Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce ...membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia.
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•Trypanosoma brucei produce extracellular vesicles (EVs) from membrane nanotubes•EVs transfer the serum resistance-associated protein (SRA) to trypanosomes•EV transfer of SRA coveys resistance to human innate immunity•EVs fusion with RBCs transfers lipids and proteins and causes anemia
African trypanosomes, pathogens responsible for human sleeping sickness, exchange virulence factors through extracellular vesicles formed via membrane nanotubes. These factors facilitate resistance to host innate immune responses and cause anemia.
Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds ...to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.
African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the ...GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG
extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life
of ∼26 h) but dropped significantly in the absence of GPI-PLC (
of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis.
African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the "life cycle" of this critical virulence factor.
The editing of trypanosome mitochondrial mRNAs produces transcripts necessary for mitochondrial functions including electron transport and oxidative phosphorylation. Precursor-mRNAs are often ...extensively edited by specific uridine insertion or deletion that is directed by small guide RNAs (gRNAs). Recently, it has been shown that cytochrome c oxidase subunit III (COXIII) mRNAs can be alternatively edited to encode a novel mitochondrial membrane protein composed of a unique hydrophilic N-terminal sequence of unknown function and the C-terminal hydrophobic segment of COXIII. To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes. Using this data, we show that alternative editing of COXIII, ATPase subunit 6 (A6), and NADH dehydrogenase subunits 7, 8 and 9 (ND7, 8, 9) mRNAs can produce novel open reading frames (ORFs). Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified. These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.
NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, ...we present a cell perturbome workflow that compares NEU-4438’s molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.
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•Cell perturbome proteomics identified polypeptides destabilized after drug addition•Changes in cellular proteomes induced by drugs were used to develop hypotheses for MOA•Experimental tests verified hypotheses formulated for NEU-4438 and acoziborole•Cell perturbomics-based workflow for the prediction of MOA is applicable to all cell types
Microbiology; Parasitology; Omics; Proteomics
Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing ...haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.
To review existing methods and illustrate the use of a new, simple method for identifying inaccurate reports of dietary energy intake (rEI).
Comparison of rEI with energy requirements estimated by ...using total energy expenditure predicted (pTEE) from age, weight, height and sex using a previously published equation. Propagation of error calculations was performed and cut-offs for excluding rEI at plus or minus two standard deviations (+/-2 SD) and +/-1 SD for the agreement between rEI and pTEE were established.
Dietary survey in a US national cohort: the Continuing Survey of Food Intakes by Individuals (CSFII), 1994-96.
Men and non-pregnant, non-lactating women aged 21-45 years in the CSFII who provided two multiple-pass 24-hour recalls, height and weight (n = 3755).
Average rEI was 77% of pTEE in men, and 64% of pTEE in women. Calculated cut-offs were rEI <40% or >160% of pTEE (+/-2 SD) and <70% or >130% of pTEE (+/-1 SD), respectively. Use of only the +/-1 SD cut-offs, not the +/-2 SD cut-offs, resulted in a relationship between rEI and body weight similar to what was expected (based on an independently calculated relationship between rEI and measured TEE). Exclusion of rEI outside either the +/-2 SD (11% of subjects) or +/-1 SD (57% of subjects) cut-offs did not affect mean reported macronutrient intakes, but did markedly affect relationships between dietary composition and body mass index.
When examining relationships between diet and health, use of +/-1 SD cut-offs may be preferable to +/-2 SD cut-offs for excluding inaccurate dietary reports.
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