Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection.

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.

Apolipoproteins L1-6 share key cation channel-regulating residues but have different membrane insertion and ion conductance properties.

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.

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation.

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.

Cation channel conductance and pH gating of the innate immunity factor APOL1 are governed by pore-lining residues within the C-terminal domain.

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.

Evolution of the primate trypanolytic factor APOL1.

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.

Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

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.

Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes.

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 factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

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.

Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections.

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.

All You Ever Wanted to Know About APOL1 and TLFs and Did Not Dare Ask.

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.

Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity.

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.

Inducible Germline IgMs Bridge Trypanosome Lytic Factor Assembly and Parasite Recognition.

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.

African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat.

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-Å-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.

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense.

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite's resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.

Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes.

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.

Characterization of primate trypanosome lytic factors.

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.

Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test.

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.