All ETDs from UAB

Advisory Committee Chair

Tino Unlap

Advisory Committee Members

Thomas M Ryan

Matthew B Renfrow

Jonathan B Waugh

Chad Epps

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Tuberculosis (TB) continues to pose a significant threat to today's society and is mediated by the pathogen Mycobacterium tuberculosis (Mtb). The ability of Mtb to invade and survive within macrophages of the pulmonary granuloma is attributed to the product of the mammalian cell entry (mce) genes whose operon, mce4, encodes a cholesterol transporter that helps transport host lipids into the bacterium that allows the bacterium to survive for years during chronic infection. Currently, there are no rapid and reliable tests for the detection and complete eradication of latent TB. Therefore, we propose and tested the hypothesis that mycobacterial infection in macrophages can be detected and eradicated using siRNA molecular beacons against mce4 operon mRNA. This hypothesis was tested in U937 cells infected or not infected with E. coli stably expressing individual mce4 genes from Mycobacterium smegmatis (E. coli-Ms4) and with Mycobacterium smegmatis (Ms) in the following specific aims: 1) to demonstrate that mce4 genes confer varying degrees of virulence, 2) to demonstrate that a mce4 siRNA molecular beacon can localize mycobacterial infection in macrophages, 3) to demonstrate that a mce4 siRNA molecular beacon can inhibit the mce4 mRNA transcript, and 4) to demonstrate that a mce4 siRNA molecular beacon can attenuate mycobacterial infection in macrophages. Our results with E. coli expressing individual M. smegmatis or Mtb mce4 operon genes showed that mce4A gene conferred the greatest degree of virulence to its host in comparison to other mce4 genes in the operon. We were then able to design a siRNA molecular beacon against the mce4A mRNA of M. smegmatis which was then successfully used to localize mycobacterial infection in macrophages. Using a GFP expressing lentiviral vector we were able to successfully transduce and stably express the mce4 siRNA molecular beacon construct in macrophages infected with either E. coli expressing mce4A gene (E. Coli-4A) or M. smegmatis. Our invasion assay with macrophages stably expressing mce4 siRNA showed that the siRNA treatment attenuated E.coli-4A infection in macrophages at 3, 6, 24, and 48 hours by 0%, 77%, 59.6%, and 99.7%, respectively. Our results also showed that the siRNA treatment attenuated M. smegmatis infection in macrophages at 3, 6, 24, and 48 hours by 94.8%, 70.3%, 98.9%, and 93.4%, respectively. The degree of attenuation of E. coli-4A mce4A mRNA levels was compared between 3, 6, and 24hrs against that at 0hr and the results were found to be 0%, 81%, 40%, and 36%, respectively. The findings of these studies show proof of concept that latent TB infection can be rapidly and accurately detected and eradicated in vitro.

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