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.

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
<ol>
	<li>Culture T. brucei brucei 90-13 BSF trypanosomes in HMI-9 medium supplemented with 10% heat-inactivated FBS and 10% Nu-Serum at 37 &#176;C in 5% CO210. 
	&#8203;...

Development of pHluorin2-Based Sensor for Glycosomal pH in &lt;i&gt;Trypanosoma brucei&lt;/i&gt;

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+carbon+metabolism+in+the+macrophage+phagolysosome-+feast+or+famine.">Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine.</a> F1000Res. 4, 938 (2015).</li><li>Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes:+Parasites+and+the+divergence+of+peroxisomal+purpose.">Glycosomes: Parasites and the divergence of peroxisomal purpose.</a> Molecular Microbiology. 53 (3), 717-724 (2004).</li><li>Parsons, M., Furuya, T., Pal, S., Kessler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+function+of+peroxisomes+and+glycosomes.">Biogenesis and function of peroxisomes and glycosomes.</a> Molecular and Biochemical Parasitology. 115 (1), 19-28 (2001).</li><li>Haanstra, J. R., Gonzalez-Marcano, E. B., Gualdron-Lopez, M., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+maintenance+and+dynamics+of+glycosomes+in+trypanosomatid+parasites.">Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.</a> Biochimica et Biophysica Acta. 1863 (5), 1038-1048 (2016).</li><li>Allmann, S., Bringaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes:+A+comprehensive+view+of+their+metabolic+roles+in+t.+Brucei.">Glycosomes: A comprehensive view of their metabolic roles in t. Brucei.</a> International Journal of Biochemistry and Cell Biology. 85, 85-90 (2017).</li><li>Dodson, H. C., Morris, M. T., Morris, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+3-phosphate+alters+Trypanosoma+brucei+hexokinase+activity+in+response+to+environmental+change.">Glycerol 3-phosphate alters Trypanosoma brucei hexokinase activity in response to environmental change.</a> The Journal of Biological Chemistry. 286 (38), 33150-33157 (2011).</li><li>Lin, S., Morris, M. T., Ackroyd, P. C., Morris, J. C., Christensen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+targeted+delivery+of+pH+sensor+for+quantitative+measurements+of+intraglycosomal+pH+in+live+Trypanosoma+brucei.">Peptide targeted delivery of pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei.</a> Biochemistry. 52 (21), 3629-3637 (2013).</li><li>Mahon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phluorin2:+An+enhanced,+ratiometric,+pH-sensitive+green+florescent+protein.">Phluorin2: An enhanced, ratiometric, pH-sensitive green florescent protein.</a> Advances in Bioscience and Biotechnology. 2 (3), 132-137 (2011).</li><li>Wirtz, E., Leal, S., Ochatt, C., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tightly+regulated+inducible+expression+system+for+conditional+gene+knock-outs+and+dominant-negative+genetics+in+Trypanosoma+brucei.">A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.</a> Molecular and Biochemical Parasitology. 99 (1), 89-101 (1999).</li><li>. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+brucei+pex13.2+is+an+accessory+peroxin+that+functions+in+the+import+of+peroxisome+targeting+sequence+type+2+proteins+and+localizes+to+subdomains+of+the+glycosome.">Trypanosoma brucei pex13.2 is an accessory peroxin that functions in the import of peroxisome targeting sequence type 2 proteins and localizes to subdomains of the glycosome.</a> mSphere. 5 (1), e00744 (2020).</li><li>Kucejova, B., Kucej, M., Petrezselyova, S., Abelovska, L., Tomaska, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screen+for+nigericin-resistant+yeast+mutants+revealed+genes+controlling+mitochondrial+volume+and+mitochondrial+cation+homeostasis.">A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis.</a> Genetics. 171 (2), 517-526 (2005).</li><li>Huynh, M. H., Carruthers, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+excretion+of+glycolytic+products+is+associated+with+acidification+of+the+parasitophorous+vacuole+during+parasite+egress.">Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress.</a> PLoS Pathogens. 18 (5), e1010139 (2022).</li><li>Lehoux, S., Abe, J., Florian, J. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=14-3-3+binding+to+Na+/H++exchanger+isoform-1+is+associated+with+serum-dependent+activation+of+Na+/H++exchange.">14-3-3 binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange.</a> TheJournal of Biological Chemistry. 276 (19), 15794-15800 (2001).</li><li>Jankowski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+measurements+of+the+ph+of+mammalian+peroxisomes+using+the+fluorescent+protein+phluorin.">In situ measurements of the ph of mammalian peroxisomes using the fluorescent protein phluorin.</a> The Journal of Biological Chemistry. 276 (52), 48748-48753 (2001).</li><li>Jankowski, A., Grinstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+fluorimetric+procedure+for+measurement+of+membrane+potential.+Quantification+of+the+NADPH+oxidase-induced+depolarization+in+activated+neutrophils.">A noninvasive fluorimetric procedure for measurement of membrane potential. Quantification of the NADPH oxidase-induced depolarization in activated neutrophils.</a> The Journal of Biological Chemistry. 274 (37), 26098-26104 (1999).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Lin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ph+regulation+in+glycosomes+of+procyclic+form+Trypanosoma+brucei.">Ph regulation in glycosomes of procyclic form Trypanosoma brucei.</a> The Journal of Biological Chemistry. 292 (19), 7795-7805 (2017).</li><li>Ha, D. S., Schwarz, J. K., Turco, S. J., Beverley, S. 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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 tran...

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&#160;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.

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	<tbody>
		<tr>
			<td>50 mL Tissue Culture Flasks (Non-treated, sterile)</td>
			<td>VWR</td>
			<td>10861-572</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 Tissue Culture Flask (Non-Treated, sterile)</td>
			<td>Corning</td>
			<td>431464U</td>
			<td></td>
		</tr>
		<tr>
			<td>80 &#181;L flat-bottom 384-well plate</td>
			<td>BrandTech&#160;</td>
			<td>781620</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Human T Cell Nucleofector&#160; Kit</td>
			<td>Lonza</td>
			<td>VPA-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>invitrogen by Thermo Fisher Scientific</td>
			<td>A24858</td>
			<td>FlowJo software</td>
		</tr>
		<tr>
			<td>BRANDplates 96-Well, flat bottom plate</td>
			<td>Millipore Sigma</td>
			<td>BR781662</td>
			<td></td>
		</tr>
		<tr>
			<td>Coloc 2 plugin of ImageJ</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/plugins/coloc-2</td>
		</tr>
		<tr>
			<td>CytKick Max Auto Sampler</td>
			<td>invitrogen by Thermo Fisher Scientific</td>
			<td>A42973</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Flow Cytometer</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences 16% Paraformaldehyde Aqueous Solution, EM Grade, 10 mL Ampoule</td>
			<td>Fisher Scientific</td>
			<td>50-980-487</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td></td>
			<td></td>
			<td>statistical software</td>
		</tr>
		<tr>
			<td>Nigericin (sodium salt)</td>
			<td>Cayman Chemical</td>
			<td>11437</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b</td>
			<td>Lonza</td>
			<td>Discontinued Product</td>
			<td></td>
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			<td>OP2 Liquid Handler</td>
			<td>opentrons</td>
			<td>OP2</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine, 0.1% (w/v) in H2O</td>
			<td>Sigma Life Science</td>
			<td>CAS:25988-63-0</td>
			<td>Pipetting robot for HTS assay</td>
		</tr>
		<tr>
			<td>Thiazole Red (TO-PRO-3)</td>
			<td>biotium</td>
			<td>#40087</td>
			<td>We machined a custom acrylic plate stand so this brand of plate could be detected and used on our CytKick Max Auto Sampler</td>
		</tr>
		<tr>
			<td>valinomycin</td>
			<td>Cayman Chemical</td>
			<td>10009152</td>
			<td>Pipetting robot for HTS assay</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>For pH calibration</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>For pH calibration</td>
		</tr>
	</tbody>
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measuring dynamic glycosomal ph changes in living trypanosoma brucei

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&#160;in the live cell setting.

Author Spotlight: Advancements in Glycosomal pH Monitoring in &lt;i&gt;Trypanosoma brucei&lt;/i&gt; Using pHluorin2 Biosensor

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for Trypanosoma brucei, yet little is known about how the cells sense and respond to changing glucose levels in the environment. We have developed parasites expressing a pH biosensor that allows us to monitor glycosomal pH in live parasites, which will aid in the understanding of parasites'dynamic responses to nutrient fluctuation. A challenge for this experiment would be the availability of a flow cytometer equipped with a microtiter plate reader, as well as the appropriate filter sets to measure biosensor fluorescence.Additionally, T.brucei is a risk group two organism, so the cytometer must be housed within a biosafety level two facility. It has been discovered that the pH level of glycosomes can affect glycolysis as hexokinase and phosphofructokinase are pH sensitive. However, the pH probes that were previously used did not accurately target to the glycosome in the bloodstream form.A better tool was needed to study the relationship between glycosomal pH and glycolysis in this parasite life stage. The pHluorin2-PTS1 sensor is a tool that is better at localizing to the glycosome than chemical pH probes in this life stage. It's user friendly, as you only need to induce expression of the pH sensor overnight, and the cells will make and localize pHluorin2 to the glycosome.Using flow cytometry to measure pHluorin2 increases throughput and improves statistical measurements compared to microscopy.

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&#39;s correlation coefficient of colocalization between anti-TbAldolase and pHluorin2-PTS1 was 0.895, indicating that pHluorin2-PTS1 wa...

Watch this Scientific Journal Video about Author Spotlight: Advancements in Glycosomal pH Monitoring in Trypanosoma brucei Using pHluorin2 Biosensor at JoVE.com

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. ...

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

Research

JoVE Journal

Immunology and Infection

Sample Preparation for Flow Cytometry to Measure pHluorin2 Fluorescence in Living Trypanosoma brucei

Flow Cytometry Procedure to Measure Relative pH of the Glycosome in Living Trypanosoma brucei

Analysis of Flow Cytometry Data to Measure the Relative pH of the Glycosome in Living Trypanosoma brucei

305 Views.  Brigham Young University.  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.

Development of pHluorin2-Based Sensor for Glycosomal pH in Trypanosoma brucei

Summary