Calflagin Inhibition Prolongs Host Survival and Suppresses Parasitemia in Trypanosoma brucei Infection

African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, a fatal disease endemic to regions throughout sub-Saharan Africa. Incidence rates of human T. brucei infection have risen dramatically in the past 30 to 50 years, leading to renewed emphasis by the World Health Organization on disease surveillance and control among the millions of people at risk of infection. T. brucei also infects cattle to cause nagana, a disease which renders vast regions unsuitable for livestock and poses a major barrier to economic development in afflicted areas (30).T. brucei is transmitted to its mammalian host via the bite of the infected tsetse fly, when parasites are introduced into the host circulation during a blood meal. To thrive in both the insect vector and the mammalian host, T. brucei has evolved digenetic life cycle stages; the two most commonly studied life cycle stages are the procyclic stage from the fly midgut and the bloodstream form found in the mammalian host. Programmed differentiation between these stages regulates broad aspects of parasite biology, enabling adaptation to either environment. In the mammalian host, avoiding clearance by the humoral immune response is particularly important, and T. brucei has evolved sophisticated mechanisms to this end. Bloodstream form parasites are covered by a thick monolayer of variant surface glycoprotein (VSG) that blocks the access of host antibodies to underlying invariant antigens (2). VSG is highly immunogenic, and VSG-specific antibodies facilitate the clearance of parasites from the blood. However, the parasite undergoes antigenic variation, a process whereby the parasite spontaneously switches from the expression of a single VSG type to that of another of the hundreds in its genomic repertoire. The new parasite clone is resistant to the existing antibodies and persists until antibodies to the new VSG are produced, thus selecting for another antigen variant and propagating the cycle. T. brucei has coevolved with primates for millions of years, and antigenic variation is not its sole means of immune evasion. Additional mechanisms such as host immunosuppression (20), motility-driven internalization and degradation of surface-bound antibodies (13, 22), and the shedding of VSG molecules (7) are each likely to contribute to the survival of T. brucei in the hostile environment of its mammalian host. However, the signaling and genetic pathways by which T. brucei regulates stage-specific adaptations to its environment remain poorly understood.Calcium signaling plays critical roles in virtually every eukaryotic cell type, and trypanosomes are no exception. Regulated changes in intracellular calcium are important for trypanosome replication, differentiation, cell invasion, and virulence (24). The importance of calcium regulation in trypanosomes is further underscored by the presence of a specialized organelle, the acidocalcisome, which contains a major intracellular reservoir of calcium (9). Cellular responses to calcium fluctuations are mediated by calcium-binding proteins. In addition to calmodulin, T. brucei expresses a family of EF-hand proteins named the calflagins. They were discovered as the predominant T. brucei proteins to bind a hydrophobic resin in a calcium-dependent manner (35, 36). These proteins specifically localize to the flagellum, an organelle which, in addition to its obvious role in cellular motility, compartmentalizes signaling proteins, including adenylate cyclases and phosphodiesterases (26, 27). In the flagellum, calflagins associate with lipid raft microdomains, which in many cell types serve as recruitment platforms for signaling molecules (32). We have recently elucidated the role of acylation in calflagin trafficking and raft association and identified the calflagin-specific palmitoyl acyltransferase (11). However, the precise functions of these abundant proteins remain unknown. The calflagins show homology with Trypanosoma cruzi FCaBP, an immunodominant protein with similar flagellar enrichment yet an unidentified biologic function (12). Outside of the common EF-hand domains, the calflagins display only minimal homology to proteins in organisms outside the kinetoplastid lineage.To elucidate the function of T. brucei calflagins, we assessed the consequences of calflagin inhibition to T. brucei in vivo during host infection and in vitro. We can now report for the first time that calflagin expression influences the outcome of parasite infection, as mice infected with calflagin-deficient cells demonstrate a sudden decrease in parasitemia approximately 1 week postinfection. This drop is associated with prolonged host survival and outgrowth of parasites expressing alternative VSG molecules, indicating selective pressure against the initial dominant VSG molecule and suggesting that the primary deficit of calflagin-deficient cells is one of enhanced sensitivity to the host adaptive immune response. We demonstrate that the molecular mechanisms underlying this effect are not due to altered parasite proliferation, motility, or clearance of surface-bound antibodies.