Elucidating "Consumption" : Using Fluorescent Steroid Probes to Understand Host Cholesterol Utilization by Mycobacterium spp.

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, is the leading cause of death due to bacterial infection worldwide, claiming nearly two million lives each year. Often, the immune system cannot eradicate the bacteria and the disease progresses into a latent state characterized by the presence of granulomas, which are lesions formed in order to prevent bacterial dissemination. This state of infection can persist for years. Current research has shown that the utilization of host cholesterol by M. tuberculosis plays a crucial role in the tenacity of this organism during latent infection. Understanding the mechanisms of cholesterol acquisition and degradation could provide researchers with novel platforms for the strategic development of anti-mycobacterial drugs. In order to further understand the pathways employed by M. tuberculosis during cholesterol utilization, a series of fluorescently labeled cholesterol analogues were synthesized by tethering cholesterol chloroformate to 1,8-naphthalimide moieties via a carbamate linkage. The analogues and their 4-amino-1,8-naphthalimide precursors were found to be intensely fluorescent in nonpolar environments such as dichloromethane and liposomes comprised of POPC. Their in vitro effects were evaluated using the Mtb research model M. smegmatis. Bacterial growth curves were consistent with active incorporation and degradation of the fluorescently labeled cholesterol compounds, while inhibitory effects were observed for the unconjugated naphthalimide fluorophores. Current studies are focused on the isolation and identification of cholesterol/fluorophore metabolites to help elucidate the mechanism of uptake and decomposition of these compounds. Preliminary results of these metabolite experiments are discussed with relation to the observed effects on M. smegmatis growth.