The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway

The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine‐5′‐monophosphate (GMP) formation: conversion from xanthosine‐5′‐monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche.     

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based pro-drug activation.     

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.     

ATG24 Represses Autophagy and Differentiation and Is Essential for Homeostasy of the Flagellar Pocket in Trypanosoma brucei

We have previously identified homologs for nearly half of the approximately 30 known yeast Atg’s in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite’s glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role.     

Thinking outside the blood: Perspectives on tissue-resident Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). In the mammalian host, the parasite lives entirely extracellularly, in both the blood and interstitial spaces in tissues. Although most T. brucei research has focused on the biology of blood- and central nervous system (CNS)-resident parasites, a number of recent studies have highlighted parasite reservoirs in the dermis and adipose tissue, leading to a renewed interest in tissue-resident parasite populations. In light of this renewed interest, work describing tissue-resident parasites can serve as a valuable resource to inform future investigations of tissue-resident T. brucei. Here, we review this body of literature, which describes infections in humans, natural hosts, and experimental animal models, providing a wealth of information on the distribution and biology of extravascular parasites, the corresponding immune response in each tissue, and resulting host pathology. We discuss the implications of these studies and future questions in the study of extravascular T. brucei.     

Mycobacterium tuberculosis IMPDH in Complexes with Substrates,Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.     

Orally Active and Selective Tubulin Inhibitors as Anti-Trypanosome Agents

Objectives

There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis.

Methods

We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host model. Growth rates of both trypanosomes and mammalian cells were determined as a means to screen compounds that selectively inhibit the proliferation of parasites. Furthermore, we examined the cell cycle profile of the parasite and compared tubulin polymerization dynamics before and after the treatment using identified compounds. Last, in vivo anti-parasite activities of these compounds were determined in T. brucei-infected mice.

Results

Three compounds were selected that are 100 fold more effective against the growth of T. brucei cells than mammalian cells. These compounds caused cell cycle progression defects in T. brucei cells. Western analyses indicated that these compounds decreased tubulin polymerization in T. brucei cells. The in vivo investigation revealed that these compounds, when admitted orally, inhibited T. brucei cell proliferation in mouse blood. However, they were not potent enough to clear up the infection completely.

Conclusions

These compounds are promising lead compounds as orally active agents for drug development of anti-trypanosome agents. A more detail structure activity relationship (SAR) was summarized that will be used to guide future lead optimization to improve the selectivity and potency of the current compounds.     

Crystal structure of human guanosine monophosphate reductase 2 (GMPR2) in complex with GMP

Guanosine monophosphate reductase (GMPR) catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in re-utilization of free intracellular bases and purine nucleosides. Here, we report the first crystal structure of human GMP reductase 2 (hGMPR2) in complex with GMP at 3.0 A resolution. The protein forms a tetramer composed of subunits adopting the ubiquitous (alpha/beta)8 barrel fold. Interestingly, the substrate GMP is bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this makes the conformation of the adjacent flexible binding region (residues 268-289) fixed, much like a door on a hinge. Structure comparison and sequence alignment analyses show that the conformation of the active site loop (residues 179-187) is similar to those of hGMPR1 and inosine monophosphate dehydrogenases (IMPDHs). We propose that Cys186 is the potential active site, and that the conformation of the loop (residues 129-133) suggests a preference for the coenzyme NADPH over NADH. This structure provides important information towards understanding the functions of members of the GMPR family.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes

Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 ? from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.     

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area,a historical sleeping sickness focus of Côte d’Ivoire

BackgroundThe existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s.MethodsFor the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations.ResultsNo HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively.DiscussionFree-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.     

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.     

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.     

Discovery of a quinoline-based phenyl sulfone derivative as an antitrypanosomal agent

A series of natural products-based phenyl sulfone derivative and their property-based analogues were investigated as potential growth inhibitors of Trypanosoma brucei. Trypanosoma brucei is a kinetoplastid protozoan parasite that causes trypanosomiasis. In this work, we found that nopol- and quinoline-based phenyl sulfone derivative were the most active and selective for T. brucei, and they were not reactive towards the active thiol of T. brucei’s cysteine protease rhodesain. A thiol reactive variant of the quinoline-based phenyl sulfone was subsequently investigated and found to be a moderate inhibitor of rhodesain. The quinoline-based compound that is not reactive towards rhodesain can serve a template for phenotypic-based lead discovery while its thiol-active congener can serve as template for structure-based investigation of new antitrypanosomal agents.     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.