Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei

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 (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against T. b. brucei infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosoma brucei rhodesiense, which is morphologically and physiologically indistinguishable from T. b. brucei, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected T. b. brucei with an epitope-tagged SRA gene. Transfected T. b. brucei expressed SRA mRNA at levels comparable to those in T. b. rhodesiense and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.     

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.     

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.     

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.     

Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes

Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.     

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.     

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.     

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.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

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.     

Trypanosoma brucei: immunogenicity of the variant surface coat glycoprotein of virulent and avirulent subspecies

Comparative analyses were made to define the immunogenic role in mice of the variant surface coat glycoprotein (VSG) of African trypanosomes. Less than 10 micrograms of the glycoprotein fixed to trypanosomes or covalently linked to sheep erythrocytes were 100 times more immunogenic than soluble VSG. Therefore, although VSG is present on the parasites and in the blood of infected hosts, the cell-bound form most likely elicits immunity. Intravenous administration of soluble or cell-bound VSG was a better route of immunization than the subcutaneous route. Therefore, although parasites grow at the site of infection, in tissue spaces, and in the blood, control of blood parasitemia is best developed if the antigen is introduced to the vascular bed. Full protection against homologous challenge occurred by 4 days and was maintained through 30 days. Trypanosome-agglutinating antibody titers could be measured at 3 days, peaked at 5 days, and remained high through 14 days after immunization. Therefore, mice immunized with an optimal dosage of VSG, 2 days before challenge, should have had ample time to elicit a protective response. Most of these mice, however, developed patent infections, and one-third died during the first peak of parasitemia at about the same time as untreated control mice. This indicates that active infection inhibits the early phases of induction of immunity. Mice, suboptimally immunized against and challenged with an avirulent isolate of Trypanosoma brucei gambiense, survived at higher rates than mice immunized and challenged with a virulent clone of T. b. rhodesiense. Cell-fixed and soluble VSG from both parasites elicited similar agglutinating-antibody titers, indicating that the two trypanosomes were equally antigenic. Results from neutralization tests, however, revealed that, per unit of immune mouse serum, 400 times more T. b. gambiense became noninfective than T. b. rhodesiense. Apparently, virulence is related to relative sensitivity of the trypanosomes to immunological assault.     

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.     

Trypanosoma brucei sspp.: cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.     

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.     

African trypanosome interactions with an in vitro model of the human blood-brain barrier

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.