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The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein–associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated nonselective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted helix-loop-helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with interspecies chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that whereas neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain, tyrosine 351 and glutamate 355, that influence pH gating properties, as well as a single residue, aspartate 348, that determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C terminus as the pore-lining segment of this novel channel-forming protein.

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei., Author summary African trypanosomes are relevant pathogens and a cause of great morbidity and economic burden in endemic countries. Anemia during infection by African Trypanosomes is one of the most common pathologies associated with the disease, but the mechanisms leading to it are poorly understood. Here, we report evidence of autoimmunity, particularly autoantibodies against the lipid phosphatidylserine that are secreted by autoimmune/atypical B-cells, in Trypanosoma brucei infected mice and that are also found in Human African Trypanosomiasis patients and correlate with levels of anemia. These findings point to a relation between autoimmunity and anemia during infection with African Trypanosomes, possibly through the secretion of autoantibodies against phosphatidylserine.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania., Author summary Leishmaniasis, the disease caused by parasites of the genus Leishmania, can be divided into cutaneous, muco-cutaneous and visceral leishmaniasis depending on the parasite species and the clinical outcome of the disease. Of the three, cutaneous leishmaniasis is the most common form, which is usually characterized by a localized lesion due to the infection of immune cells, primarily dermal and lymph node-resident macrophages. The time between infection and lesion appearance ranges from weeks to years, while some individuals never develop lesions. The length of this subclinical stage of leishmaniasis depends on a variety of factors: parasite virulence, infectious dose, and the host immune response. Therefore, it remains crucial to develop our understanding of each component of the host-parasite interface and assess the role that each component plays in the clinical outcome. Here, we analyze the interaction between L. major, a cutaneous strain, and the host innate immune factor Trypanosome Lytic Factor (TLF), a sub-class of circulating High-Density Lipoprotein (HDL). TLF provides sterile immunity to most extracellular African Trypanosomes by osmotically lysing the parasites. Lysis is driven by the primate specific protein apolipoprotein L-1 (APOL1), a cation channel-forming protein that is activated by a series of pH-dependent conformational changes. APOL1 inserts into cellular membranes at acidic pH and forms a closed ion channel that subsequently opens when re-exposed to neutral pH, resulting in ion flux. Using transgenic mice producing primate TLF, we show that both human and baboon TLFs ameliorate cutaneous Leishmania major infection and that this reduction in parasite burden correlates with: 1. infectious dose of metacyclic promastigotes 2. the concentration of circulating TLF in plasma and 3. early recruitment of neutrophils at the site of infection. Our results show that the acidification step is essential for TLF-mediated lysis of axenic metacyclic promastigotes of Leishmania in vitro. The susceptibility of metacyclic promastigotes to TLF-mediated lysis is governed by the surface glycoconjugates of Leishmania. We find that surface glycoconjugate-deficient Leishmania are resistant to TLF-mediated killing. Based on these data, we conclude that the shedding of surface glycoconjugates while transitioning from metacyclic promastigotes to amastigotes, results in parasite resistance to TLF-mediated lysis. Whether TLF is effective at killing metacyclic promastigotes of other experimentally tractable Leishmania sp., such as L. infantum and L. donovani, which have different surface glycoconjugate structures is yet to be tested. Our data raise the possibility that TLF may have lytic activity against a broader range of pathogens such as bacteria, viruses, fungi and parasites with surface glycoconjugates that transit through intracellular acidic compartments.

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel's pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.

The human apolipoprotein L gene family encodes the apolipoprotein L1–6 (APOL1–6) proteins, which are effectors of the innate immune response to viruses, bacteria and protozoan parasites. Due to a high degree of similarity between APOL proteins, it is often assumed that they have similar functions to APOL1, which forms cation channels in planar lipid bilayers and membranes resulting in cytolytic activity. However, the channel properties of the remaining APOL proteins have not been reported. Here, we used transient overexpression and a planar lipid bilayer system to study the function of APOL proteins. By measuring lactate dehydrogenase release, we found that APOL1, APOL3, and APOL6 were cytolytic, whereas APOL2, APOL4, and APOL5 were not. Cells expressing APOL1 or APOL3, but not APOL6, developed a distinctive swollen morphology. In planar lipid bilayers, recombinant APOL1 and APOL2 required an acidic environment for the insertion of each protein into the membrane bilayer to form an ion conductance channel. In contrast, recombinant APOL3, APOL4, and APOL5 readily inserted into bilayers to form ion conductance at neutral pH, but required a positive voltage on the side of insertion. Despite these differences in membrane insertion properties, the ion conductances formed by APOL1-4 were similarly pH-dependent and cation-selective, consistent with conservation of the pore-lining region in each protein. Thus, despite structural conservation, the APOL proteins are functionally different. We propose that these proteins interact with different membranes and under different voltage and pH conditions within a cell to effect innate immunity to different microbial pathogens.

Recently evolved alleles of Apolipoprotein L-1 (APOL1) provide increased protection against African trypanosome parasites while also significantly increasing the risk of developing kidney disease in humans. APOL1 protects against trypanosome infections by forming ion channels within the parasite, causing lysis. While the correlation to kidney disease is robust, there is little consensus concerning the underlying disease mechanism. We show in human cells that the APOL1 renal risk variants have a population of active channels at the plasma membrane, which results in an influx of both Na+and Ca2+. We propose a model wherein APOL1 channel activity is the upstream event causing cell death, and that the activate-state, plasma membrane-localized channel represents the ideal drug target to combat APOL1-mediated kidney disease.

Interest in trypanosome lytic factors (TLFs) and apolipoprotein L1, the ion channel-forming protein component of TLFs, has increased tenfold since 2010. This is due to the association of African variants of APOL1 with kidney disease such that interest has reached circles beyond parasitology. We have extensive experience purifying and working with these proteins and protein complexes. Herein we describe our detailed purification protocols to aid the new burgeoning field by providing an opportunity for consistency in reagents used across laboratories. We emphasize that it is imperative to maintain APOL1 protein intact (~42 kDa) to analyze the active ion channel-forming component/protein.

Interest in trypanosome lytic factors (TLFs) and apolipoprotein L1, the ion channel-forming protein component of TLFs, has increased tenfold since 2010. This is due to the association of African variants of APOL1 with kidney disease such that interest has reached circles beyond parasitology. We have extensive experience purifying and working with these proteins and protein complexes. Herein we describe our detailed purification protocols to aid the new burgeoning field by providing an opportunity for consistency in reagents used across laboratories. We emphasize that it is imperative to maintain APOL1 protein intact (~42 kDa) to analyze the active ion channel-forming component/protein.

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and ‘switching’ to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-A-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact. Trypanosoma brucei parasites switch their variant surface glycoprotein (VSG) coats to escape immune recognition. The crystal structure of VSG3 reveals that additional modifications, such as O-glycosylation, further expand antigenic variation and potentiate immune evasion.

Trypanosomiasis is a devastating neglected tropical disease that affects millions of livestock and thousands of humans each year. Only two subspecies of Trypanosoma brucei can cause disease in humans, while immunity to most trypanosomes is mediated in primates by HDL-associated circulating complexes called trypanosome lytic factors (TLFs) 1 and 2. Both TLFs are similar in composition, though TLF2 uniquely contains non-covalently associated IgM antibodies, the role and origin of which remain unclear. Here, we show that these TLF2-IgMs interact with TLF components and trypanosome surface proteins, including variant surface glycoprotein (VSG), likely facilitating complex biogenesis and TLF uptake into parasites. We also demonstrate that TLF2-IgMs are natural antibodies that, while present at basal concentrations in healthy individuals, are elicited by trypanosome infection in both murine models and human sleeping sickness patients. These data suggest that poly- and selfreactive natural antibodies such as TLF2-IgMs may play a role in antimicrobial immunity.

Trypanosomiasis is a devastating neglected tropical disease affecting livestock and humans. Humans are susceptible to two Trypanosoma brucei subspecies but protected from other trypanosomes by circulating high-density lipoprotein (HDL) complexes called trypanosome lytic factors (TLFs) 1 and 2. TLFs contain apolipoprotein L-1 contributing to lysis and haptoglobin-related protein (HPR), which can function as a ligand for a parasite receptor. TLF2 also uniquely contains non-covalently associated immunoglobin M (IgM) antibodies, the role and origin of which remain unclear. Here, we show that these TLF2-associated IgMs interact with both HPR and alternate trypanosome surface proteins, including variant surface glycoprotein, likely facilitating complex biogenesis and TLF uptake into parasites. TLF2-IgMs are germline antibodies that, while present at basal concentrations in healthy individuals, are elicited by trypanosome infection in both murine models and human sleeping sickness patients. These data suggest that poly- and self-reactive germline antibodies such as TLF2-associated IgMs play a role in antimicrobial immunity.

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na+by K+also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca2+and Cl−fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin–hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.

Several species of African trypanosomes cause fatal disease in livestock, but most cannot infect humans due to innate trypanosome lytic factors (TLFs). Human TLFs are pore forming high-density lipoprotein (HDL) particles that contain apolipoprotein L-I (apoL-I) the trypanolytic component, and haptoglobin-related protein (Hpr), which binds free hemoglobin (Hb) in blood and facilitates the uptake of TLF via a trypanosome haptoglobin-hemoglobin receptor. The human-infective Trypanosoma brucei rhodesiense escapes lysis by TLF by expression of serum resistance-associated (SRA) protein, which binds and neutralizes apoL-I. Unlike humans, baboons are not susceptible to infection by T. b. rhodesiense due to previously unidentified serum factors. Here, we show that baboons have a TLF complex that contains orthologs of Hpr and apoL-I and that full-length baboon apoL-I confers trypanolytic activity to mice and when expressed together with baboon Hpr and human apoA-I, provides protection against both animal infective and the human-infective T. brucei rhodesiense in vivo. We further define two critical lysines near the C terminus of baboon apoL-1 that are necessary and sufficient to prevent binding to SRA and thereby confer resistance to human-infective trypanosomes. These findings form the basis for the creation of TLF transgenic livestock that would be resistant to animal and human-infective trypanosomes, which would result in the reduction of disease and the zoonotic transmission of human infective trypanosomes.

Trypanosome lytic factors (TLFs) are high-density lipoproteins and components of primate innate immunity. TLFs are characterized by their ability to kill extracellular protozoon parasites of the genus Trypanosoma. Two subspecies of Trypanosoma brucei have evolved resistance to TLFs and can consequently infect humans, resulting in the disease African sleeping sickness. The unique protein components of TLFs are a hemoglobin-binding protein, haptoglobin-related protein and a pore-forming protein, apoL-I. The recent advances in our understanding of the roles that these proteins play in the mechanism of TLF-mediated lysis are highlighted in this article. In light of recent data, which demonstrate that TLFs can ameliorate infection by the intracellular pathogen Leishmania, we also discuss the broader function of TLFs as components of innate immunity.

Humans are one of the few species that resist infection by Trypanosoma brucei brucei because the parasites are killed by lytic factors found in human serum. Trypanosome lytic factors (TLFs) are protein/lipid complexes that contain apolipoprotein A-I (apoA-I), and are therefore a class of high density lipoproteins (HDLs). Haptoglobin-related protein (Hpr) is a unique protein component of TLFs, and its expression has only been demonstrated in humans. Trypanolytic activity has only been found in the sera of five primates: humans, gorillas, mandrills, baboons and sooty mangabeys. We describe here previously unidentified components of highly purified human TLF1: apolipoprotein L-I (apoL-I), human cathelicidin antimicrobial peptide 18 (hCAP18) and glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). However, we found that hCAP18 and GPI-PLD, along with apoA-I, are common components of both lytic and non-lytic primate HDLs. In contrast, Hpr, which has been previously implicated as the main lytic component of TLF1, was a unique component of all trypanolytic primate HDLs. Furthermore, a polyclonal antiserum to Hpr neutralized the lytic activity from humans and baboons. ApoL-I, a candidate lytic component of human serum, was not immunologically or genetically detectable in two primate species with lytic activity. Polyclonal antiserum to apoL-I also did not neutralize TLF activity in a total human HDL preparation. These findings suggest that apoL-I is not essential in all primate TLFs, and apoL-I alone is not sufficient for optimal trypanosome lytic activity in human TLF.

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.

This review focuses on the epidemiology of human African trypanosomiasis: why it occurs in humans, the current methods of surveillance, and the drugs available to treat it. Emphasis is placed on the identification of human-infective trypanosomes by the blood incubation infectivity test. This test distinguishes between trypanosomes that are non-infective for humans and those that are potentially infective. Currently the test requires incubation of parasites with human serum before injection into mice; any surviving parasites are considered human-infective. The factors in serum that kill all non-human-infective parasites are known as trypanosome lytic factors. The paper details the biochemistry of these factors and recommends standardization of the test based on current knowledge. This test can be used to screen animals with trypanosomiasis, in order to evaluate their role during endemic and epidemic human African trypanosomiasis.

ApolipoproteinL1 (APOL1) protects humans and some primates against several African trypanosomes. APOL1 genetic variants strongly associated with kidney disease in African Americans have additional trypanolytic activity against Trypanosoma brucei rhodesiense, the cause of acute African sleeping sickness. We combined genetic, physiological, and biochemical studies to explore coevolution between the APOL1 gene and trypanosomes. We analyzed the APOL1 sequence in modern and archaic humans and baboons along with geographic distribution in present day Africa to understand how the kidney risk variants evolved. Then, we tested Old World monkey, human, and engineered APOL1 variants for their ability to kill human infective trypanosomes in vivo to identify the molecular mechanism whereby human trypanolytic APOL1 variants evade T. brucei rhodesiense virulence factor serum resistance-associated protein (SRA). For one APOL1 kidney risk variant, a two-residue deletion of amino acids 388 and 389 causes a shift in a single lysine residue that mimics the Old World monkey sequence, which augments trypanolytic activity by preventing SRA binding. A second human APOL1 kidney risk allele, with an amino acid substitution that also restores sequence alignment with Old World monkeys, protected against T. brucei rhodesiense due in part to reduced SRA binding. Both APOL1 risk variants induced tissue injury in murine livers, the site of transgenic gene expression. Our study shows that both genetic variants of human APOL1 that protect against T. brucei rhodesiense have recapitulated molecular signatures found in Old World monkeys and raises the possibility that APOL1 variants have broader innate immune activity that extends beyond trypanosomes.

African trypanosomes are the causative agents of sleeping sickness in humans and of Nagana in cattle. The infectivity of African trypanosome species for humans appears to be defined by their susceptibility to two lytic factors in human serum; trypanosome lytic factor (TLF)1, a subclass of human high density lipoprotein (HDL) and TLF2, a high molecular weight protein complex. Available evidence indicates that following receptor mediated uptake, TLF is targeted to the lysosome where the low pH triggers a TLF-dependant peroxidase activity resulting in the formation of reactive oxygen radicals with consequent lipid peroxidation and destruction of the lysosomal membrane. Nearly all previous work on the mechanism of parasite lysis has been performed using TLF1. In this study, we directly test the hypothesis that TLF1 and TLF2 kill Trypanosoma brucei by a mechanism involving oxidative stress. We found no evidence for lipid peroxidation in trypanosomes exposed to high concentrations of trypanolytic HDL (impure TLF1), although lipid peroxidation was detected in parasites exposed to low concentrations of low molecular weight peroxides. Neither HDL, TLF1 nor TLF2 generated detectable levels of intracellular reactive oxygen intermediates. Various antioxidants also had no effect on TLF1 or TLF2-mediated lysis, although the antioxidants catalase and superoxide dismutase were effective at inhibiting peroxide generation and parasite lysis in control systems. Various metal chelating agents and protease inhibitors were also tested without effect. These data provide strong evidence against a peroxidative mechanism being involved in TLF-mediated lysis.

Natural resistance of humans to the cattle pathogen Trypanosoma brucei brucei has been attributed to the presence in human serum of nonimmune factors that lyse the parasite. Normal human serum contains two trypanosome lytic factors (TLFs). TLF1 is a 500-kDa lipoprotein, which is reported to contain apolipoprotein A-I (apoA-I), haptoglobin-related protein (Hpr), hemoglobin, paraoxonase, and apoA-II, whereas TLF2 is a larger, poorly characterized particle. We report here a new immunoaffinity-based purification procedure for TLF2 and TLF1, as well as further characterization of the components of each purified TLF. Immunoaffinitypurified TLF1 has a specific activity 10-fold higher than that of TLF1 purified by previously described methods. Moreover, we find that TLF1 is a lipoprotein particle that contains mainly apoA-I and Hpr, trace amounts of paraoxonase, apoA-II, and haptoglobin, but no detectable hemoglobin. Characterization of TLF2 reveals that it is a 1,000-kDa protein complex containing mainly immunoglobulin M, apoA-I, and Hpr but less than 1% detectable lipid. African trypanosomes are unicellular eukaryotic protozoans that infect both animals and humans. They are usually transmitted by the bite of the tsetse fly, and they live in the bloodstream of their mammalian hosts. The trypanosome continuously evades the immune system, systematically changing its surface coat by switching expression among a thousand distinct genes encoding the variant surface glycoprotein (6). Nevertheless, humans are resistant to the widespread Trypanosoma subspecies T. brucei brucei due to the presence of nonimmune serum factors that lyse the invading trypanosome (12). In contrast, two other subspecies, T. b. gambiense and T. b. rhodesiense, infect humans and cause sleeping sickness because they are resistant to lysis by human serum. A factor from human serum that lyses T. b. brucei was characterized in 1978 (3) as a subset of high-density lipoprotein (HDL) and is now known as trypanosome lytic factor 1 (TLF1). More recently, Smith et al. (39) identified Hpr (haptoglobin [Hp]-related protein) as a unique component of TLF1 that is not found in nontrypanolytic HDL. Hpr shares over 90% amino acid sequence identity with Hp, an abundant serum protein (0.2 to 2 mg/ml) that binds free hemoglobin (Hb) and facilitates its clearance via receptors in the liver (5). The physiological role and biological properties of Hpr are unknown. The current model explaining trypanolysis requires binding of TLF1 to a receptor in the trypanosome flagellar pocket, followed by endocytosis and subsequent delivery to lysosomes (10). The low lysosomal pH is hypothesized to activate an Hpr-dependent peroxidase activity that results in lipid peroxidation, lysis of the lysosomal membrane, and autodigestion of the parasite (25, 39). However, the physiological significance of TLF1 has been questioned due to the presence of a natural inhibitor within serum that may mask TLF1 activity in normal human serum (NHS). The inhibitor of TLF1 has been identi

Complement-dependent destruction of invading micro-organisms is a crucial first-line defense against infection, yet both African and American trypanosomes are able to resist attack by complement. African trypanosomes resist non-specific complement attack by virtue of a thick glycoprotein surface coat, and the host range of certain African trypanosomes is believed to be defined by their susceptibility to a subclass of human high density lipoprotein (HDL) and/or a high molecular weight protein complex present in human serum. In the first part of this review, Stephen Tomlinson and Jayne Raper look at the properties and mechanisms of action of these trypanolytic factors on African trypanosomes, and discuss briefly the possible mechanisms whereby these human pathogens resist lysis by human serum. The mechanisms that enable the American trypanosome Trypanosoma cruzi to resist complement attack are reviewed in the second part of this article.

Natural immunity of humans to the cattle pathogen Trypanosoma brucei brucei has been attributed to the presence in normal human serum (NHS) of lytic factors for the parasites. We and others have shown that NHS contains two trypanolytic factors (herein termed TLF1 and TLF2) that can be separated by gel filtration. TLF1 copurifies with a subclass of high density lipoprotein (HDL), whereas TLF2 has a much higher molecular weight and does not appear to be a lipoprotein. We find that the trypanolytic activity of purified TLF1 is totally inhibited by exogenous haptoglobin (Hp) at concentrations (0.1 mg/ml) lower than those present in NHS (0.2-2 mg/ml). In contrast, exogenous Hp (up to 2.5 mg/ml) has no effect on the lytic activity of either NHS or isolated TLF2. Hp-depleted sera from patients with intravascular hemolysis is severalfold more trypanolytic than NHS. These sera contain only TLF1, and their lytic activity is totally abolished upon the addition of Hp (0.1 mg/ml). When NHS containing different Hp allotypes is fractionated by gel filtration, TLF1 activity is either revealed or remains masked, depending on whether it coelutes with Hp. Masked TLF1 activity in the column fractions is revealed if Hp is removed by density gradient ultracentrifugation. We conclude that endogenous Hp inhibits TLF1 activity, and that TLF2 is the main trypanolytic factor in NHS.

In view of the importance of the low-density lipoprotein (LDL)-receptor in Trypanosoma brucei, we have examined whether other bloodstream trypanosomes of medical and veterinary importance (T.b. rhodesiense, T. equiperdum, T. vivax, T. congolense), but also related parasites developing in mammalian (Leishmania donovani) and non-mammalian hosts (Crithidia luciliae and Phytomonas sp. isolated from Euphorbia), would possess an LDL-receptor of their own. (1) All these parasites specifically accumulate human 125I-LDL with a relatively 2.5-fold higher rate for bloodstream trypanosomes. (2) A mixture of monoclonal antibodies raised against T.b. brucei LDL-receptor inhibit binding of LDL to all species but with different efficiency. (3) A single glycoprotein of similar M(r) (gp145) is isolated by LDL-affinity chromatography from all the above species, as well as from both human serum-resistant and sensitive strain of T.b. rhodesiense, and from the bodonid member of the Kinetoplastida Trypanoplasma borelli. (4) Several control experiments including 35S-metabolic labeling of procyclic T.b. brucei and of C. luciliae followed by LDL-affinity chromatography or immunoprecipitation demonstrate that gp145 is indeed synthesised by the parasites and is not a contaminant of the experimental system. (5) In immunoblots and ELISA, these gp145 cross-react with the polyclonal and monoclonal antibodies raised against the LDL-receptor of T.b. brucei, the highest degree of cross-reactivity being found among the members of the Trypanozoon subgroup. (6) Finally, immunisation of mice with the purified LDL-receptor from one strain of T.b. brucei is not sufficient to confer durable protection against another strain of this parasite.

Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable. Published version

The variant surface glycoprotein of African trypanosomes has a glycosyl phosphatidylinositol (GPI) anchor that is unusual in that its fatty acids are exclusively myristate. We showed previously that the myristate is added to a free GPI in a fatty acid remodeling reaction involving deacylation and reacylation, forming glycolipid A, the anchor precursor. We now demonstrate that trypanosomes have a second pathway for GPI anchor myristoylation distinct from the fatty acid remodeling pathway, which we call "myristate exchange." This reaction involves exchange of myristate into both the sn-1 and sn-2 positions of glycolipid A, which already contain myristate. Myristoyl-CoA, the probable myristate donor in the exchange reaction, has an apparent Km of about 6 nM. We have now identified a lyso-GPI, named theta', which has myristate as its sole fatty acid; the kinetics of formation and utilization of theta' are consistent with it being an intermediate in exchange. Myristate exchange and fatty acid remodeling appear to occur in different subcellular compartments, and the two reactions have different sensitivities to inhibitors. The myristate exchange reaction may be a proofreading system to ensure that the fatty acids on variant surface glycoproteins are exclusively myristate.

The glycosylphosphatidylinositol membrane anchor of variant surface glycoprotein of the African trypanosome Trypanosoma brucei contains several mannosyl residues for which dolichol phosphoryl mannose is supposed to be the precursor; this itself is probably synthesised by a dolichol-dependent mannosyltransferase. We have characterised and localised a mannosyltransferase activity of T. brucei which transfers mannose from GDP-[14C]mannose to exogenously added dolichyl phosphate. The enzyme was saturable for both its substrates and had a Km of 7.8 microM and 3.3 microM, respectively, for dolichyl phosphate and GDP-mannose. Mannosyltransferase was labile at 37 degrees C in the presence of Triton X-100, but its activity remained constant for at least 60 min at temperatures between 10-15 degrees C. The enzyme was inhibited by amphomycin and this inhibition was potentiated by the presence of 10 mM CaCl2. After subcellular fractionation of cell homogenates by differential centrifugation, mannosyltransferase was recovered mainly in the microsomal fraction and its distribution was very similar to that of RNA, a marker for the rough endoplasmic reticulum. After isopycnic centrifugation in a linear sucrose gradient the distribution of mannosyltransferase also resembled that of RNA. Both constituents exhibited a shift towards lower densities after pre-treatment of microsomal membranes with inorganic pyrophosphate, while other membrane markers such as acid phosphatase and nucleoside diphosphatase did not. It is concluded that the formation of dolichol phosphoryl mannose from GDP-mannose and dolichyl phosphate in T. brucei occurs mainly in the rough endoplasmic reticulum.

Background The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites. Methodology Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed. Conclusions Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2., Author Summary The sub-species of Trypanosoma brucei are important pathogens of humans and animals in sub-Saharan Africa. T. b. brucei is not able to infect humans due to sensitivity to trypanosome lytic factors (TLF) containing the lytic protein apolipoprotein L1 (APOL1). T. b. gambiense is the most prevalent human infective sub-species, although there are two distinct sub-groups of T. b. gambiense that differ genetically and in terms of their human serum resistance phenotype stability. How they resist lysis by TLF is unknown. Previous data indicates that some group 1 T. b. gambiense parasites are able to avoid lysis due in part to reduced expression and activity of the gene encoding a TLF receptor (HpHbR). The authors showed that despite group 1 T. b gambiense displaying avoidance of some TLF particles, this sub-species group is resistant to the lytic protein of TLF, APOL1. This suggests that avoidance is not the complete story in this sub-species, as previously theorised. Group 2 T. b. gambiense is also innately resistant to APOL1 and there is no evidence of avoidance of TLF in resistant parasites. Group 2 T. b. gambiense variably displays the human serum resistance phenotype that does not appear to correlate with expression of HbHpR.

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system., Author Summary Innate immunity (present from birth) is the first line of defense against microorganisms and provides an initial barrier against disease. Here we show that a minor sub-fraction of human high-density lipoprotein (the good cholesterol), known as Trypanosome Lytic Factor (TLF), not only kills the parasite Trypanosoma brucei, but is also a more broadly acting antimicrobial component of the innate immune system in humans. As TLF is activated under acidic conditions, we evaluated the activity of TLF against the intracellular parasite Leishmania, which infects and grows within acidic compartments of macrophages, cells in our blood that normally destroy invading microorganisms. Here we show that TLF acts directly on Leishmania parasites, causing them to swell, thereby decreasing their infectivity. Furthermore, microscopy of macrophages infected with Leishmania reveal that TLF is taken up and delivered to the same compartment as Leishmania, concomitant with a reduction in the intracellular parasite number. Finally, we made mice that expressed the genes for human TLF; these mice reduced the pathogen burden and thereby controlled the Leishmania infection better than unmodified mice. In contrast, TLF mice were not protected from infection by Trypanosoma cruzi, a related parasite, which transiently passes through acidic compartments within cells.

Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals.

Glycosyl phosphatidylinositols (GPIs) serve as membrane anchors for many plasma membrane proteins in all eukaryotic cells. African trypanosomes provide an ideal system for investigating GPIs, because these parasites produce extremely large quantities to anchor their major surface protein, the variant surface glycoprotein. Through radiolabeling methods and use of a cell-free system, the major features of the trypanosome GPI biosynthetic pathway have been elucidated. It is likely that these techniques, successful in the study of trypanosome GPIs, will be applicable to other eukaryotic cells.

Trypanosome lytic factor 1 (TLF1) is a subclass of human high-density lipoprotein that kills some African trypanosomes thereby protecting humans from infection. We have shown that TLF1 is a 500 kDa HDL complex composed of lipids and at least seven different proteins. Here we present evidence outlining a new paradigm for the mechanism of lysis; TLF1 forms cation-selective pores in membranes. We show that the replacement of external Na+ (23 Da) with the larger tetramethylammonium+, choline+ and tetraethylammonium+ ions (74 Da, 104 Da and 130 Da) ameliorates the osmotically driven swelling and lysis of trypanosomes by TLF1. Confirmation of cation pore-formation was obtained using small unilamellar vesicles incubated with TLF1; these showed the predicted change in membrane potential expected from an influx of sodium ions. Using planar lipid bilayer model membranes made from trypanosome lipids, which allow the detection of single channels, we found that TLF1 forms discrete ion-conducting channels (17 pS) that are selective for potassium ions over chloride ions. We propose that the initial influx of extracellular Na+ down its concentration gradient promotes the passive entry of Cl- through preexisting Cl- channels. The net influx of both Na+ and Cl- create an osmotic imbalance that leads to passive water diffusion. This loss of osmoregulation results in cytoplasmic vacuolization, cell swelling and ultimately trypanosome lysis.

Pathogens and their hosts engage in perpetual molecular arms races. In one such evolutionary stand-off, the protagonists are trypanosome parasites and a human immune complex based on a high-density lipoprotein. See Letter p.430 Humans are normally protected against trypanosomal pathogens by the serum antiparasitic molecule apolipoprotein LI (APOL1). But two Trypanosoma brucei subspecies — rhodesiense and gambiense — can resist APOL1 and it is these that cause almost all human sleeping sickness cases. This paper describes the mechanism by which T. b. gambiense resists APOL1. Three complementary processes are involved: reduced APOL1 uptake, increased APOL1 degradation and APOL1 target membrane modification by a specific resistance factor. This resistance can be bypassed under some conditions, opening up new possibilities for intervention against the parasite.

A novel trypanosome lytic factor (TLF) has been characterized that protects humans from infection by Trypanosoma brucei brucei. The mechanism of trypanolysis is unknown; contrary to one hypothesis, TLF does not kill trypanosomes by generating oxygen radicals. However, these trypanosomes become human-infective when they express a serum-resistance-associated gene.

The survival of Trypanosoma cruzi, the causative agent of Chagas' disease, depends vitally on proteins and glycoconjugates that mediate the parasite/host interaction. Since most of these molecules are attached to the membrane by glycosylphosphatidylinositol (GPI), alternative means of chemotherapeutic intervention might emerge from GPI biosynthesis studies. The structure of the major 1G7 antigen GPI has been fully characterized by us (Guther, M. L. S., Cardoso de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J. (1992) J. Biol. Chem. 267, 6820-6828; Heise, N., Cardoso de Almeida, M. L., and Ferguson, M. A. J. (1995) Mel. Biochem. Parasitol. 70, 71-84), and based on its properties we now report the complete precursor glycolipids predicted to be transferred to the nascent protein. Migrating closely to Trypanosoma brucei glycolipid A on TLC, such species, named glycolipids A-like 1 and A-like 2, were labeled with tritiated palmitic acid, myo-inositol, glucosamine, and mannose, but surprisingly only the less polar glycolipid A-like 1 incorporated ethanolamine. The predicted products following nitrous acid deamination and digestion with phospholipases A(2), C, and D confirmed their GPI nature. Evidence that they may represent the anchor transferred to the 1G7 antigen came from the following analyses: (i) alpha-mannosidase treatments indicated that only one mannose was amenable to removal; (ii) their lipid moiety was identified as sn-l-alkyl-a-acylglycerol due to their sensitivity to phospholipase A(2) (PLA(2)), mild base and by direct high performance TLC analysis of the corresponding benzoylated diradylglycerol components; and (iii) both glycolipids incorporated H-3-fatty acid only in the sn-2- and not in the sn-l-alkyl position as previously found in the GPI of the mature 1G7 antigen. Based on the differential [H-3]ethanolamine incorporation pattern and the recent report that an aminoethylphosphonic acid (AEP) replaces ethanolamine phosphate (EtNH(2)-PO4) in the GPI in epimastigote sialoglycoproteins (Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendonca-Previato, L. (1995) J. Biol. Chem. 270, 7241-7250) it is proposed that glycolipid A-like 2 contains AEP and A-like 1 EtNH(2)-PO4. In the in vitro cell-free system both glycolipids were synthesized simultaneously and do not seem to bear a precursor/product relationship. Among the various components synthesized in vitro a glycolipid C-like corresponding to a form of glycolipid A-like 1 acylated on the inositol was also characterized. Phenylmethylsulfonyl fluoride, an inhibitor known to block the addition of ethanolamine phosphate in T. brucei but not in mammalian cells, also inhibits the synthesis of glycolipids A like and C-like in T. cruzi, indicating that the putative trypanosome EtNH(2)-PO4/AEP transferase(s) might represent a potential target for chemotherapy. UNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,BR-04023062 SAO PAULO,SP,BRAZIL JOHNS HOPKINS UNIV,SCH MED,DEPT BIOL CHEM,BALTIMORE,MD 21205 UNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,BR-04023062 SAO PAULO,SP,BRAZIL Web of Science

The natural immunity of humans to the cattle pathogen Trypanosoma brucei brucei, but not to the morphologically indistinguishable human pathogens T. brucei gambiense and T. brucei rhodesiense, is due to the selective killing of the parasite by normal human serum. The factor in human serum that mediates lysis of T. brucei brucei has long been attributed to a minor subclass of high density lipoprotein (HDL). Evidence indicates that the trypanolytic activity of isolated human HDL is due to peroxidase activity of an associated haptoglobin-related protein-hemoglobin complex. However, recent data suggest that the trypanolytic activity of HDL may be completely inhibited in whole human serum, and that trypanolytic activity of norman human serum is due to a second, less well-defined factor of high molecular weight. Current research aimed at understanding the mechanisms of cytotoxicity and the affected metabolic pathways may open new approaches for the development of specific drugs and vaccines against trypanosomiasis.

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phospatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuringiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma brucei, the protozoan parasite responsible for African sleeping sickness, evades the host immune response through the process of antigenic variation. The variant antigen, known as the variant surface glycoprotein (VSG), is anchored to the cell surface by a glycosyl phosphatidylinositol (GPI) structure that contains myristate (n-tetradecanoate) as its only fatty acid component. The utilization of heteroatom-containing analogs of myristate was studied both in a cell-free system and in vivo. Results indicated that the specificity of fatty acid incorporation depends on chain length rather than on hydrophobicity. One analog, 10-(propoxy)decanoic acid, was highly toxic to trypanosomes in culture although it is nontoxic to mammalian cells.