African trypanosomes: intracellular trafficking of host defense molecules

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.     

Endosomal localization of the serum resistance-associated protein in African trypanosomes confers human infectivity

Trypanosoma brucei rhodesiense is the causative agent of human African sleeping sickness. While the closely related subspecies T. brucei brucei is highly susceptible to lysis by a subclass of human high-density lipoproteins (HDL) called trypanosome lytic factor (TLF), T. brucei rhodesiense is resistant and therefore able to establish acute and fatal infections in humans. This resistance is due to expression of the serum resistance-associated (SRA) gene, a member of the variant surface glycoprotein (VSG) gene family. Although much has been done to establish the role of SRA in human serum resistance, the specific molecular mechanism of SRA-mediated resistance remains a mystery. Thus, we report the trafficking and steady-state localization of SRA in order to provide more insight into the mechanism of SRA-mediated resistance. We show that SRA traffics to the flagellar pocket of bloodstream-form T. brucei organisms, where it localizes transiently before being endocytosed to its steady-state localization in endosomes, and we demonstrate that the critical point of colocalization between SRA and TLF occurs intracellularly.     

Hemoglobin is a co-factor of human trypanosome lytic factor

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.     

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense. T. b. rhodesiense expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT. A truncated apoL-I (Tr-apoL-I) has been engineered by deleting its SRA-interacting domain, which makes it lytic for T. b. rhodesiense. Here, we conjugated Tr-apoL-I with a single-domain antibody (nanobody) that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of trypanosomes to generate a new manmade type of immunotoxin with potential for trypanosomiasis therapy. Treatment with this engineered conjugate resulted in clear curative and alleviating effects on acute and chronic infections of mice with both NHS-resistant and NHS-sensitive trypanosomes.     

The trypanolytic factor of human serum

African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei - Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense - allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein - known as apolipoprotein L1 - that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum.     

Selective pressure can influence the resistance of Trypanosoma congolense to normal human serum

Resistance and sensitivity to normal human serum (NHS) of Trypanosoma congolense, a parasite believed to cause disease in animals only, were investigated in vivo as well as in vitro. Our results indicate that like Trypanosoma brucei, T. congolense can be grouped into three different phenotypes according to its resistance to NHS. Some strains are completely resistant to NHS, like Trypanosoma brucei gambiense and the resistant form of Trypanosoma brucei rhodesiense. Other strains show a very low degree of resistance comparable to the sensitive form of T. b. rhodesiense, and some are completely sensitive to NHS. Continuous passaging in mice in the presence or absence of NHS shows that the resistance and sensitivity of T. congolense can be reversed like in T. b. rhodesiense. Our data suggest that T. congolense might be able to infect man in regions where animals may serve as reservoirs for the infection.     

Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance

Trypanosoma brucei rhodesiense (Tbr) and T. b. gambiense (Tbg), causative agents of Human African Trypanosomiasis (sleeping sickness) in Africa, have evolved alternative mechanisms of resisting the activity of trypanosome lytic factors (TLFs), components of innate immunity in human serum that protect against infection by other African trypanosomes. In Tbr, lytic activity is suppressed by the Tbr-specific serum-resistance associated (SRA) protein. The mechanism in Tbg is less well understood but has been hypothesized to involve altered activity and expression of haptoglobin haemoglobin receptor (HpHbR). HpHbR has been shown to facilitate internalization of TLF-1 in T.b. brucei (Tbb), a member of the T. brucei species complex that is susceptible to human serum. By evaluating the genetic variability of HpHbR in a comprehensive geographical and taxonomic context, we show that a single substitution that replaces leucine with serine at position 210 is conserved in the most widespread form of Tbg (Tbg group 1) and not found in related taxa, which are either human serum susceptible (Tbb) or known to resist lysis via an alternative mechanism (Tbr and Tbg group 2). We hypothesize that this single substitution contributes to reduced uptake of TLF and thus may play a key role in conferring serum resistance to Tbg group 1. In contrast, similarity in HpHbR sequence among isolates of Tbg group 2 and Tbb/Tbr provides further evidence that human serum resistance in Tbg group 2 is likely independent of HpHbR function.     

The trypanosome lytic factor of human serum and the molecular basis of sleeping sickness

Trypanosoma brucei brucei infects a wide range of mammals but is unable to infect humans because this subspecies is lysed by normal human serum (NHS). The trypanosome lytic factor is associated with High Density Lipoproteins (HDLs). Several HDL-associated components have been proposed as candidate lytic factors, and contradictory hypotheses concerning the mechanism of lysis have been suggested. Elucidation of the process by which Trypanosoma brucei rhodesiense resists lysis and causes human sleeping sickness has indicated that the HDL-bound apolipoprotein L-I (apoL-I) could be the long-sought after lytic component of NHS. This research also allowed the identification of a specific diagnostic DNA probe for T. b. rhodesiense, and may lead to the development of novel anti-trypanosome strategies for use in the field.     

High-density lipoprotein-mediated lysis of trypanosomes

Nearly 90 years after the discovery that certain African trypanosornes were killed by normal human serum, we still do not understand how this innate trypanocidal factor works. Biochemical studies have provided us with an unlikely candidate: human high-density lipoprotein (HDL). This trypanosome lytic factor (TLF) from human serum is important since its activity restricts the host range of Trypanosoma brucei brucei, and the expression of this natural killing factor in cattle would represent a novel approach to the control of bovine tryponosomiasis. Here, Steve Hajduk, Kristin Hager and Jeffrey Esko discuss evidence for the TLF being a minor subclass of serum HDL and propose a mechanism for lysis based on the binding, endocytosis and lysosomal targeting of TLF.     

A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T. b. rhodesiense

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.     

Mutual self-defence: the trypanolytic factor story

Around 1900 Laveran and Mesnil discovered that African trypanosomes (prototype: Trypanosoma brucei brucei) do not survive in the blood of some primates and humans. The nature of the trypanolytic factor present in these sera has been the focus of a long-standing debate between different groups, but recent developments have allowed the proposal of a coherent model incorporating most seemingly divergent views and providing an interesting example of the complex interplay that continuously occurs between hosts and parasites. Possibly as an adaptation to their natural environment, great African apes and humans have acquired a new member of the apolipoprotein-L family, termed apoL1. This protein is the only one of the family to be secreted in the blood, where it binds to a subset of HDL particles that also contain another human-specific protein, haptoglobin-related protein or Hpr. T. b. brucei possesses a specific surface receptor for the haptoglobin-hemoglobin (Hp-Hb) complex, as a way to capture heme into hemoproteins that contribute to cell growth and resistance to the oxidative stress of the host. As this receptor does not discriminate between Hp and Hpr, Hpr-containing HDL particles of human serum are efficiently taken up by the parasite, leading to the simultaneous internalization of apoL1, Hpr and Hb-derived heme. Once in the lysosome, apoL1 is targeted to the lysosomal membrane, where its colicin-like anionic pore-forming activity triggers an influx of chloride ions from the cytoplasm. Osmotic effect linked to this ionic flux leads to uncontrolled swelling of the lysosome, ultimately causing the death of the parasite. Two T. brucei clones, termed Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, have managed to resist this lysis mechanism and, therefore, cause sleeping sickness in humans. While the mechanism of this resistance is still not known in the case of T. b. gambiense, the dominant factor responsible for resistance of T. b. rhodesiense has been identified. This protein, named SRA for Serum Resistance-Associated, is a truncated version of the major and variable surface antigen of the parasite, the Variant Surface Glycoprotein or VSG. Presumably due to its defective nature, SRA is not targeted to the plasma membrane as do regular VSGs, but ends up in the late endosomal compartment. In this location SRA is thought to neutralize apoL1 through coiled-coil interactions between alpha-helices. We discuss the potential of these discoveries in terms of fight against the disease.     

Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes

The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF- mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.     

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.     

Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.     

Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Haptoglobin-related protein mediates trypanosome lytic factor binding to trypanosomes

Trypanosome lytic factor (TLF-1) is an unusual high density lipoprotein (HDL) found in human serum that is toxic to Trypanosoma brucei brucei and may be critical in preventing human infections by this parasite. TLF-1 is composed of four major apolipoproteins: apolipoprotein AI, apolipoprotein AII, paraoxonase, and the primate-specific haptoglobin-related protein (Hpr). Hpr is greater than 90% homologous to haptoglobin (Hp), an abundant acute phase serum protein. Killing of trypanosomes by TLF-1 requires cell surface binding, endocytosis, and subsequent lysosomal targeting. Low temperature binding studies reveal two receptors for TLF-1: one that is high affinity/low capacity (K(d) approximately 12 nm, 350 receptors per cell) and another that binds with low affinity/high capacity (K(d) approximately 1 microm, 60,000 receptors per cell). The low affinity binding is competed by nonlytic human HDL and is likely to be apolipoprotein AI-mediated. Purified human Hpr and human Hp bind to trypanosomes, are internalized, and are targeted to the lysosome. Furthermore, Hpr shows competition for TLF-1 binding, and a monoclonal antibody against Hpr prevents both TLF-1 uptake and trypanosome killing. Based on these results, we propose that Hpr mediates the high affinity binding of TLF-1 to T. b. brucei through a haptoglobin-like receptor.     

Localization of serum resistance‐associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human‐infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance‐associated protein (SRA protein), protects against ApoL1‐mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA‐mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well‐characterized endosomal markers. By three‐dimensional deconvolved immunofluorescence single‐cell analysis, combined with double‐labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.     

The trypanolytic factor of human serum: many ways to enter the parasite,a single way to kill

Humans have developed a particular innate immunity system against African trypanosomes, and only two Trypanosoma brucei clones (T. b. gambiense, T. b. rhodesiense) can resist this defence and cause sleeping sickness. The main players of this immunity are the primate‐specific apolipoprotein L‐I (apoL1) and haptoglobin‐related protein (Hpr). These proteins are both associated with two serum complexes, a minor subfraction of HDLs and an IgM/apolipoprotein A‐I (apoA1) complex, respectively, termed trypanosome lytic factor (TLF) 1 and TLF2. Although the two complexes appear to lyse trypanosomes by the same mechanism, they enter the parasite through various modes of uptake. In case of TLF1 one uptake process was characterized. When released in the circulation, haemoglobin (Hb) binds to Hpr, hence to TLF1. In turn the TLF1–Hpr–Hb complex binds to the trypanosome haptoglobin (Hp)–Hb receptor, whose original function is to ensure haem uptake for optimal growth of the parasite. This binding triggers efficient uptake of TLF1 and subsequent trypanosome lysis. While Hpr is involved as TLF ligand, the lytic activity is due to apoL1, a Bcl‐2‐like pore‐forming protein. We discuss the in vivo relevance of this uptake pathway in the context of other potentially redundant delivery routes.