Greenwood, Daniel Joseph; (2019) Visualising the subcellular distribution of antibiotics against tuberculosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), remains the world’s deadliest infectious disease. Although treatable, effective chemotherapy requires at least six months of treatment with a minimum of four antibiotics. Novel antibiotics are needed to quell the pandemic. However, we do not fully understand why current treatments take so long to work in patients. Mtb has a dynamic intracellular lifestyle, and this thesis tests the hypothesis that not all antibiotics penetrate into, or are effective within, all compartments containing Mtb during infection. Our understanding of the intracellular pharmacokinetics of drugs against TB has been limited by a lack of technologies for studying the subcellular distribution of antibiotics. This work developed a correlative imaging workflow incorporating fluorescence, electron and nanoscale ion microscopy (CLEIM) to map the subcellular distribution of two antibiotics, bedaquiline (BDQ) and pyrazinamide (PZA), at sub-micrometre resolution in Mtb-infected human macrophages. This workflow was complemented with orthogonal methods, including high-content live-cell imaging, to study the dynamic processes that contribute to antibiotic activity. BDQ accumulated primarily in host cell lipid droplets (LD), but heterogeneously in Mtb within a variety of intracellular compartments. Surprisingly, LD did not sequester the antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Lipid binding therefore facilitated drug trafficking by host organelles to an intracellular target. PZA is a pro-drug, and the accumulation of its active metabolite pyrazinoic acid has been hypothesised to depend on the bacteria being in an acidic environment. Direct analysis of antibiotic accumulation by ion microscopy, combined with live-cell imaging at the single cell level, revealed that, whilst acidic intracellular environments support PZA activity, they are not necessary for antibiotic efficacy. Many intracellular pathogens interact with LD or reside in partially acidified vacuoles, and these results therefore have broad implications for our understanding of antibiotic activity.

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