ORCID

http://orcid.org/0000-0001-8603-0670

Date of Award

Spring 5-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The presence of Leishmania RNA virus 1 (LRV1) in parasites of the Leishmania (Viannia) subgenus increases the virulence of the parasite in mouse models of leishmaniasis and is correlated with treatment failure, relapse, and the development of mucocutaneous disease in humans. LRV1 is not shed or infectious; rather, the infection is persistent, and as yet it is unknown how the parasite controls virus levels. Many eukaryotic organisms use RNA interference (RNAi) to limit virus replication, and Leishmania (Viannia) parasites have an active RNAi pathway. To determine whether Leishmania are capable of using RNAi to control LRV1, we sequenced sRNAs from LRV1-containing L. braziliensis and L. guyanensis and found that these cells have abundant LRV1-derived sRNAs. Further, I targeted LRV1 using an RNAi transgene in these species, which resulted in a loss of virus. Together, these data suggest that RNAi can limit LRV1 replication. In contrast, knockout of the RNAi effector protein gene Argonaute1 resulted in only a small increase in LRV1 levels, as opposed to the expected dramatic increase. While we did not find evidence of a role for Dicer1/2 or Piwi in control of LRV1, we cannot rule out that such a role exists. These studies suggest that RNAi may play a role in control of LRV1, but that other mechanisms may contribute more or be redundant. In addition to these studies, I also developed a new genetic tool for the manipulation of Leishmania in the laboratory. These "popout constructs" use GFP expression to facilitate the removal of the construct after it has been integrated into the parasite genome, and will allow short-term expression of genes and RNAi transgenes in Leishmania (Viannia) species. Finally, I present investigations into the effect of RNAi transgenes on parasite biology and virulence. I found that the presence of an RNAi transgene impairs knockdown of an unrelated target, results in an accumulation of stable dsRNA and transposable element transcripts, and may increase parasite virulence. These findings suggest that caution is warranted when using these constructs.

Language

English (en)

Chair and Committee

Stephen M. Beverley

Committee Members

Michael Diamond, Sergej Djuranovic, Tamara Doering, Daniel Goldberg,

Comments

Permanent URL: https://doi.org/10.7936/K7M61HQW