The Trypanosoma brucei-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells.

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

The protozoan parasite Trypanosoma brucei is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of T. brucei is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.

Genetic and immunological basis of human African trypanosomiasis.

Human African trypanosomiasis, or sleeping sickness, results from infection by two subspecies of the protozoan flagellate parasite Trypanosoma brucei, termed Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, prevalent in western and eastern Africa respectively. These subspecies escape the trypanolytic potential of human serum, which efficiently acts against the prototype species Trypanosoma brucei brucei, responsible for the Nagana disease in cattle. We review the various strategies and components used by trypanosomes to counteract the immune defences of their host, highlighting the adaptive genomic evolution that occurred in both parasite and host to take the lead in this battle. The main parasite surface antigen, named Variant Surface Glycoprotein or VSG, appears to play a key role in different processes involved in the dialogue with the host.

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

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

Queuine Micronutrient Deficiency Promotes Warburg Metabolism and Reversal of the Mitochondrial ATP Synthase in Hela Cells.

Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F1FO-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.

Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages.

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

Metabolic reprogramming during the Trypanosoma brucei life cycle.

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion.

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1.

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion.

Detailed Biological Profiling of a Photoactivated and Apoptosis Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells.

Ruthenium polypyridyl complexes show great promise as new photodynamic therapy (PDT) agents. However, a lack of detailed understanding of their mode of action in cells poses a challenge to their development. We have designed a new Ru(II) PDT candidate that efficiently enters cells by incorporation of the lipophilic aromatic pdppz ([2,3-h]dipyrido[3,2-a:2',3'-c]phenazine) ligand and exhibits photoactivity through incorporation of 1,4,5,8-tetraazaphenanthrene ancillary ligands. Its photoreactivity toward biomolecules was studied in vitro, where light activation caused DNA cleavage. Cellular internalization occurred via an energy dependent mechanism. Confocal and transmission electron microscopy revealed that the complex localizes in various organelles, including the mitochondria. The complex is nontoxic in the dark, with cellular clearance within 96 h; however, upon visible light activation it induces caspase-dependent and reactive-oxygen-species-dependent apoptosis, with low micromolar IC50 values. This investigation greatly increases our understanding of such systems in cellulo, aiding development and realization of their application in cancer therapy.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

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

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

Preparation, anti-trypanosomal activity and localisation of a series of dipeptide-based vinyl sulfones.

An improved, Weinreb amide-based, synthesis of anti-trypanosomal lysine-containing vinyl sulfones is described incorporating, as a feature, diversity at the ε-lysine amino group. Members of this family demonstrated moderate to good efficacy as anti-trypanosomal agents and a fluorescent dansyl (19) derivative was used to investigate subcellular localisation of the compound class.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

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

Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: design, synthesis, biological and computational evaluation.

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei . These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1', and P2 positions. The most potent compound, 29 (EC50 = 70 nM, T. b. brucei whole cell assay), displayed minimal toxicity (>785 times selectivity) when assayed for cytotoxicity against the human promyelocytic leukemia (HL-60) cell line. Cells treated with compound 29, as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in TbCatB/rhodesain and trypanocidal activity in vitro.

Inhibition of late-stage autophagy synergistically enhances pyrrolo-1,5-benzoxazepine-6-induced apoptotic cell death in human colon cancer cells.

The pyrrolo-1,5-benzoxazepines (PBOXs) are a novel group of selective apoptotic agents displaying promising therapeutic potential in both ex vivo chemotherapy-refractory patient samples and in vivo murine carcinoma models. In this report, we present novel data concerning the induction of autophagy by the PBOXs in adenocarcinoma-derived colon cancer cells. Autophagy is a lysosome-dependent degradative pathway recently associated with chemotherapy. However, whether autophagy facilitates cell survival in response to chemotherapy or contributes to chemotherapy-induced cell death is highly controversial. Autophagy was identified by enhanced expression of LC3B-II, an autophagosome marker, an increase in the formation of acridine orange-stained cells, indicative of increased vesicle formation and electron microscopic confirmation of autophagic structures. The vacuolar H+ ATPase inhibitor bafilomycin-A1 (BAF-A1) inhibited vesicle formation and enhanced the apoptotic potential of PBOX-6. These findings suggest a cytoprotective role of autophagy in these cells following prolonged exposure to PBOX-6. Furthermore, BAF-A1 and PBOX-6 interactions were determined to be synergistic and caspase-dependent. Potentiation of PBOX-6-induced apoptosis by BAF-A1 was associated with a decrease in the levels of the anti-apoptotic protein, Mcl-1. The data provide evidence that autophagy functions as a survival mechanism in colon cancer cells to PBOX-6-induced apoptosis and a rationale for the use of autophagy inhibitors to further enhance PBOX­6­induced apoptosis in colon cancer.

Identification and characterization of a stage specific membrane protein involved in flagellar attachment in Trypanosoma brucei.

Flagellar attachment is a visibly striking morphological feature of African trypanosomes but little is known about the requirements for attachment at a molecular level. This study characterizes a previously undescribed membrane protein, FLA3, which plays an essential role in flagellar attachment in Trypanosoma brucei. FLA3 is heavily N-glycosylated, locates to the flagellar attachment zone and appears to be a bloodstream stage specific protein. Ablation of the FLA3 mRNA rapidly led to flagellar detachment and a concomitant failure of cytokinesis in the long slender bloodstream form but had no effect on the procyclic form. Flagellar detachment was obvious shortly after induction of the dsRNA and the newly synthesized flagellum was often completely detached after it emerged from the flagellar pocket. Within 12 h most cells possessed detached flagella alongside the existing attached flagellum. These results suggest that proteins involved in attachment are not shared between the new and old attachment zones. In other respects the detached flagella appear normal, they beat rapidly although directional motion was lost, and they possess an apparently normal axoneme and paraflagellar rod structure. The flagellar attachment zone appeared to be disrupted when FLA3 was depleted. Thus, while flagellar attachment is a constitutive feature of the life cycle of trypanosomes, attachment requires stage specific elements at the protein level.

The vascular targeting agent Combretastatin-A4 directly induces autophagy in adenocarcinoma-derived colon cancer cells.

Recent clinical data demonstrated that the vascular targeting agent Combretastatin-A4 phosphate (CA-4P) prolonged survival of patients with advanced anaplastic thyroid cancer without any adverse side effects. However, as a single agent CA-4 failed to reduce tumour growth in the murine CT-26 adenocarcinoma colon cancer model. Furthermore, the molecular mechanism of the innate resistance of HT-29 human adenocarcinoma cells to CA-4 is largely unknown. In this report, we demonstrate for the first time that prolonged exposure to CA-4 and an azetidinone cis-restricted analogue, CA-432 (chemical name; 4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one) induced autophagy in adenocarcinoma-derived CT-26, Caco-2 and HT-29 cells but not in fibrosarcoma-derived HT-1080 cells. Autophagy is a fundamental self-catabolic process which can facilitate a prolonged cell survival in spite of adverse stress by generating energy via lysosomal degradation of cytoplasmic constituents. Autophagy was confirmed by acridine orange staining of vesicle formation, electron microscopy and increased expression of LC3-II. Combretastatin-induced autophagy was associated with a loss of mitochondrial membrane potential and elongation of the mitochondria. Furthermore, inhibition of autophagy by the vacuolar H(+)ATPase inhibitor Bafilomycin-A1 (BAF-A1) significantly enhanced CA-432 induced HT-29 cell death. Both CA-4 and its synthetic derivative, CA-432 induced the formation of large hyperdiploid cells in Caco-2 and CT-26 cells. The formation of these polyploid cells was significantly inhibited by autophagy inhibitor, BAF-A1. Results presented within demonstrate that autophagy is a novel response to combretastatin exposure and may be manipulated to enhance the therapeutic efficacy of this class of vascular targeting agents.

Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

Alanine aminotransferase of Trypanosoma brucei--a key role in proline metabolism in procyclic life forms.

African trypanosomes possess high levels of alanine aminotransferase (EC 2.6.1.2), although the function of their activity remains enigmatic, especially in slender bloodstream forms where the metabolism of ketoacids does not occur. Therefore, the gene for alanine aminotransferase enzyme in Trypanosoma brucei (TbAAT) was characterized and its function assessed using a combination of RNA interference and gene knockout approaches. Surprisingly, as much as 95% or more of the activity appears to be unnecessary for growth of either bloodstream or procyclic forms respiring on glucose. A combination of RNA interference and NMR spectroscopy revealed an important role for the activity in procyclic forms respiring on proline. Under these conditions, the major end product of proline metabolism is alanine, and a reduction in TbAAT activity led to a proportionate decrease in the amount of alanine excreted along with an increase in the doubling time of the cells. These results provide evidence of a role for alanine aminotransferase in the metabolism of proline in African trypanosomes by linking glutamate produced by the initial oxidative steps of the pathway with pyruvate produced by the final oxidative step of the pathway. This step appears to be essential when proline is the primary carbon source, which is likely to be the physiological situation in the tsetse fly vector.