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1.  Correction: A Novel Rho-Like Protein TbRHP Is Involved in Spindle Formation and Mitosis in Trypanosomes 
PLoS ONE  2011;6(11):10.1371/annotation/b4b38a99-0b4c-483a-ad21-4dd721974b97.
doi:10.1371/annotation/b4b38a99-0b4c-483a-ad21-4dd721974b97
PMCID: PMC3228669
2.  Life and times: synthesis, trafficking, and evolution of VSG 
Trends in Parasitology  2014;30(5):251-258.
Highlights
•Variant surface glycoprotein (VSG) is a paradigm for antigenic variation.•VSG provides a mechanism for immune evasion.•Rapid transport, turnover, and endocytosis contribute to VSG function.•VSG has provided, and continues to offer, important insights into trypanosome biology.
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. In this review, we discuss the biosynthesis, trafficking, and turnover of VSG, emphasising those unusual mechanisms that act to maintain coat integrity and to protect against immunological attack. We also highlight new findings that suggest the presence of unique or highly divergent proteins that may offer therapeutic opportunities, as well as considering aspects of VSG biology that remain to be fully explored.
doi:10.1016/j.pt.2014.03.004
PMCID: PMC4007029  PMID: 24731931
Trypanosoma brucei; protein sorting; exocytosis; endocytosis; protein turnover; variant surface glycoprotein; evolution
3.  The Ancient Small GTPase Rab21 Functions in Intermediate Endocytic Steps in Trypanosomes 
Eukaryotic Cell  2014;13(2):304-319.
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. brucei Rab21 is a member of an ancient, pan-eukaryotic, endocytic Rab clade that is retained by trypanosomes. We show that T. brucei Rab21 (TbRab21) localizes to endosomes, partially colocalizing with TbRab5A, TbRab28, and TbVps23, the latter two being present at late endosomes. TbRab21 expression is essential for cellular proliferation, and its suppression results in a partial block in traffic to the lysosome. RNA interference (RNAi)-mediated knockdown of TbRab21 had no effect on TbRab5A expression or location but did result in decreased in trans expression of ESCRT (trypanosome endosomal sorting complex required for transport) components and TbRab28, while knockdown of ESCRT subunit TbVps23 resulted in decreased TbRab21 expression. These data suggest that TbRab21 acts downstream of TbRab5A and functions in intimate connection with the trypanosome ESCRT system.
doi:10.1128/EC.00269-13
PMCID: PMC3910970  PMID: 24376004
4.  Production of 2-Aminophenoxazin-3-one by Staphylococcus aureus Causes False-Positive Results in β-Galactosidase Assays 
Journal of Clinical Microbiology  2012;50(11):3780-3782.
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. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative β-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) should be used for testing staphylococci.
doi:10.1128/JCM.02299-12
PMCID: PMC3486264  PMID: 22972831
5.  High-throughput decoding of anti-trypanosomal drug efficacy and resistance 
Nature  2012;482(7384):232-236.
Summary
The concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.
doi:10.1038/nature10771
PMCID: PMC3303116  PMID: 22278056
DFMO; eflornithine; ISG75; nifurtimox; RNAi
6.  Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes ▿ † ‡ 
Eukaryotic Cell  2011;10(7):916-931.
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. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic and trans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulating trans-membrane domain surface proteins in trypanosomes.
doi:10.1128/EC.05012-11
PMCID: PMC3147413  PMID: 21571921
7.  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. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.
doi:10.1016/j.bbrc.2010.05.045
PMCID: PMC2908739  PMID: 20471365
GTPases; Prenylation; Statin; Trafficking; Rab proteins
8.  Evidence that low endocytic activity is not directly responsible for human serum resistance in the insect form of African trypanosomes 
BMC Research Notes  2010;3:63.
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. Here we asked if endocytic down-regulation in the procyclic form was associated with resistance to innate trypanolytic immune factors in the blood meal or tsetse fly midgut.
Findings
Using a well-characterized procyclic parasite with augmented endocytic flux mediated via TbRab5A overexpression, we found that insect stage parasites were able to grow both in the presence of trypanosome lytic factor (TLF) provided in human serum, and also in tsetse flies. Additionally, by placing blood stage parasites in restricted glucose medium, we observed that enlargement of the flagellar pocket, a key morphology associated with defective endocytosis, manifests in parallel with loss of cellular ATP levels.
Conclusions
These observations suggest that a high rate of endocytosis per se is insufficient to render insect form parasites sensitive to TLF or tsetse-derived trypanocidal factors. However, the data do suggest that endocytosis is energetically burdensome, as endocytic activity is rapidly compromised on energy depletion in bloodstream stages. Hence an important aspect of endocytic modulation in the nutrient-poor tsetse midgut is likely energetic conservation.
doi:10.1186/1756-0500-3-63
PMCID: PMC2848055  PMID: 20205710
9.  The Trypanosome Rab-Related Proteins RabX1 and RabX2 Play No Role in IntraCellular Trafficking but May Be Involved in Fly Infectivity 
PLoS ONE  2009;4(9):e7217.
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. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.
Methodology/Principal Findings
Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.
Conclusions/Significance
RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.
doi:10.1371/journal.pone.0007217
PMCID: PMC2748683  PMID: 19787065

Results 1-9 (9)