Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate
            N
            -Acetyltransferase from Trypanosoma brucei

ABSTRACT A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N -acetyltransferase ( TbGNA1 ; EC 2.3.1.4) was cloned and expressed in Escherichia coli . The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly- N -acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.

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The GPI-Phospholipase C of Trypanosoma brucei Is Nonessential But Influences Parasitemia in Mice

The Journal of Cell Biology ◽

10.1083/jcb.139.1.103 ◽

1997 ◽

Vol 139 (1) ◽

pp. 103-114 ◽

Cited By ~ 66

Author(s):

Helena Webb ◽

Nicola Carnall ◽

Luc Vanhamme ◽

Sylvie Rolin ◽

Jakke Van Den Abbeele ◽

...

Keyword(s):

Phospholipase C ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Covalent Attachment ◽

Mammalian Host ◽

Surface Coat ◽

Null Mutant

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

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Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.737 ◽

1989 ◽

Vol 108 (2) ◽

pp. 737-746 ◽

Cited By ~ 179

Author(s):

I Roditi ◽

H Schwarz ◽

T W Pearson ◽

R P Beecroft ◽

M K Liu ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Tsetse Fly ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Mammalian Host ◽

Cylindrical Shape ◽

Surface Coat ◽

Insect Vector ◽

Molecular Arrangement

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

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Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase

Glycobiology ◽

10.1093/glycob/cwq115 ◽

2010 ◽

Vol 20 (12) ◽

pp. 1619-1630 ◽

Cited By ~ 22

Author(s):

K. Marino ◽

M. L. S. Guther ◽

A. K. Wernimont ◽

M. Amani ◽

R. Hui ◽

...

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Subcellular Localization ◽

Trypanosoma Brucei ◽

Biochemical Properties

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Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42769-x ◽

1992 ◽

Vol 267 (8) ◽

pp. 5324-5329

Author(s):

M.C. Field ◽

A.K. Menon ◽

G.A. Cross

Keyword(s):

Trypanosoma Brucei ◽

Surface Glycoprotein ◽

Gpi Anchor ◽

Developmental Variation ◽

In Vitro Biosynthesis

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Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates TargetingMycobacterium tuberculosisandMycobacterium abscessus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01696-17 ◽

2018 ◽

Vol 62 (5) ◽

Cited By ~ 6

Author(s):

Rashmi Gupta ◽

Carolina Rodrigues Felix ◽

Matthew P. Akerman ◽

Kate J. Akerman ◽

Cathryn A. Slabber ◽

...

Keyword(s):

Mycobacterium Abscessus ◽

Cross Resistance ◽

Human Pathogens ◽

Intrinsic Resistance ◽

Content Type ◽

Starting Point ◽

Scalable Synthesis ◽

High Level ◽

Novel Drug

ABSTRACTMycobacterium tuberculosisand the fast-growing speciesMycobacterium abscessusare two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistantM. tuberculosisstrains and the high level of intrinsic resistance ofM. abscessuscall for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), againstM. abscessusandM. tuberculosis. We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicatingin vitroconditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverseM. tuberculosisandM. abscessusclinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target theM. tuberculosisgyrase.In vitroenzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity againstM. tuberculosisandM. abscessusthat act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.

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In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

Eukaryotic Cell ◽

10.1128/ec.00116-06 ◽

2006 ◽

Vol 5 (8) ◽

pp. 1276-1286 ◽

Cited By ~ 15

Author(s):

Sara D. Faulkner ◽

Monika W. Oli ◽

Rudo Kieft ◽

Laura Cotlin ◽

Justin Widener ◽

...

Keyword(s):

High Density Lipoprotein ◽

Trypanosoma Brucei ◽

Density Lipoprotein ◽

High Density ◽

Surface Glycoprotein ◽

Trypanosoma Brucei Brucei ◽

African Trypanosomes ◽

Expression Site ◽

Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

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Escaping the immune system by DNA repair and recombination in African trypanosomes

Open Biology ◽

10.1098/rsob.190182 ◽

2019 ◽

Vol 9 (11) ◽

pp. 190182 ◽

Cited By ~ 3

Author(s):

Núria Sima ◽

Emilia Jane McLaughlin ◽

Sebastian Hutchinson ◽

Lucy Glover

Keyword(s):

Immune Response ◽

Dna Repair ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Surface Coat ◽

African Trypanosomes ◽

Active Expression ◽

Expression Site

African trypanosomes escape the mammalian immune response by antigenic variation—the periodic exchange of one surface coat protein, in Trypanosoma brucei the variant surface glycoprotein (VSG), for an immunologically distinct one. VSG transcription is monoallelic, with only one VSG being expressed at a time from a specialized locus, known as an expression site. VSG switching is a predominantly recombination-driven process that allows VSG sequences to be recombined into the active expression site either replacing the currently active VSG or generating a ‘new’ VSG by segmental gene conversion. In this review, we describe what is known about the factors that influence this process, focusing specifically on DNA repair and recombination.

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Synergistic Lethality of a Binary Inhibitor ofMycobacterium tuberculosisKasA

mBio ◽

10.1128/mbio.02101-17 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 14

Author(s):

Pradeep Kumar ◽

Glenn C. Capodagli ◽

Divya Awasthi ◽

Riju Shrestha ◽

Karishma Maharaja ◽

...

Keyword(s):

Crystal Structure ◽

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Substrate Binding ◽

Content Type ◽

X Ray ◽

Structure Activity Relationships ◽

Structure Activity ◽

Transcriptional Pattern

ABSTRACTWe report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) inMycobacterium tuberculosis. Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented thein vitroactivity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model ofM. tuberculosisinfection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCECell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors ofMycobacterium tuberculosisKasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

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A New NonpolarN-Hydroxy Imidazoline Lead Compound with Improved Activity in a Murine Model of Late-Stage Trypanosoma brucei brucei Infection Is Not Cross-Resistant with Diamidines

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03958-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 890-904 ◽

Cited By ~ 11

Author(s):

Carlos H. Ríos Martínez ◽

Florence Miller ◽

Kayathiri Ganeshamoorthy ◽

Fabienne Glacial ◽

Marcel Kaiser ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Late Stage ◽

Parent Compound ◽

Central Nervous System Infection ◽

Sleeping Sickness ◽

Intrinsic Activity ◽

Cross Resistance ◽

Content Type

ABSTRACTTreatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four newN-hydroxy and 12 newN-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluatedin vitroagainstTrypanosoma brucei rhodesienseandin vivoin murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters,in vitroBBB permeability, and microsomal stability also were determined. TheN-hydroxy imidazoline analogues were the most effectivein vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was foundin vitro, suggesting thatN-hydroxy imidazolines are metabolically stable and have intrinsic activity againstT. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d inT. bruceiwas independent of known drug transporters (i.e.,T. bruceiAT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, theN-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.

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