We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.
Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics in the live cell setting.
Parasitic kinetoplastids, like most living organisms, rely on glucose as a fundamental component of central carbon metabolism. This group includes medically important organisms, such as the African trypanosome, Trypanosoma brucei; the American trypanosome, T. cruzi; and parasites of the genus Leishmania. Glucose metabolism is critical to parasite growth in the pathogenic lifecycle stages. For example, when deprived of glucose, the bloodstream form (BSF) of the African trypanosome dies rapidly. Notably, glycolysis serves as the sole source of ATP during this stage of infection1. Leishmania parasites are likewise dependent on glucose in the human host, with the amastigote lifecycle stage that resides in host macrophages reliant on this carbon source for growth2.
While these parasites have distinct lifestyles involving different insect vectors, they share many commonalities in how they respond to and consume glucose. For example, these parasites localize most glycolytic enzymes into modified peroxisomes called glycosomes. This kinetoplastid-specific organelle is related to mammalian peroxisomes based on conserved biosynthetic mechanisms and morphology3,4,5,6.
The compartmentalization of most of the glycolytic pathway enzymes into the glycosome offers parasite-specific means of regulation of the pathway. Using a chemical pH probe, we established that nutrient deprivation triggers a rapid acidification of procyclic form (PF) parasite glycosomes that results in altered glycolytic enzyme activity through exposure of an allosteric regulator binding site on the key glycolytic enzyme hexokinase7,8. In our previous work, the chemical probe required constant delivery for use, limiting its utility in other applications. Additionally, challenges maintaining the probe distribution in the glycosome in the BSF limited the utility of the approach for investigating glycosomal pH in that life stage.
In this study, we have used the fluorescent protein biosensor pHluorin2 to monitor glycosomal pH change in BSF T. brucei in response to environmental cues including glucose starvation9 (Figure 1). Results from this work suggest that BSF T. brucei acidifies glycosomes rapidly in response to starvation in a reversible fashion, similar to responses we have observed in PF parasites. We expect this biosensor will improve our understanding of glycolytic regulation in T. brucei and related parasites.
Using T. brucei brucei 90-13 BSF trypanosomes, a monomorphic parasite line, requires consideration of safety as they are considered Risk Group 2 organisms that should be handled in biosafety level 2 facilities.
1. Trypanosome culture and transfection
2. Immunofluorescence colocalization of pHlourin2-PTS1
3. Sample preparation for flow cytometry
4. Flow cytometry
NOTE: Prepare the experiment on a flow cytometer containing the following lasers: 405 nm (violet), 488 nm (blue), and 561 nm (yellow) or 638 nm (red). See Supplemental Table S1 for common names for channels discussed below.
5. Data analysis of flow cytometry results
NOTE: This data analysis workflow uses FlowJo software. If other flow cytometry analysis software is used, continue to follow the key steps described below, using software-appropriate tools. To visualize the plots and gating, see Supplemental Figure S3 and Supplemental Figure S4.
6. pH biosensor calibration
NOTE: To convert measured fluorescence ratios to pH units, calibrate pHL-expressing cells using nigericin and valinomycin. Nigericin is a K+/H+ antiporter, an ionophore that can equilibrate pH across membranes when there is sufficient K+ in the buffer15. Nigericin has been commonly used to calibrate pHluorin and other pH sensors16,17. As peroxisomally localized pHluorin was previously calibrated using 10 µM nigericin18, we chose to treat with that concentration. Valinomycin is a potassium ionophore and has been used (at 4 µM) to equilibrate pH across mitochondrial membranes19. We used 10 µM valinomycin to assist the pH equilibration activity of nigericin by ensuring K+ ions were equilibrated across the membranes. While we used a nigericin-valinomycin combination, nigericin may be sufficient to equilibrate organellar pH.
7. Glucose starvation and addback time-courses
8. Optimizing the assay for drug screening
Figure 1: Diagram of the method for scoring glycosomal pH in live BSF trypanosomes. (A) Depiction of cell lines expressing glycosomally located pHluorin2 sensor. The inclusion of a peroxisomal targeting sequence provides control over the localization. NOTE: We anticipate that elimination of the PTS-1 would lead to cytosolic localization, allowing future analysis of pH in that subcellular compartment. (B) Depiction of the sensor validation assay. Abbreviation: BSF = bloodstream form. Please click here to view a larger version of this figure.
NOTE: The Z-factor statistic is used to determine how suitable an assay is for HTS. Values between 0.5 and 1.0 generally mean the assay quality is acceptable for HTS.
pHLuorin2-PTS1 localization to glycosomes in BSF T. brucei
To assess the subcellular localization of the pHluorin2-PTS1, parasites were subjected to immunofluorescence assays. Signal from the transgene colocalized with anti-sera raised against a glycosome-resident protein, aldolase (TbAldolase) (Figure 2A). The average Pearson's correlation coefficient of colocalization between anti-TbAldolase and pHluorin2-PTS1 was 0.895, indicating that pHluorin2-PTS1 was primarily localized to glycosomes. With pHluorin2-PTS1 localized to the glycosome, we proceeded to investigate BF glycosome pH.
Figure 2: Localization of pHluorin2-PTS1 to glycosomes in BSF Trypanosoma brucei. (A) Colocalization of pHluorin2-PTS1 with the glycosomal-resident protein TbAldolase. BF 90-13 parasites were transfected with pLEWpHluorin2-PTS1 and expression was induced with Doxycycline (1 µg/mL). TbAldolase was localized using anti-TbAldolase sera, followed by incubation with goat anti-rabbit Alexa fluor 568. The average Pearson's correlation coefficient was 0.895 (30 cells). (B) Calibration of pHluorin2-PTS1 in BSF T. brucei. Scale bars = 4 µm. Abbreviation: BSF = bloodstream form. Please click here to view a larger version of this figure.
pHluorin2-PTS1 calibration
Changes in pH alter the excitation spectrum of pHluorin2-PTS1. Under neutral pH, pHluorin2-PTS1 excitation at 405 nm is greater than at 488 nm; as pH falls, the reverse is true9,21. To measure the relative pH of the glycosome by flow cytometry, we measured emission when excited by the 405 nm laser (VL2 channel) and emission when excited by the 488 nm laser (BL1 channel), using the ratio of VL2/BL1 (fluorescence ratio) to measure the relative glycosomal pH. To convert the fluorescence ratio to pH, we equilibrated intracellular and glycosomal pH with extracellular pH using the ionophores valinomycin and nigericin17,18 followed by flow cytometry analysis to find the fluorescence ratio. As expected, the fluorescence ratio increased as extracellular pH increased with an intracellular Kd of pH 6.5 (Figure 2B). This Kd was slightly lower than the reported in vitro Kd of pHluorin29. Interestingly, the BSF glycosomal pH in the presence of glucose was ~pH 8.0, which was more basic than PF glycosomes in the presence of glucose22. We used this calibration curve to convert the fluorescence ratio to pH in subsequent experiments.
Glycosome acidification due to glucose starvation
While T. brucei PF parasites acidify their glycosomes when starved of glucose22, how BSF glycosomes respond to glucose is unknown. To explore this, we washed BSF parasites expressing pHluorin2-PTS1 in PBS plus 10 mM glucose to remove the culture medium and then resuspended the cells in PBS without glucose. The sensor response to this perturbation was measured immediately and then every 10 min thereafter for 1.5 h by flow cytometry. Responses were compared to cells in PBS plus 10 mM glucose (un-starved).
In response to starvation, we observed a gradual mild acidification over time, which plateaued by ~90 min (Figure 3A). This change in glycosomal pH was statistically significant (p < 0.0001) and repeatable across three separate experiments. This suggests that BSF cells mildly acidify their glycosomes when starved of glucose, similar to the response observed in the PF life stage9.
Figure 3: Reversible acidification of glycosomes of BSF T. brucei when deprived of glucose. (A) Glycosomal pH of cells grown in the absence (starved, blue) or presence (un-starved, red) of glucose. The starved cells were resuspended in PBS without glucose ~2 min prior to the first measurement on an Attune NxT flow cytometer. Three biological replicates of the time course were performed. Un-starved parasites were incubated in PBS plus 10 mM glucose. An unpaired two-tailed Student's t-test was performed comparing the starved and un-starved 90 min time points, ***p = 0.0001. (B) Time course of glycosomal pH change starved (blue) and un-starved (red) BSF parasites with 10 mM glucose reintroduced at 90 min (green dotted line). Five biological replicates of the time course were performed. NS = not significant (p = 0.25, unpaired two-tailed Student's t-test). Please click here to view a larger version of this figure.
Reversible glycosomal acidification in response to glucose
We next tested if BSF glycosome acidification was reversible by starving the cells and then reintroducing glucose. Parasites were incubated in the absence of glucose for 90 min. Glucose (10 mM) was then added and the sensor response was measured by cytometry for another 90 min (Figure 3B). We observed that after glucose was reintroduced, glycosomal pH returned to pre-starvation levels in ~30 min. These results suggest that BSF glycosomal pH is dynamic and regulable in response to glucose, similar to the pH response observed in PF parasites.
Adaptation of the pHluorin2 assay for high-throughput drug screening
Glycosomes are essential organelles for the trypanosome, as they house key metabolic pathways. The importance of glycosomes suggests that inhibitors of their homeostasis could hold promise as potential therapeutic leads. Here, we have adapted the assay for glycosomal pH to a high-throughput format, which will allow adaptation to drug screens to identify inhibitors of glycosomal pH. We anticipate disruption of the regulation of this response could be detrimental to the parasite, given the known impact of pH on glycosome-resident protein function7.
To establish the high-throughput format, we scored the assay robustness. To complete this, parasites induced to express the pHluorin2-PTS1 were plated in a 384-well microtiter plate in either 5 mM glucose (high controls) or no glucose (low controls) and then incubated for 90 min at room temperature. The plate was then analyzed by flow cytometry. As shown in Figure 4, there was low variability between replicate measurements and the high and low controls are well-separated, features that resulted in an acceptable Z-factor of 0.645. Assays with values > 0.5 are generally considered robust enough for adaptation to high-throughput screening campaigns. Given the success here, we anticipate that this sensor and approach will be used in future high-throughput drug screens.
Figure 4: Assay to assess the suitability of the pHluorin2-PTS1 sensor-bearing BSF for future HTS campaigns. Cells were incubated for 90 min with glucose (high control, red, 5 mM glucose) or without the hexose (low control, blue, no additional glucose). The calculated Z-factor was 0.645. Please click here to view a larger version of this figure.
Supplemental Figure S1: Cloning pHluorin2-PTS1 gene into the inducible T. brucei expression vector pLEW100v5. (A) Both vectors were double restriction digested by HindIII and BamHI and then purified. The pHluorin2-PTS1 gene fragment was ligated into pLEW100v5 using T4 DNA ligase. (B) pLEW100-pHlourin2-PTS1. Please click here to download this File.
Supplemental Figure S2: Colocalization of pHL with TbAldolase BF 90-13 parasites transfected with pLEWpHluorin2-PTS1. Expression was induced with doxycycline (1 µg/mL). TbAldolase was localized using anti-TbAldolase sera (diluted 1:500 in block), followed by incubation with goat anG-rabbit Alexa fluor 568. The average Pearson's correlation coefficient was 0.895 (30 cells). Scale bars = 10 µm. Please click here to download this File.
Supplemental Figure S3: Representative gating and dot plots for calibration of BSF pHL sensor cell-line using the pH 8 calibration buffer sample as an example. Samples were stained with the viability dye PI to assess how pH affected viability using the YL2-H channel, but viability was not used in the gating scheme. A wide gate on FSC-A vs SSC-A was used to gate for cells as both live and dead cells were used in the calibration. After gating for cells, single cells (singlets) were gated using FSC-A vs FSC-H. Last, a stringent gate was used for events fluorescent for pHL in the BL1-H and VL2-H channels. Abbreviations: BSF = bloodstream form; PI = propidium iodide; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height. Please click here to download this File.
Supplemental Figure S4: Representative gating and dot plots for glucose starvation and add-back time course assays. The Starved 0 min sample is used as an example. Live cells were gated on the YL2-H channel since PI was used. Cells were gated using FSC-A vs SSC-A to exclude debris and aggregates. Singlets were gated using FSC-A vs FSC-H. Channels BL1-H and VL2-H were used to gate for pHL+ events. A WT control was used to exclude auto-fluorescent events when setting this gate. Abbreviations: PI = propidium iodide; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area. Please click here to download this File.
Supplemental Table S1: Channel and common name used for flow cytometry. The channel name, common name, and the laser and emission filter used are provided. Please click here to download this File.
Supplemental Table S2: Results from pHL calibration using nigericin and valinomycin in different pH buffers. This table includes the exported statistics from the FlowJo analysis of the .fcs files. These values were used to find the fluorescence ratio (VL2-H/BL1-H) for calibrating pHL, as shown in Figure 2. These pH calibration results were also used to interpolate pH for the glucose starvation and add-back time-course experiments (Figure 3). The following statistics were used for quality control: Total event count, pHL+ count, PI- (%), PI+ (%), and pHL+ (%). Data for each biological replicate is in a separate tab, and the tab labeled "Summarized Results" contains the fluorescence ratios for each pH treatment and replicate. Please click here to download this File.
Supplemental Table S3: Analyzed data from BSF pHL glucose starvation time-course assay presented in Figure 3A. This table includes exported statistics from .fcs files analyzed in FlowJo software. Fluorescence ratios were calculated by taking the ratio of median VL2-H and median BL1-H (both from the pHL+ population). These ratios were compiled in the tab labeled "Summarized Results". PI- (%) was used to determine the impact of glucose starvation on viability over time. Total count and pHL+ count were used for quality control. Please click here to download this File.
Supplemental Table S4: Analyzed data from the BSF pHL glucose add-back time-course assay presented in Figure 3B. This table contains exported statistics from .fcs files analyzed in FlowJo software. The pHL+ Median VL2/BL1 values were calculated from pHL+ median VL2-H and BL1-H in Excel. The other statistics were used for quality control. Please click here to download this File.
Supplemental Table S5: Resultant data from FlowJo analysis of the Z-Factor trial high-throughput screening assay using pHL. Tabs labeled "0 mM Glucose" (Low) and "5 mM Glucose" (High) include data from cells treated with 0 or 5 mM glucose, respectively. These fluorescence ratios are presented in Figure 4. In the "Pooled Analysis" tab, the fluorescence ratios for both treatments were compiled and the mean and standard deviation of the sample (SD) were calculated for each. The Z-factor statistic was calculated using equation 3. The ratio of mean High/Low was calculated to measure the separation of the means. Please click here to download this File.
Environmental perception and response mechanisms in the African trypanosome are poorly understood. Changes in nutrient availability are known to trigger diverse responses in the parasite, including acidification of glycosomes. Here, we have described a method to study glycosomal pH response to environmental perturbations in living cells using a heritable protein sensor, pHluorin2, and flow cytometry.
There are several critical steps in the use of the sensor. First, the characterization of transfected parasites that express the transgene is important, as there can be cell-to-cell variation in expression. Expression levels of some transgenes can fall over time. While that has not been observed with the pHluorin2-bearing cells to date, if sensor responsiveness becomes highly variable (e.g., after induction, the sensor signal is not robust), it may be necessary to clone out the culture, as some members of the population may express the protein at higher levels than others. Alternatively, retransformation and selection have proven useful for generating new cell lines with higher (albeit possibly transient) expression. If re-transfection is required, we recommend cloning out individual lines, as the expression of the transgene of interest could impart a subtle growth disadvantage leading to loss of high expressors in the population over time.
Additionally, it is essential to monitor parasite viability-both during the assay and while routinely maintaining cultures. We have included either PI or thiazole red for this purpose during the assay, and these reporters ensure that data only reflects responses in living cells. Ensuring that cell doubling time during normal culture is constant, particularly prior to initiation of large-scale assays (e.g., HTS-type assays) limits concern about initiating assays with cells that are not thriving, which could negatively alter high and low control signals and impact overall robustness of the screen.
The approach we have described here for measuring glycosomal pH is robust and sensitive. However, there are limitations to the approach. First, access to cytometers with appropriate capabilities, including samplers capable of accepting microtiter plates, is required. While ratiometric data can be collected by fluorescence microscopy, the limited number of samples that can be analyzed that way limits the utility of approaches such as screens (and time course-based assays).
A second potential limitation is that the assays involve an agent of biosafety concern. It is possible that core facilities that house the instruments needed for the work may not allow living parasites on their equipment. These limitations, along with the technical challenges of generating and maintaining transgenic trypanosomes, can be overcome through collaboration between groups with appropriate expertise in parasite biology and cell analyses.
The work described here focuses on using the glycosomal pH sensor in African trypanosomes but the tool could be adapted for use in other kinetoplastid parasites. While it is unclear what role acidification of the organelle might have in the biology of Leishmania spp. or Trypanosoma cruzi, is it possible that pHLuorin2-PTS1 could be expressed as a transgene in the cells given that parasite-specific expression vectors are available and that the glycosomal import machinery is similar in the organisms5,23,24.
One potential application of the sensor-bearing cells is the identification of small molecules that alter the cellular capacity to acidify the glycosome. These would likely be noxious to the parasite, as altered pH has been found to be a possible means of regulation of hexokinase, a glycosome-resident protein that is essential in the pathogenic life cycle stage of the African trypanosome7. The assay described here has been adapted to be high throughput (Figure 4), allowing interrogation of large chemical collections for this activity.
In summary, this tool offers the opportunity to dissect mechanisms involved in dynamic responses in a parasite-specific organelle. Using the sensor lines in combination with forward and reverse genetics it is likely that unique parasite-specific pathways will be identified. Further, the sensor lines offer the opportunity for the identification of small molecule inhibitors of the response, opening the door to new drug target discovery.
The authors have nothing to disclose.
pHluorin2-PTS1 was cloned into pLEW100v5 by Twist Bioscience who provided the construct in a high-copy cloning vector; pLEW100v5 was a gift from Dr. George Cross. Antiserum raised against T. brucei aldolase is available from Dr. Meredith T. Morris, Clemson University, upon request. Work from the JCM and KAC laboratories was partially supported by an award from the National Institutes of Health (R01AI156382). SSP was supported by NIH 3R01AI156382.
50 mL Tissue Culture Flasks (Non-treated, sterile) | VWR | 10861-572 | |
75 cm2 Tissue Culture Flask (Non-Treated, sterile) | Corning | 431464U | |
80 µL flat-bottom 384-well plate | BrandTech | 781620 | |
Amaxa Human T Cell Nucleofector Kit | Lonza | VPA-1002 | |
Attune NxT Flow Cytometer | invitrogen by Thermo Fisher Scientific | A24858 | FlowJo software |
BRANDplates 96-Well, flat bottom plate | Millipore Sigma | BR781662 | |
Coloc 2 plugin of ImageJ | https://imagej.net/plugins/coloc-2 | ||
CytKick Max Auto Sampler | invitrogen by Thermo Fisher Scientific | A42973 | |
CytoFLEX Flow Cytometer | Beckman-Coulter | ||
Electron Microscopy Sciences 16% Paraformaldehyde Aqueous Solution, EM Grade, 10 mL Ampoule | Fisher Scientific | 50-980-487 | |
GraphPad Prism | statistical software | ||
Nigericin (sodium salt) | Cayman Chemical | 11437 | |
Nucleofector 2b | Lonza | Discontinued Product | |
OP2 Liquid Handler | opentrons | OP2 | |
poly-L-lysine, 0.1% (w/v) in H2O | Sigma Life Science | CAS:25988-63-0 | Pipetting robot for HTS assay |
Thiazole Red (TO-PRO-3) | biotium | #40087 | We machined a custom acrylic plate stand so this brand of plate could be detected and used on our CytKick Max Auto Sampler |
valinomycin | Cayman Chemical | 10009152 | Pipetting robot for HTS assay |
For pH calibration | |||
For pH calibration |