measuring-dynamic-glycosomal-ph-changes-in-living-trypanosoma-brucei

Measuring Dynamic Glycosomal pH Changes in Living <i>Trypanosoma brucei</i>

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

Brigham Young University

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form  When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na/H exchangers are required for glycosomal pH regulation.

pH regulation in glycosomes of procyclic form .

A new drug delivery strategy was investigated for the development of potent anti-parasitic compounds against Trypanosoma brucei, the causative agent of African sleeping sickness. Thus, potent in vitro hexokinase inhibitors were rendered cytotoxic by appending a tripeptide peroxosomal targeting sequence that facilitated delivery of the molecular cargo to the appropriate organelle in the parasite.

A targeted delivery strategy for the development of potent trypanocides.

Trypanosoma brucei, which causes human African typanosomiasis (HAT), derives cellular ATP from glucose metabolism while in the mammalian host. Targeting glucose uptake or regulation in the parasite has been proposed as a potential therapeutic strategy. However, few methods have been described to identify and characterize potential inhibitors of glucose uptake and regulation. Here, we report development of a screening assay that identifies small molecule disrupters of glucose levels in the cytosol and glycosomes. Using an endogenously expressed fluorescent protein glucose sensor expressed in cytosol or glycosomes, we monitored intracellular glucose depletion in the different cellular compartments. Two glucose level disrupters were identified, one of which only exhibited inhibition of glycosomal glucose and did not affect cytosolic levels. In addition to inhibiting glucose uptake with relatively high potency (EC = 700 nM), the compound also showed modest bloodstream form parasite killing activity. Expanding this assay will allow for identification of candidate compounds that disrupt parasite glucose metabolism.

FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei.

The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700μδ6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.

A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites.

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.

A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation.

We previously showed that a small molecule of natural origin, 1,2,3,4,6-penta- O-galloyl-β-d-glucopyranose (PGG), binds to capillary morphogenesis gene 2 (CMG2) with a submicromolar IC and also has antiangiogenic activity in vitro and in vivo. In this work, we synthetized derivatives of PGG with different sugar cores and phenolic substituents and tested these as angiogenesis inhibitors. In a high-throughput Förster resonant energy transfer-based binding assay, we found that one of our synthetic analogues (1,2,3,4,6-penta- O-galloyl-β-d-mannopyranose (PGM)), with mannose as central core and galloyl substituents, exhibit higher (up to 10×) affinity for CMG2 than the natural glucose prototype PGG and proved to be a potent angiogenesis inhibitor. These findings demonstrate that biochemical CMG2 binding in vitro predicts inhibition of endothelial cell migration ex vivo and antiangiogenic activity in vivo. The molecules herein described, and in particular PGM, might be useful prototypes for the development of novel agents for angiogenesis-dependent diseases, including blinding eye disease and cancer.

Galloyl Carbohydrates with Antiangiogenic Activity Mediated by Capillary Morphogenesis Gene 2 (CMG2) Protein Binding.

Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. CMG2 also plays a role in angiogenic processes. However, the molecular mechanism that mediates the observed CMG2-related angiogenic effects is not fully elucidated. Previous studies have reported that CMG2 binds type IV collagen (Col-IV), a vital component of the vascular basement membrane, as well as other ECM proteins. Here, we further characterize the interaction between CMG2 and individual peptides from Col-IV and explore the effects of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (noncollagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative peptide-binding epitope onto the Col-IV structure. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with a submicromolar affinity (peptide S16,  = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. CMG2 specifically mediates endocytic uptake of S16; both CMG2-/- endothelial cells and WT cells treated with PA show markedly reduced S16 uptake. Furthermore, S16 dramatically reduces directional endothelial cell migration with no impact on cell proliferation. These data demonstrate that this canstatin-derived peptide acts via CMG2 to elicit a marked effect on a critical process required for angiogenesis.

A Canstatin-Derived Peptide Provides Insight into the Role of Capillary Morphogenesis Gene 2 in Angiogenic Regulation and Matrix Uptake.

We report a non-cytotoxic resin compatible with and designed for use in custom high-resolution 3D printers that follow the design approach described in Gong et al., Lab Chip 17, 2899 (2017). The non-cytotoxic resin is based on a poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). Both NPS-PEGDA and avobenzone-PEGDA (A-PEGDA) resins were evaluated for cytotoxicity and cell adhesion. We show that NPS-PEGDA can be made effectively non-cytotoxic with a post-print 12-hour ethanol wash, and that A-PEGDA, as-printed, is effectively non-cytotoxic. 3D prints made with either resin do not support strong cell adhesion in their as-printed state; however, cell adhesion increases dramatically with a short plasma treatment. Using A-PEGDA, we demonstrate spheroid formation in ultra-low adhesion 3D printed wells, and cell migration from spheroids on plasma-treated adherent surfaces. Given that A-PEGDA can be 3D printed with high resolution, it has significant promise for a wide variety of cell-based applications using 3D printed microfluidic structures.

Biocompatible PEGDA Resin for 3D Printing.

CD4 T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4 T cell receptor (TCR) clinical studies, CD4 T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8 T cells directly kill tumor cells, most research has focused on the attributes of CD8 TCRs. Less is known about how TCR affinity and CD4 expression affect CD4 T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4 T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4 T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4 T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs.

Pathogenic fungi reside in the intestinal microbiota but rarely cause disease. Little is known about the interactions between fungi and the immune system that promote commensalism. Here we investigate the role of adaptive immunity in promoting mutual interactions between fungi and host. We find that potentially pathogenic Candida species induce and are targeted by intestinal immunoglobulin A (IgA) responses. Focused studies on Candida albicans reveal that the pathogenic hyphal morphotype, which is specialized for adhesion and invasion, is preferentially targeted and suppressed by intestinal IgA responses. IgA from mice and humans directly targets hyphal-enriched cell-surface adhesins. Although typically required for pathogenesis, C. albicans hyphae are less fit for gut colonization and we show that immune selection against hyphae improves the competitive fitness of C. albicans. C. albicans exacerbates intestinal colitis and we demonstrate that hyphae and an IgA-targeted adhesin exacerbate intestinal damage. Finally, using a clinically relevant vaccine to induce an adhesin-specific immune response protects mice from C. albicans-associated damage during colitis. Together, our findings show that adaptive immunity suppresses harmful fungal effectors, with benefits to both C. albicans and its host. Thus, IgA uniquely uncouples colonization from pathogenesis in commensal fungi to promote homeostasis.

Adaptive immunity induces mutualism between commensal eukaryotes.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

Traditional 3D printing based on Digital Light Processing Stereolithography (DLP-SL) is unnecessarily limiting as applied to microfluidic device fabrication, especially for high-resolution features. This limitation is due primarily to inherent tradeoffs between layer thickness, exposure time, material strength, and optical penetration that can be impossible to satisfy for microfluidic features. We introduce a generalized 3D printing process that significantly expands the accessible spatially distributed optical dose parameter space to enable the fabrication of much higher resolution 3D components without increasing the resolution of the 3D printer. Here we demonstrate component miniaturization in conjunction with a high degree of integration, including 15 μm × 15 μm valves and a 2.2 mm × 1.1 mm 10-stage 2-fold serial diluter. These results illustrate our approach's promise to enable highly functional and compact microfluidic devices for a wide variety of biomolecular applications.

Spatially and optically tailored 3D printing for highly miniaturized and integrated microfluidics.

Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis.

Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

Soluble ECM promotes organotypic formation in lung alveolar model.

A series of eight new ethyl ()-benzotriazolyl acrylates 6a-d and 7a-d have been synthesized by conventional heating and microwave irradiation from ethyl benzotriazolyl acetates 3 and 4 with the corresponding aromatic aldehydes. This work reports the synthetic approach and spectroscopic characterization (H, C-NMR, HRMS) of all the synthesized compounds. X-ray diffraction analyses were performed for molecules 6a, 7a and 7d. Photophysical properties of compounds were evaluated. Finally, compound 6a was tested in a human cell line and showed low to no cytotoxicity at relevant concentrations. Initial testing demonstrates its potential use as a fluid-phase fluorescent marker for live cell imaging.

Synthesis and characterization of ethyl benzotriazolyl acrylate-based D-π-A fluorophores for live cell-based imaging applications.

We demonstrate a method to effectively 3D print microfluidic devices with high-resolution features using a biocompatible resin based on avobenzone as the UV absorber. Our method relies on spectrally shaping the 3D printer source spectrum so that it is fully overlapped by avobenzone's absorption spectrum. Complete overlap is essential to effectively limit the optical penetration depth, which is required to achieve high out-of-plane resolution. We demonstrate the high resolution in practice by 3D printing 15 μm square pillars in a microfluidic chamber, where the pillars are separated by 7.7 μm and are printed with 5 μm layers. Furthermore, we show reliable membrane valves and pumps using the biocompatible resin. Valves are tested to 1,000,000 actuations with no observable degradation in performance. Finally, we create a concentration gradient generation (CG) component and utilize it in two device designs for cell chemotaxis studies. The first design relies on an external dual syringe pump to generate source and sink flows to supply the CG channel, while the second is a complete integrated device incorporating on-chip pumps, valves, and reservoirs. Both device types are seeded with adherent cells that are subjected to a chemoattractant CG, and both show clear evidence of chemotactic cellular migration. Moreover, the integrated device demonstrates cellular migration comparable to the external syringe pump device. This demonstration illustrates the effectiveness of our integrated chemotactic assay approach and high-resolution biocompatible resin 3D printing fabrication process. In addition, our 3D printing process has been tuned for rapid fabrication, as printing times for the two device designs are, respectively, 8 and 15 min.

Article	Year
pH regulation in glycosomes of procyclic form . The Journal of biological chemistry\| PubMed ID: 28348078	2017
A targeted delivery strategy for the development of potent trypanocides. Chemical communications (Cambridge, England)\| PubMed ID: 28726862	2017
FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei. ACS infectious diseases\| PubMed ID: 29741365	2018
A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites. PLoS neglected tropical diseases\| PubMed ID: 29851949	2018
A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation. Biochemistry\| PubMed ID: 30638014	2019
Galloyl Carbohydrates with Antiangiogenic Activity Mediated by Capillary Morphogenesis Gene 2 (CMG2) Protein Binding. Journal of medicinal chemistry\| PubMed ID: 30964669	2019
A Canstatin-Derived Peptide Provides Insight into the Role of Capillary Morphogenesis Gene 2 in Angiogenic Regulation and Matrix Uptake. ACS chemical biology\| PubMed ID: 32003961	2020
Biocompatible PEGDA Resin for 3D Printing. ACS applied bio materials\| PubMed ID: 32467881	2020
CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs. Frontiers in immunology\| PubMed ID: 33542711	2020
Adaptive immunity induces mutualism between commensal eukaryotes. Nature\| PubMed ID: 34262174	2021
TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth. Nature communications\| PubMed ID: 34504101	2021
Spatially and optically tailored 3D printing for highly miniaturized and integrated microfluidics. Nature communications\| PubMed ID: 34535656	2021
Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis. Angiogenesis\| PubMed ID: 35212873	2022
Soluble ECM promotes organotypic formation in lung alveolar model. Biomaterials\| PubMed ID: 35306229	2022
Synthesis and characterization of ethyl benzotriazolyl acrylate-based D-π-A fluorophores for live cell-based imaging applications. RSC advances\| PubMed ID: 35517661	2019
Biocompatible High-Resolution 3D-Printed Microfluidic Devices: Integrated Cell Chemotaxis Demonstration. Micromachines\| PubMed ID: 37630125	2023

Kenneth A. Christensen

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