCLUSIONS: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Analysis of pir gene expression across the Plasmodium life cycle.

BACKGROUND: Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. METHODS: Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. RESULTS: Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. CONCLUSIONS: The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

Differential drivers of intraspecific and interspecific competition during malaria-helminth co-infection.

Various host and parasite factors interact to determine the outcome of infection. We investigated the effects of two factors on the within-host dynamics of malaria in mice: initial infectious dose and co-infection with a helminth that limits the availability of red blood cells (RBCs). Using a statistical, time-series approach to model the within-host 'epidemiology' of malaria, we found that increasing initial dose reduced the time to peak cell-to-cell parasite propagation, but also reduced its magnitude, while helminth co-infection delayed peak cell-to-cell propagation, except at the highest malaria doses. Using a mechanistic model of within-host infection dynamics, we identified dose-dependence in parameters describing host responses to malaria infection and uncovered a plausible explanation of the observed differences in single vs co-infections. Specifically, in co-infections, our model predicted a higher background death rate of RBCs. However, at the highest dose, when intraspecific competition between malaria parasites would be highest, these effects of co-infection were not observed. Such interactions between initial dose and co-infection, although difficult to predict a priori, are key to understanding variation in the severity of disease experienced by hosts and could inform studies of malaria transmission dynamics in nature, where co-infection and low doses are the norm.

Host circadian clocks do not set the schedule for the within-host replication of malaria parasites.

Circadian clocks coordinate organisms' activities with daily cycles in their environment. Parasites are subject to daily rhythms in the within-host environment, resulting from clock-control of host activities, including immune responses. Parasites also exhibit rhythms in their activities: the timing of within-host replication by malaria parasites is coordinated to host feeding rhythms. Precisely which host feeding-related rhythm(s) parasites align with and how this is achieved are unknown. Understanding rhythmic replication in malaria parasites matters because it underpins disease symptoms and fuels transmission investment. We test if rhythmicity in parasite replication is coordinated with the host's feeding-related rhythms and/or rhythms driven by the host's canonical circadian clock. We find that parasite rhythms coordinate with the time of day that hosts feed in both wild-type and clock-mutant hosts, whereas parasite rhythms become dampened in clock-mutant hosts that eat continuously. Our results hold whether infections are initiated with synchronous or with desynchronized parasites. We conclude that malaria parasite replication is coordinated to rhythmic host processes that are independent of the core-clock proteins PERIOD 1 and 2; most likely, a periodic nutrient made available when the host digests food. Thus, novel interventions could disrupt parasite rhythms to reduce their fitness, without interference by host clock-controlled homeostasis.

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 host's 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 host's 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.