Many gene regulatory processes depend on proteins that interact with DNA. Characterizing these interactions can shed light on the molecular mechanisms that allow cells to control which RNA is made, when it is made, and how much is made. Additionally, DNA protein interactions are essential for cell division, DNA replication, and repair. Chromatin ImmunoPrecipitation followed by sequencing (ChIP-seq) has been an invaluable tool for understanding gene regulatory processes in the eukaryotic kinetoplastid parasite Trypanosoma brucei by mapping genomic binding sites for a protein of interest. We recently sought to expand the repertoire of available techniques to interrogate protein-DNA interactions by optimizing Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for use in Trypanosoma brucei. The protocol presented here details a CUT&RUN protocol suitable for proteins in small complexes for which there is an available antibody.

The early-branching eukaryotic parasite, Trypanosoma brucei, lives extracellularly in the mammalian host, where it expresses variant surface glycoprotein (VSG). Antigenic variation of VSGs allows the parasite to evade the host immune response while in the bloodstream. Upon transition to the tsetse fly vector, the parasite remodels its surface to express procyclin protein from a limited set of EP and GPEET genes. While the environmental signals that trigger the switch from expressing VSG genes to expressing EP and GPEET genes are known, the molecular mechanism that initiates transcription of the EP and GPEET genes during parasite differentiation to the procyclic stage within the tsetse is not well understood. Previous work has shown that the chromatin-interacting bromodomain protein Bdf3 is absent from the RNA polymerase I promoter of the EP locus in bloodstream parasites, but appears there shortly after initiation of differentiation from the bloodstream to the procyclic stage. Here, we show that tethering of Bdf3 to the EP promoter using a dCas9-Bdf3 fusion protein and a guide RNA is sufficient to increase transcript levels of EP1 in bloodstream parasites, where the gene is normally silenced. This result supports the model that Bdf3 appears at the EP locus during differentiation to facilitate initiation of transcription, which is consistent with the role of Bdf3 as a transcriptional activator in other systems. Understanding gene regulatory mechanisms in this early-branching eukaryote may help build a more complete picture of the conserved and unique features of gene regulatory proteins across diverse biological systems. IMPORTANCE:The Trypanosoma brucei parasite is transmitted via the tsetse fly vector to a mammalian host , where it causes African trypanosomiasis, a fatal disease that imposes a severe human and economic burden for people living in areas of sub-Saharan Africa. While drug treatments have improved for some strains, some infections still require treatment with drugs that have serious side effects. One avenue for drug development is to try to manipulate the life cycle of the parasite to make it poorly adapted to the mammalian host. However, this requires detailed knowledge of the mechanisms by which parasites regulate their genes as they progress through the life cycle. Here, we show that the DNA-interacting bromodomain protein Bdf3 may be a key driver for turning on insect-specific genes that code for insect-stage surface proteins. This finding could aid in the development of life cycle-manipulating drugs and shed light on how gene regulatory mechanisms evolved.

Plasmodium falciparum causes most malaria deaths. Its developmental transitions and environmental adaptation are partially regulated by epigenetic mechanisms. Plasmodium falciparum GCN5 (PfGCN5) is an epigenetic regulator that acetylates lysines and can also bind to acetylated lysine residues on histones via its bromodomain (BRD). Here, we showed that PfGCN5 was essential for parasite transmission and survival in human blood and mosquitoes. PfGCN5 regulated genes important for metabolism and development and its BRD was required at euchromatic gene promoters for their proper expression and for acetylation of the variant histone Pf H2B.Z. However, PfGCN5 was most abundant in heterochromatin and loss of the PfGCN5 BRD de-repressed heterochromatic genes and increased levels of acetylated Pf H2B.Z in heterochromatin. The PfGCN5 BRD-binding compound L-45 phenocopied deletion of the PfGCN5 BRD, identifying PfGCN5 as a promising drug target for BRD inhibitors. Thus, PfGCN5 appears to directly contribute to activating euchromatic promoters, but PfGCN5 is also critical for maintaining repressive heterochromatin structure.

Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) provides insights into both the genomic location occupied by the protein of interest and the difference in DNA occupancy between experimental states. Given that ChIP-seq data are collected experimentally, an important step for determining regions with differential DNA occupancy between states is between-sample normalization. While between-sample normalization is crucial for downstream differential binding analysis, the technical conditions underlying between-sample normalization methods have yet to be examined for ChIP-seq. We identify three important technical conditions underlying ChIP-seq between-sample normalization methods: balanced differential DNA occupancy, equal total DNA occupancy, and equal background binding across states. To illustrate the importance of satisfying the selected normalization method's technical conditions for downstream differential binding analysis, we simulate ChIP-seq read count data where different combinations of the technical conditions are violated. We then externally verify our simulation results using experimental data. Based on our findings, we suggest that researchers use their understanding of the ChIP-seq experiment at hand to guide their choice of between-sample normalization method. Alternatively, researchers can use a high-confidence peakset, which is the intersection of the differentially bound peaksets obtained from using different between-sample normalization methods. In our two experimental analyses, roughly half of the called peaks were called as differentially bound for every normalization method. High-confidence peaks are less sensitive to one's choice of between-sample normalization method, and thus could be a more robust basis for identifying genomic regions with differential DNA occupancy between experimental states when there is uncertainty about which technical conditions are satisfied.

SUMMARY Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it impossible to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria uses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.

Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it difficult to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria expresses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule-binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule-binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.

Running a research program with undergraduates provides unique challenges and opportunities. Research experiences gained by undergraduates have the power to launch lifelong careers for future practicing scientists. However, getting publishable data from the hands of inexperienced students can be a challenge. In this talk, I'll discuss my projects with undergraduate students on gene regulation in the African trypanosome. These single celled eukaryotic parasites cycle between tsetse flies and mammals, where they cause the fatal disease sleeping sickness. We are interested in the role of chromatin interacting bromodomain proteins during the transition from the bloodstream parasite stage in the mammal to the procyclic parasite stage in the fly. We have recently mapped the genome location of the chromatin interacting bromodomain protein Bdf3 throughout the transition from the bloodstream parasite stage to the fly stage, showing that occupancy of this protein is dynamic throughout the process. We have also shown that inhibition of bromodomain proteins in bloodstream and procyclin stages causes spontaneous differentiation to the next stage of the life cycle. In describing these projects, I'll highlight some strategies I've developed for choosing appropriate undergraduate projects, recruiting and retaining a diverse cohort of students, and publishing papers with undergraduate student co-authors. Finally, I'll talk about leveraging the role of working at a primarily undergraduate institution to attain external funding. My hope is that this talk will provide a realistic picture of the challenges of doing research at an undergraduate institution, while at the same time highlighting the positive impact these experiences can have on the careers of trainees. Danae Schulz is supported by an NSF CAREER grant 2041395.

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT) and animal trypanosomiases, cycles between a bloodstream form in mammals and a procyclic form in the gut of its insect vector. We previously discovered that the human bromodomain inhibitor I-BET151 causes transcriptome changes that resemble the transition from the bloodstream to the procyclic form. In particular, I-BET151 induces replacement of variant surface glycoprotein (VSG) with procyclin protein. While modest binding of I-BET151 to TbBdf2 and TbBdf3 has been demonstrated, it is unknown whether I-BET151 binds to other identified T. brucei bromodomain proteins and/or other targets. To identify target(s) in T. brucei, we have synthesized I-BET151 derivatives maintaining the key pharmacophoric elements with functionality useful for chemoproteomic approaches. We identified compounds that are potent in inducing expression of procyclin, delineating a strategy towards the design of drugs against HAT and other trypanosomiases. Furthermore, these derivatives represent useful chemical probes to elucidate the molecular mechanism underlying I-BET151-induced differentiation.

The Plasmodium falciparum alternative histones Pf H2A.Z and Pf H2B.Z are enriched in the same nucleosomes in intergenic euchromatin but depleted from heterochromatin. They occupy most promoters but are only dynamically associated with expression at var genes. In other organisms, acetylation of H2A.Z is important for its functions in gene expression and chromatin structure. Here, we show that acetylated Pf H2A.Z and Pf H2B.Z are dynamically associated with gene expression at promoters. In addition, acetylated Pf H2A.Z and Pf H2B.Z are antagonized by the sirtuin class III histone deacetylases (HDAC) PfSir2A and B at heterochromatin boundaries and encroach upon heterochromatin in parasites lacking PfSir2A or B. However, the majority of acetylated Pf H2A.Z and Pf H2B.Z are deacetylated by class I or II HDACs. Acetylated Pf H2A.Z and Pf H2B.Z are also dynamically associated with promoter activity of both canonical upstream var gene promoters and var gene introns. These findings suggest that both acetylated Pf H2A.Z and Pf H2B.Z play critical roles in gene expression and contribute to maintenance of chromatin structure at the boundaries of subtelomeric, facultative heterochromatin, critical for the variegated expression of genes that enable rapid adaptation to altered host environments. IMPORTANCE The malaria parasite Plasmodium falciparum relies on variant expression of members of multi-gene families as a strategy for environmental adaptation to promote parasite survival and pathogenesis. These genes are located in transcriptionally silenced DNA regions. A limited number of these genes escape gene silencing, and switching between them confers variant fitness on parasite progeny. Here, we show that PfSir2 histone deacetylases antagonize DNA-interacting acetylated alternative histones at the boundaries between active and silent DNA. This finding implicates acetylated alternative histones in the mechanism regulating P. falciparum variant gene silencing and thus malaria pathogenesis. This work also revealed that acetylation of alternative histones at promoters is dynamically associated with promoter activity across the genome, implicating acetylation of alternative histones in gene regulation genome wide. Understanding mechanisms of gene regulation in P. falciparum may aid in the development of new therapeutic strategies for malaria, which killed 619,000 people in 2021.

The eukaryotic protozoan parasite Trypanosoma brucei is transmitted by the tsetse fly to both humans and animals, where it causes a fatal disease called African trypanosomiasis. While the parasite lacks canonical DNA sequence-specific transcription factors, it does possess histones, histone modifications, and proteins that write, erase, and read histone marks. Chemical inhibition of chromatin-interacting bromodomain proteins has previously been shown to perturb bloodstream specific trypanosome processes, including silencing of the variant surface glycoprotein (VSG) genes and immune evasion. Transcriptomic changes that occur in bromodomain-inhibited bloodstream parasites mirror many of the changes that occur as parasites developmentally progress from the bloodstream to the insect stage. We performed transcriptome sequencing (RNA-seq) time courses to determine the effects of chemical bromodomain inhibition in insect-stage parasites using the compound I-BET151. We found that treatment with I-BET151 causes large changes in the transcriptome of insect-stage parasites and also perturbs silencing of VSG genes. The transcriptomes of bromodomain-inhibited parasites share some features with early metacyclic-stage parasites in the fly salivary gland, implicating bromodomain proteins as important for regulating transcript levels for developmentally relevant genes. However, the downregulation of surface procyclin protein that typically accompanies developmental progression is absent in bromodomain-inhibited insect-stage parasites. We conclude that chemical modulation of bromodomain proteins causes widespread transcriptomic changes in multiple trypanosome life cycle stages. Understanding the gene-regulatory processes that facilitate transcriptome remodeling in this highly diverged eukaryote may shed light on how these mechanisms evolved. IMPORTANCE The disease African trypanosomiasis imposes a severe human and economic burden for communities in sub-Saharan Africa. The parasite that causes the disease is transmitted to the bloodstream of a human or ungulate via the tsetse fly. Because the environments of the fly and the bloodstream differ, the parasite modulates the expression of its genes to accommodate two different lifestyles in these disparate niches. Perturbation of bromodomain proteins that interact with histone proteins around which DNA is wrapped (chromatin) causes profound changes in gene expression in bloodstream-stage parasites. This paper reports that gene expression is also affected by chemical bromodomain inhibition in insect-stage parasites but that the genes affected differ depending on life cycle stage. Because trypanosomes diverged early from model eukaryotes, an understanding of how trypanosomes regulate gene expression may lend insight into how gene-regulatory mechanisms evolved. This could also be leveraged to generate new therapeutic strategies.

This Cleavage Under Targets and Release Using Nuclease (CUT&RUN) protocol produces genomic occupancy data for a protein of interest in the protozoan parasite Trypanosoma brucei. The data produced is analyzed in a similar way as that produced by ChIP-seq. While we describe the protocol for parasites carrying an epitope tag for the protein of interest, antibodies against the native protein could be used for the same purpose.

The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Trypanosoma brucei, the causative agent of human and animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism during adaptation to each host environment. These changes are reflected in the different transcriptomes of parasites living in each host. However, it remains unclear whether chromatin-interacting proteins help mediate these changes. Bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stages remains unknown. To address this question, we performed cleavage under target and release using nuclease (CUT&RUN) against bromodomain protein 3 (Bdf3) in parasites differentiating from bloodstream to insect forms. We found that Bdf3 occupancy at most loci increased at 3 h following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes with altered transcript levels during differentiation, such as procyclins, procyclin-associated genes, and invariant surface glycoproteins. Most Bdf3-occupied sites are observed throughout differentiation. However, one site appears de novo during differentiation and lies proximal to the procyclin gene locus housing genes essential for remodeling surface proteins following transition to the insect stage. These studies indicate that occupancy of chromatin-interacting proteins is dynamic during life cycle stage transitions and provide the groundwork for future studies on the effects of changes in bromodomain protein occupancy. Additionally, the adaptation of CUT&RUN for Trypanosoma brucei provides other researchers with an alternative to chromatin immunoprecipitation (ChIP). IMPORTANCE The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Existing drugs have significant side effects. Thus, these parasites impose a significant human and economic burden in sub-Saharan Africa, where trypanosomiasis is endemic. T. brucei cycles between the mammalian host and a tsetse fly vector, and parasites undergo huge changes in morphology and metabolism to adapt to different hosts. Here, we show that DNA-interacting bromodomain protein 3 (Bdf3) shows changes in occupancy at its binding sites as parasites transition from the bloodstream to the insect stage. Additionally, a new binding site appears near the locus responsible for remodeling of parasite surface proteins during transition to the insect stage. Understanding the mechanisms behind host adaptation is important for understanding the life cycle of the parasite.

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. We screened a library of 1,585 U.S. or foreign-approved drugs and identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wished to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites lack the immune evasion mechanisms prevalent in bloodstream forms, making them vulnerable to the host immune system. To identify drugs that increase transcript levels of an invariant, insect-stage specific surface protein called procyclin, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction or stabilization of the procyclin transcript. Using these bloodstream reporter strains in combination with automated flow cytometry, we identified eflornithine, spironolactone, and phenothiazine as small molecules that increase abundance of procyclin transcript. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. While we failed to identify compounds that increase levels of procyclin protein on the cell surface, this study is proof of principle that these fluorescent reporter parasites represent a useful tool for future small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions.

Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

Trypanosoma brucei is an early branching protozoan that causes Human and Animal African Trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of Trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here we have generated a T. brucei ORFeome consisting of over 90% of the targeted genome and used it to make an inducible Gain-of-Function library for broadly applicable forward genetic screening. Using a critical drug of last resort, melarsoprol, we conducted a proof of principle genetic screen. Hits arising from this screen support the significance of trypanothione, a key player in redox metabolism, as a target of melarsoprol and implicate novel proteins of the flagellum and mitochondria in drug resistance. This study has produced two powerful new genetic tools for kinetoplastida research, which are expected to promote major advances in kinetoplastida biology and therapeutic development in the years to come.

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wish to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites do not vary proteins on their cell surface, making them vulnerable to the host immune system. To identify drugs that trigger differentiation of the parasite from bloodstream to insect stages, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction of the invariant insect-stage specific procyclin transcript. Using these bloodstream reporter strains in combination with high-throughput flow cytometry, we screened a library of 1,585 U.S. or foreign-approved drugs and identified eflornithine, spironolactone, and phenothiazine as small molecules that induce transcription of procylin. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. We further identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. Finally, this study is proof of principle that fluorescent reporters are a useful tool for small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions. Author Summary African trypanosomes are unicellular parasites that infect humans and animals, causing a fatal disease known as sleeping sickness in humans and nagana in cattle. These diseases impose a severe economic burden for people living in Sub-Saharan Africa, where parasites are transmitted to humans and animals through the bite of the tsetse fly. Parasites living outside cells in humans and animals are attacked by the antibodies of the host immune system, but they can evade this attack by varying the proteins on their cell surface. In contrast, because flies do not have an antibody-mediated immune response, parasites living in flies do not vary the proteins on their cell surface. In this study, we performed a small molecule screen to identify compounds that might force bloodstream parasites to move forward in their life cycle to become more similar to parasites living in flies, causing them to express a protein on their cell surface that does not vary. This invariant protein on the surface of bloodstream parasites would make bloodstream parasites vulnerable to the host antibodies. We found 3 compounds that increased RNA levels for an invariant insect-stage surface protein and 154 compounds that inhibit parasite growth. We hope these compounds might have potential as novel trypanosomiasis therapeutics.

Parasites have long been known to influence host responses to infection through the secretion of virulence factors. Extracellular vesicles are emerging as important mediators of these manipulations, and a new study by Szempruch et al. suggests they could play a crucial role in host responses to African trypanosome infections.

The African trypanosome, Trypanosoma brucei , is a causative agent of African Trypanosomiasis (also known as “sleeping sickness” in humans and “nagana” in cattle) and imposes an enormous economic burden in regions of Sub-Saharan Africa. T. brucei is transmitted to the mammalian host through the bite of the tsetse fly. In the mammal, it survives extracellularly in the bloodstream, eventually migrating into the central nervous system and causing coma and death. To survive in the bloodstream of its mammalian host, T. brucei must evade the host immune system. The parasite is covered by a dense, variant surface glycoprotein (VSG) coat that shields other epitopes on the cell surface from antibodies produced by the host immune system (reviewed in refs. 1 and 2). A strong antibody response is mounted against the VSG upon entry of the parasite into the bloodstream. However, the parasite harbors thousands of variants of the VSG gene and periodically switches the particular variant that is expressed in a process called antigenic variation (known colloquially in T. brucei as “switching”) (1). VSG genes are transcribed from one of ∼15 telomeric bloodstream expression sites (BESs), only one of which is transcriptionally active at any given time. Transcription of VSG genes occurs within a discrete nuclear structure, called the expression site body (ESB) and is driven by Pol I (1). Switching from expression of one VSG to a new VSG can occur by multiple mechanisms, including transcriptional activation of a new BES and recombinatorial mechanisms (1). Analysis of T. brucei populations in mouse models of infection has revealed that a population of trypanosomes can contain parasites expressing as many as 66 different VSGs at any given time (3). However, although the repertoire of VSGs expressed in a population of trypanosomes is quite diverse, a single trypanosome expresses just one … [↵][1]1To whom correspondence may be addressed. Email: dschulz{at}hmc.edu or papavas{at}rockefeller.edu. [1]: #xref-corresp-1-1

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.