Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Papavasiliou Laboratory

Abstract

Trypanosoma brucei, a causative agent of African sleeping sickness in humans and nagana in animals, constantly changes its dense variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of its mammalian host, using an extensive repertoire of dedicated genes. Although this process, referred to as antigenic variation, is the major mechanism of pathogenesis for T. brucei, the dynamics of VSG expression in T. brucei during an infection are poorly understood. In this thesis, I describe the development of VSG-seq, a method for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Using VSG-seq, I monitored VSG expression dynamics in vivo during both acute and chronic mouse infections. My experiments revealed unexpected diversity within parasite populations, and the expression of as much as one-third of the functional genomic VSG repertoire after only one month of infection. In addition to suggesting that the host-pathogen interaction in T. brucei infection is substantially more dynamic and nuanced than previously expected, this observed diversity highlighted the importance of the mechanisms by which T. brucei diversifies its genome-encoded VSG repertoire. During infection, the parasite can form mosaic VSGs, novel variants that arise through recombination events within the parasite genome during infection. Though these novel variants had been identified previously, little was known about the mechanisms by which they form. VSG-seq facilitated the identification of mosaic VSGs during the infection, which allowed me to track their formation over time. My results provide the first temporal data on the formation of these variants and suggest that mosaic VSGs likely form at sites of VSG transcription. VSG-seq, which is based on the de novo assembly of VSGs, obviates the requirement for a reference genome for the analysis of expressed VSG populations. This allows the method to be used for the high-resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. To this end, I have applied VSG-seq to samples grown in vitro, parasites isolated from natural infections, and extravascular parasites occupying various tissues in vivo. These extensions of the method reveal new aspects of T. brucei biology and demonstrate the potential of high-throughput approaches for studying antigenic variation, both in trypanosomes and in any pathogen that uses antigenic variation as a means of immune evasion.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Friday, April 27, 2018

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