This is a competing renewal application of R01 AI51622 entitled ?Chemical Mycobacteriology?. The broad objectives of this project are (1) to study the effects of tuberculosis drugs on mycobacterial cell wall dynamics by in vivo imaging in the Mycobacterium marinum/zebrafish infection model; and (2) to develop a new point-of- care method for clinical detection of live Mtb in patient sputum samples. Tuberculosis (TB) is a chronic pulmonary disease caused by infection with Mycobacterium tuberculosis (Mtb). A variety of drugs have been identified that rapidly kill Mtb and its relatives in vitro, yet clinical treatment requires at least 6 months of combination therapy and resistance is rampant. The reasons that antibiotics are less effective in vivo remain unclear, and this knowledge gap is exacerbated by our inability to directly study the molecular effects of TB drugs on bacteria during infection. To do so would require an infection model amenable to noninvasive monitoring, and probes that report on bacterial systems affected by drug action. In the previous granting period, we developed chemical methods for imaging components of the mycobacterial cell wall, a target of several frontline TB drugs. We used metabolic and bioorthogonal labeling methods to image trehalose glycolipids of the mycomembrane, an essential cell wall layer disrupted by the TB drugs isoniazid and ethambutol. In parallel, we developed reagents for imaging newly synthesized peptidoglycan (PG) in bacteria residing within human host cells. These methods offer a newfound capability of monitoring how the cell wall responds to drug treatment during the course of infection. Our proposal for the next granting period comprises three specific aims.
Aim 1 : We will investigate the effects of TB drugs on cell wall dynamics in vitro and in vivo, with an eye for identifying stages of infection that perturb drug responses. We will image changes in trehalose mycolate production, subcellular localization and mobility as a function of infection stage and drug treatment using the M. marinum/zebrafish infection model, a natural and experimentally tractable host-pathogen system.
Aim 2 : We will develop a new method for point-of- care detection of Mtb in patient sputum samples using solvatochromic mycomembrane imaging agents. In collaboration with Prof. Bavesh Kana at Univ. Witswatersrand, South Africa, we will field test the method by analysis of sputum samples collected from HIV-1 coinfected and uninfected TB patients as well as nave and drug-treated TB patients.
Aim 3 : Finally, we will explore how mycobacteria's unusual property of asymmetric growth contributes to virulence and drug sensitivity in vivo by imaging PG in the M. marinum/zebrafish infection model. Achievement of these aims will provide new insights into TB drug action and resistance, and deliver a new clinical tool for accurate diagnosis and drug efficacy monitoring.
Tuberculosis (TB) is a global health crisis that has frustrated efforts to treat and contain, and, unlike other bacterial infections that can be treated with a week-long course of a single antibiotic, TB therapy requires several drugs in combination for at least 6 months and often even this regimen does not work. Several front- line TB drugs target a structure called the mycomembrane and in this project we will: (1) study how TB drugs alter the structure of the mycomembrane using a model of tuberculosis in zebrafish, and (2) develop a better detection method for active TB in patient sputum samples through collaboration with a group in South Africa that works with TB-infected and HIV/TB-coinfected patients. Our new method for clinical TB diagnosis should make it easier to determine whether TB drugs are working in patients; additionally, if they are not working well, new insight gleaned from this work may help us to better understand why.
| Walton, Eric M; Cronan, Mark R; Cambier, C J et al. (2018) Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling. Cell Host Microbe 24:514-525.e6 |
| Rodriguez-Rivera, Frances P; Zhou, Xiaoxue; Theriot, Julie A et al. (2018) Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs. Angew Chem Int Ed Engl 57:5267-5272 |
| Kamariza, Mireille; Shieh, Peyton; Ealand, Christopher S et al. (2018) Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe. Sci Transl Med 10: |
| Kamariza, Mireille; Shieh, Peyton; Bertozzi, Carolyn R (2018) Imaging Mycobacterial Trehalose Glycolipids. Methods Enzymol 598:355-369 |
| Schump, Michael D; Fox, Douglas M; Bertozzi, Carolyn R et al. (2017) Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis. Antimicrob Agents Chemother 61: |
| Tapia, Hugo; Young, Lindsey; Fox, Douglas et al. (2015) Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 112:6122-7 |
| Shieh, Peyton; Dien, Vivian T; Beahm, Brendan J et al. (2015) CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum. J Am Chem Soc 137:7145-51 |
| Seeliger, Jessica C; Topp, Shana; Sogi, Kimberly M et al. (2012) A riboswitch-based inducible gene expression system for mycobacteria. PLoS One 7:e29266 |
| Hatzios, Stavroula K; Bertozzi, Carolyn R (2011) The regulation of sulfur metabolism in Mycobacterium tuberculosis. PLoS Pathog 7:e1002036 |
| Hatzios, Stavroula K; Schelle, Michael W; Newton, Gerald L et al. (2011) The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth. Bioorg Med Chem Lett 21:4956-9 |
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