Trypanosoma musculi Infection in Mice Critically Relies on Mannose Receptor-Mediated Arginase Induction by a TbKHC1 Kinesin H Chain Homolog.

Arginase activity induction in macrophages is an escape mechanism developed by parasites to cope with the host's immune defense and benefit from increased host-derived growth factor production. We report that arginase expression and activity were induced in macrophages during mouse infection by Trypanosoma musculi, a natural parasite of this host. This induction was reproduced in vitro by excreted/secreted factors of the parasite. A mAb directed to TbKHC1, an orphan kinesin H chain from Trypanosoma brucei, inhibited T. musculi excreted/secreted factor-mediated arginase induction. Anti-TbKHC1 Ab also inhibited T. musculi growth, both in vitro and in vivo. Induction of arginase activity and parasite growth involved C-type lectin receptors, because mannose injection decreased arginase activity induction and parasite load in vitro and in vivo. Accordingly, the parasite load was reduced in mice lacking mannose receptor C-type 1. The T. musculi KHC1 homolog showed high similarity with TbKHC1. Bioinformatics analysis revealed the presence of homologs of this gene in other trypanosomes, including pathogens for humans and animals. Host metabolism dysregulation represents an effective parasite mechanism to hamper the host immune response and modify host molecule production to favor parasite invasion and growth. Thus, this orphan kinesin plays an important role in promoting trypanosome infection, and its neutralization or the lock of its partner host molecules offers promising approaches to increasing resistance to infection and new developments in vaccination against trypanosomiasis.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus.

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth.

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24ß-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 µM from lipid-depleted media) with small amounts of ergosterol (1.2 µM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 µM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Sequencing rare and common APOL1 coding variants to determine kidney disease risk.

A third of African Americans with sporadic focal segmental glomerulosclerosis (FSGS) or HIV-associated nephropathy (HIVAN) do not carry APOL1 renal risk genotypes. This raises the possibility that other APOL1 variants may contribute to kidney disease. To address this question, we sequenced all APOL1 exons in 1437 Americans of African and European descent, including 464 patients with biopsy-proven FSGS/HIVAN. Testing for association with 33 common and rare variants with FSGS/HIVAN revealed no association independent of strong recessive G1 and G2 effects. Seeking additional variants that might have been under selection by pathogens and could represent candidates for kidney disease risk, we also sequenced an additional 1112 individuals representing 53 global populations. Except for G1 and G2, none of the 7 common codon-altering variants showed evidence of selection or could restore lysis against trypanosomes causing human African trypanosomiasis. Thus, only APOL1 G1 and G2 confer renal risk, and other common and rare APOL1 missense variants, including the archaic G3 haplotype, do not contribute to sporadic FSGS and HIVAN in the US population. Hence, in most potential clinical or screening applications, our study suggests that sequencing APOL1 exons is unlikely to bring additional information compared to genotyping only APOL1 G1 and G2 risk alleles.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum.

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.

BACKGROUND: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. METHODOLOGY/PRINCIPAL FINDINGS: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. CONCLUSION: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Apolipoproteins L1 and L3 control mitochondrial membrane dynamics.

Apolipoproteins L1 and L3 (APOLs) are associated at the Golgi with the membrane fission factors phosphatidylinositol 4-kinase-IIIB (PI4KB) and non-muscular myosin 2A. Either APOL1 C-terminal truncation (APOL1Δ) or APOL3 deletion (APOL3-KO [knockout]) reduces PI4KB activity and triggers actomyosin reorganization. We report that APOL3, but not APOL1, controls PI4KB activity through interaction with PI4KB and neuronal calcium sensor-1 or calneuron-1. Both APOLs are present in Golgi-derived autophagy-related protein 9A vesicles, which are involved in PI4KB trafficking. Like APOL3-KO, APOL1Δ induces PI4KB dissociation from APOL3, linked to reduction of mitophagy flux and production of mitochondrial reactive oxygen species. APOL1 and APOL3, respectively, can interact with the mitophagy receptor prohibitin-2 and the mitophagosome membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). While APOL1 conditions PI4KB and APOL3 involvement in mitochondrion fission and mitophagy, APOL3-VAMP8 interaction promotes fusion between mitophagosomal and endolysosomal membranes. We propose that APOL3 controls mitochondrial membrane dynamics through interactions with the fission factor PI4KB and the fusion factor VAMP8.

Heme-deficient metabolism and impaired cellular differentiation as an evolutionary trade-off for human infectivity in Trypanosoma brucei gambiense.

Resistance to African trypanosomes in humans relies in part on the high affinity targeting of a trypanosome lytic factor 1 (TLF1) to a trypanosome haptoglobin-hemoglobin receptor (HpHbR). While TLF1 avoidance by the inactivation of HpHbR contributes to Trypanosoma brucei gambiense human infectivity, the evolutionary trade-off of this adaptation is unknown, as the physiological function of the receptor remains to be elucidated. Here we show that uptake of hemoglobin via HpHbR constitutes the sole heme import pathway in the trypanosome bloodstream stage. T. b. gambiense strains carrying the inactivating mutation in HpHbR, as well as genetically engineered T. b. brucei HpHbR knock-out lines show only trace levels of intracellular heme and lack hemoprotein-based enzymatic activities, thereby providing an uncommon example of aerobic parasitic proliferation in the absence of heme. We further show that HpHbR facilitates the developmental progression from proliferating long slender forms to cell cycle-arrested stumpy forms in T. b. brucei. Accordingly, T. b. gambiense was found to be poorly competent for slender-to-stumpy differentiation unless a functional HpHbR receptor derived from T. b. brucei was genetically restored. Altogether, we identify heme-deficient metabolism and disrupted cellular differentiation as two distinct HpHbR-dependent evolutionary trade-offs for T. b. gambiense human infectivity.

C-terminal mutants of apolipoprotein L-I efficiently kill both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense.

Apolipoprotein L-I (apoL1) is a human-specific serum protein that kills Trypanosoma brucei through ionic pore formation in endosomal membranes of the parasite. The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. In the case of T. b. rhodesiense, resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1. We undertook a mutational and deletional analysis of the C-terminal helix of apoL1 to investigate the linkage between interaction with SRA and lytic potential for different T. brucei subspecies. We confirm that the C-terminal helix is the SRA-interacting domain. Although in E. coli this domain was dispensable for ionic pore-forming activity, its interaction with SRA resulted in inhibition of this activity. Different mutations affecting the C-terminal helix reduced the interaction of apoL1 with SRA. However, mutants in the L370-L392 leucine zipper also lost in vitro trypanolytic activity. Truncating and/or mutating the C-terminal sequence of human apoL1 like that of apoL1-like sequences of Papio anubis resulted in both loss of interaction with SRA and acquired ability to efficiently kill human serum-resistant T. b. rhodesiense parasites, in vitro as well as in transgenic mice. These findings demonstrate that SRA interaction with the C-terminal helix of apoL1 inhibits its pore-forming activity and determines resistance of T. b. rhodesiense to human serum. In addition, they provide a possible explanation for the ability of Papio serum to kill T. b. rhodesiense, and offer a perspective to generate transgenic cattle resistant to both T. b. brucei and T. b. rhodesiense.

Diversification of function by different isoforms of conventionally shared RNA polymerase subunits.

Eukaryotic nuclei contain three classes of multisubunit DNA-directed RNA polymerase. At the core of each complex is a set of 12 highly conserved subunits of which five--RPB5, RPB6, RPB8, RPB10, and RPB12--are thought to be common to all three polymerase classes. Here, we show that four distantly related eukaryotic lineages (the higher plant and three protistan) have independently expanded their repertoire of RPB5 and RPB6 subunits. Using the protozoan parasite Trypanosoma brucei as a model organism, we demonstrate that these distinct RPB5 and RPB6 subunits localize to discrete subnuclear compartments and form part of different polymerase complexes. We further show that RNA interference-mediated depletion of these discrete subunits abolishes class-specific transcription and hence demonstrates complex specialization and diversification of function by conventionally shared subunit groups.

Multiparametric Atomic Force Microscopy Identifies Multiple Structural and Physical Heterogeneities on the Surface of Trypanosoma brucei.

A unique feature of the African trypanosome Trypanosoma brucei is the presence of an outer layer made of densely packed variable surface glycoproteins (VSGs), which enables the cells to survive in the bloodstream. Although the VSG coat is critical to pathogenesis, how exactly the glycoproteins are organized at the nanoscale is poorly understood. Here, we show that multiparametric atomic force microscopy is a powerful nanoimaging tool for the structural and mechanical characterization of trypanosomes, in a label-free manner and in buffer solution. Directly correlated images of the structure and elasticity of trypanosomes enable us to identify multiple nanoscale mechanical heterogeneities on the cell surface. On a ∼250 nm scale, regions of softer (Young's modulus ∼50 kPa) and stiffer (∼100 kPa) elasticity alternate, revealing variations of the VSG coat and underlying structures. Our nanoimaging experiments show that the T. brucei cell surface is more heterogeneous than previously anticipated and offer promising prospects for the design of trypanocidal drugs targeting cell surface components.

The Trypanosoma Brucei KIFC1 Kinesin Ensures the Fast Antibody Clearance Required for Parasite Infectivity.

Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin.

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins.

The intracellular parasite Toxoplasma gondii develops within a nonfusogenic vacuole containing a network of elongated nanotubules that form connections with the vacuolar membrane. Parasite secretory proteins discharged from dense granules (known as GRA proteins) decorate this intravacuolar network after invasion. Herein, we show using specific gene knockout mutants, that the unique nanotubule conformation of the network is induced by the parasite secretory protein GRA2 and further stabilized by GRA6. The vacuolar compartment generated by GRA2 knockout parasites was dramatically disorganized, and the normally tubular network was replaced by small aggregated material. The defect observed in Deltagra2 parasites was evident from the initial stages of network formation when a prominent cluster of multilamellar vesicles forms at a posterior invagination of the parasite. The secretory protein GRA6 failed to localize properly to this posterior organizing center in Deltagra2 cells, indicating that this early conformation is essential to proper assembly of the network. Construction of a Deltagra6 mutant also led to an altered mature network characterized by small vesicles instead of elongated nanotubules; however, the initial formation of the posterior organizing center was normal. Complementation of the Deltagra2 knockout with mutated forms of GRA2 showed that the integrity of both amphipathic alpha-helices of the protein is required for correct formation of the network. The induction of nanotubues by the parasite protein GRA2 may be a conserved feature of amphipathic alpha-helical regions, which have also been implicated in the organization of Golgi nanotubules and endocytic vesicles in mammalian cells.

APOLs with low pH dependence can kill all African trypanosomes.

The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.

Characterization of RNA polymerase II subunits of Trypanosoma brucei.

The Trypanosoma brucei homolog of the RNA polymerase II (RNA Pol II) subunit RPB9 was cloned and characterized. Contrary to what occurs in Saccharomyces cerevisiae, in T. brucei this protein was found to be essential since the knock down of its expression by RNAi led to lethality in both bloodstream and procyclic forms of the parasite. As expected, TbRPB9 knock down specifically inhibited transcription by RNA Pol II, but not by RNA Pol I and III. TbRPB9 was used as bait to isolate the RNA Pol II core complex by tandem affinity purification. Nine subunits homologous to the other eukaryotic RNA Pol II, namely RPB1, RPB2, RPB3, RPB4, RPB5, RPB6, RPB7, RPB8 and RPB11, were identified in the purified complex. Interestingly, the RPB5 homolog associated with RNA Pol II was different from the one previously found in RNA Pol I. Analysis of the genome database revealed the presence of genes for all purified subunits plus RPB10. As in the case of TbRPB5, two genes coding for different isoforms of TbRPB6 were identified, suggesting the existence of polymerase-specific isoforms for both TbRPB5 and TbRPB6.

Apolipoprotein L1 Variant Associated with Increased Susceptibility to Trypanosome Infection.

UNLABELLED: African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE: Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis.

Characterization of subunits of the RNA polymerase I complex in Trypanosoma brucei.

The Trypanosoma brucei homologue of the RNA polymerase I (RNA Pol I) subunit Rpa12p of Saccharomyces cerevisiae was cloned and characterized. This protein did not appear to be essential for growth in either bloodstream or procyclic forms of the parasite. Trypanosomes expressing a C-terminal tagged version of TbRPA12 were generated in order to purify RNA Pol I from both developmental stages. Tandem affinity purification (TAP) revealed a number of proteins associating with TbRPA12, some of which appeared to be stage-specific. Mass spectrometry allowed the identification of four subunits in addition to TbRPA12, namely TbRPA1, TbRPA2, TbRPC40 and one isoform of TbRPB5 (Tb1RPB5), as well as an unknown 30kDa protein and histones H2A and H3. Whereas these studies demonstrated that TbRPA1 was phosphorylated, no evidence for phosphorylation of TbRPA2 was found.