Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Summary

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Abstract

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.

Introduction

The protozoan parasite, Trypanosoma brucei, is a causative agent of diseases that affect humans (via Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense) and animals (via Trypanosoma brucei brucei) throughout Sub-Saharan Africa. It is transmitted to the mammalian host through the saliva of the tsetse fly vector. Both Human and Animal African Trypanosomiasis cause a severe economic burden in endemic regions, and few drugs are available or in development to treat either disease. Understanding mechanisms of immune evasion is critical for the development of drugs for trypanosomiasis. Antigenic variation of the dense, Variant Surface ....

Protocol

NOTE: Throughout the procedure, it is necessary to keep cells on ice. Cold media should also be used throughout.

1. Sample Harvesting

1. Grow Lister 427 strain bloodstream trypanosomes to a density of 0.5 - 1 million/ml. It is best to start cultures with a small number of parasites. Spin down 50 x 10⁶ cells/sample for 10 min at 1,500 x g. Be sure to leave 1 x 10⁶ cells in culture for later use as positive and negative antibody controls.
   NOTE: This protocol can also be used on trypanosomes isolated from animal blood (see discussion for details).
2. Pipette or pour off most of the supernatant (leav....

Results

The method described here was used to demonstrate that double-strand breaks within the VSG expression site increased the number of switchers in a population⁸. Here, we show representative results from a population of trypanosomes that have been similarly induced to generate a double-strand break at the expression site. We compare these trypanosomes to those that have not been induced to generate a double-strand break. Figure 1 shows representative flow cytometr.......

Discussion

With respect to experimental technique, the most critical component of the protocol is keeping all the samples cold. Trypanosomes very rapidly internalize antibody bound to their surface⁹, but this process is motility dependent, and does not influence the assay as long as the cells are kept at 4 °C. All samples should always be kept on ice, and pipetting should be done quickly to minimize exposure to the 25 °C lab environment. Cold HMI-9 with serum should be available at the start of the experiment a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
unlabeled anti-VSG antibody	n/a	n/a	VSG antibody is made in house
fluorophore labeled anti-VSG antibody	n/a	n/a	VSG antibody is made in house
HMI-9 media	n/a	n/a	HMI-9 is made in house
Propidium Iodide	BD Pharmingen	556463
CountBright absolute counting beads	Thermofisher Scientific	C36950
LD columns	Miltenyi Biotech	130-042-901
MACS magnet	Miltenyi Biotech	130-042-303
MACS magnetic separator	Miltenyi Biotech	130-042-302
vortex adapter-60	ThermoFisher scientific	AM10014
flow cytometer	Coulter	n/a
flow cytometer analysis software	FloJo	n/a