Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite , occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism ( polypharmacology).

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles.

Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei.

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes.

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance.

NMR-based metabolomic urinalysis: a rapid screening test for urinary tract infection.

Background: Urinary tract infection (UTI) is one of the most common bacterial infections in humans; however, there is no accurate and fast quantitative test to detect UTI. Dipstick urinalysis is semi-quantitative with a limited diagnostic accuracy, while urine culture is accurate but takes time. We described a quantitative biochemical method for the diagnosis of bacteriuria using a single marker.

Life and times: synthesis, trafficking, and evolution of VSG.

Evasion of the acquired immune response in African trypanosomes is principally mediated by antigenic variation, the sequential expression of distinct variant surface glycoproteins (VSGs) at extremely high density on the cell surface. Sequence diversity between VSGs facilitates escape of a subpopulation of trypanosomes from antibody-mediated killing. Significant advances have increased understanding of the mechanisms underpinning synthesis and maintenance of the VSG coat.

The cell biology of the endocytic system from an evolutionary perspective.

Evolutionary cell biology can afford an interdisciplinary comparative view that gives insights into both the functioning of modern cells and the origins of cellular systems, including the endocytic organelles. Here, we explore several recent evolutionary cell biology studies, highlighting investigations into the origin and diversity of endocytic systems in eukaryotes. Beginning with a brief overview of the eukaryote tree of life, we show how understanding the endocytic machinery in a select, but diverse, array of organisms provides insights into endocytic system origins and predicts the likely configuration in the last eukaryotic common ancestor (LECA).

The ancient small GTPase Rab21 functions in intermediate endocytic steps in trypanosomes.

Endocytosis is an essential process in nearly all eukaryotic cells, including the African trypanosome Trypanosoma brucei. Endocytosis in these organisms is exclusively clathrin mediated, although several lineage-specific features indicate that precise mechanisms are distinct from those of higher eukaryotes. T.

Production of 2-aminophenoxazin-3-one by Staphylococcus aureus causes false-positive results in β-galactosidase assays.

Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of β-galactosidase activity. This is commonly tested using o-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent β-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively.

High-throughput decoding of antitrypanosomal drug efficacy and resistance.

The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown.

Organochlorine isotopic pattern-enhanced detection and quantification of triclosan and its metabolites in human serum by ultra-high-performance liquid chromatography/quadrupole time-of-flight/mass spectrometry.

Organochlorines possess special isotopic patterns that obey the chlorine rule. In the case of triclosan (TCS), which contains three chlorine atoms, the isotopic patterns are composed of seven obvious peaks with the calculated masses ranging from 286.9435 to 292.

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways.

Early endosomal cargo is typically targeted to either a degradative or recycling pathway. Despite established functions for the retromer and ESCRT complexes at late endosomes/multivesicular bodies, the mechanisms integrating and coordinating these functions remain largely unknown. Rab family GTPases are key membrane trafficking organizers and could contribute.

A novel Rho-like protein TbRHP is involved in spindle formation and mitosis in trypanosomes.

Background: In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.

Ubiquitylation and developmental regulation of invariant surface protein expression in trypanosomes.

The cell surface of Trypanosoma brucei is dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, but trans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines.

Non-invasive screening of HLA-DPA1 and HLA-DPB1 alleles for persistent hepatitis B virus infection: susceptibility for vertical transmission and toward a personalized approach for vaccination and treatment.

Background: Polymorphisms in the major histocompatibility complex (MHC) and non-MHC genes were recently reported to be associated with persistent hepatitis B virus (HBV) infection and host response to hepatitis B vaccine in Asian populations. We aimed to confirm the associations in Chinese population and develop a non-invasive screening method for the risk loci.

Methods: We genotyped 2 risk alleles on the MHC loci, HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277535), and 1 risk allele near a non-MHC gene, FOXP1 (rs6789153) using high-resolution melting curve analysis.

A novel statin-mediated "prenylation block-and-release" assay provides insight into the membrane targeting mechanisms of small GTPases.

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus.

Evidence that low endocytic activity is not directly responsible for human serum resistance in the insect form of African trypanosomes.

Background: In Trypanosoma brucei, the African trypanosome, endocytosis is developmentally regulated and substantially more active in all known mammalian infective stages. In both mammalian and insect stages endocytic activity is likely required for nutrient acquisition, but in bloodstream forms increased endocytosis is involved in recycling the variant surface glycoprotein and removing host immune factors from the surface. However, a rationale for low endocytic activity in insect stages has not been explored.

The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

Background: Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized.

Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes.

The surface of Trypanosoma brucei is dominated by glycosyl-phosphatidylinositol (GPI)-anchored proteins, and endocytosis is clathrin dependent. The vast majority of internalized GPI-anchored protein is efficiently recycled, while the processes by which transmembrane domain (TMD) proteins are internalized and sorted are unknown. We demonstrate that internalization of invariant surface glycoprotein (ISG)65, a trypanosome TMD protein, involves ubiquitylation and also requires clathrin.

Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage.

Lysosomal targeting of ubiquitylated endocytic cargo is mediated in part by the endosomal sorting complex required for transport (ESCRT) complexes, a system conserved between animals and fungi (Opisthokonta). Extensive comparative genomic analysis demonstrates that ESCRT factors are well conserved across the eukaryotic lineage and complexes I, II, III and III-associated are almost completely retained, indicating an early evolutionary origin. The conspicuous exception is ESCRT 0, which functions in recognition of ubiquitylated cargo, and is restricted to the Opisthokonta, suggesting that a distinct mechanism likely operates in the vast majority of eukaryotic organisms.

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation.

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs.

Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases.

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes.