# Regulators of VSG allelic exclusion and switching in Trypanosoma brucei

> **NIH NIH R21** · RBHS-NEW JERSEY MEDICAL SCHOOL · 2020 · $234,000

## Abstract

PROJECT SUMMARY
Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and related
diseases in animals, mainly in sub-Saharan Africa. T. brucei undergoes life-cycle-specific differentiation between
the insect vector (tsetse) and mammalian hosts. In the mammalian hosts, ‘Bloodstream Form (BF)’
trypanosomes proliferate extracellularly in the vascular system and the surfaces of trypanosome cells are coated
with Variant Surface Glycoprotein (VSG). BF trypanosomes escape the host immune response through
sequential expression of VSGs, a process known as ‘antigenic variation’. Two mechanisms that underlie T.
brucei antigenic variation, monoallelic VSG expression and periodic VSG switching, represent a prototype of
host-evasion systems found in several pathogenic parasites. However, our knowledge of how antigenic variation
operates in trypanosomes remains limited in three vital areas: 1) how expression of a single active VSG is
maintained, 2) how the remainder of thousands of VSGs remain silenced, and 3) how their expression status
switches. The methodology developed in this proposal by integrating basic genetic concepts and state-of-the-art
techniques will efficiently identify genes that regulate specific aspects of monoallelic VSG expression and VSG
switching. I will utilize a dual-color reporter strain containing a GFP gene (green) integrated at the transcriptionally
active VSG locus and a tdTomato (red) at a silent VSG locus. Two genome-wide libraries, overexpression ORF
library containing ~ 7,500 genes (Aim 1) and RNAi library (Aim 2), will be screened for differential expression of
GFP and/or tdTomato reporters. If a gene is a critical factor for monoallelic VSG expression and/or VSG
switching, depletion or overexpression of this gene should change the expression of reporters. To maximize
chances of obtaining true positives, tailored approaches will be used in ORF and RNAi library screens. The
overexpression ORF library consists of 30 mini libraries, each containing about 150-350 independent ORFs. Due
to this low complexity, ORF library can be screened in a traditional way. ORF library transfected cells will be
distributed in 96-well plates. Upon inducing overexpression, each well will be analyzed by flow cytometry and
positive clones will be identified by PCR and sequencing. Because RNAi library represents the genome of T.
brucei with about 100,000 clones (too many to be screened by manual plating), positive clones will be enriched
using two different methods: (i) enrichment of candidate populations exhibiting altered reporter expression by
FACS sorting and (ii) enrichment of candidate population by depleting ‘non-candidate (parental)’ population by
Magnet-Activated Cell Sorting (MACS) technique. High-throughput sequencing will clone the genes responsible
for differential expression of the reporters from these sorted or depleted populations. Using these integrative
approaches, I will identify genes necessary for...

## Key facts

- **NIH application ID:** 9953640
- **Project number:** 1R21AI151261-01
- **Recipient organization:** RBHS-NEW JERSEY MEDICAL SCHOOL
- **Principal Investigator:** Hee-Sook Kim
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $234,000
- **Award type:** 1
- **Project period:** 2020-02-13 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9953640

## Citation

> US National Institutes of Health, RePORTER application 9953640, Regulators of VSG allelic exclusion and switching in Trypanosoma brucei (1R21AI151261-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9953640. Licensed CC0.

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