Projects

BrkB's role in TB

"Due to the rise of antibiotic resistant tuberculosis and the long treatment time of current anti-tuberculosis therapies, there is an immediate need for new drug targets against tuberculosis. My lab has identified the mycobacterial BrkB ortholog as a likely drug target. We have already observed that mutation of mycobacterial BrkB greatly attenuates growth of our model organism Mycobacterium marinum in its natural host, the zebrafish. Furthermore, we have observed that this organism is attenuated for growth in both intracellular and extracellular spaces, making it particularly attractive because extracellular mycobacteria are especially present in late-stage TB and contribute to TB transmission, and because there is a specific need to target mycobacteria in the extracellular niche. While BrkB is found throughout the bacterial kingdom, the function of BrkB remains elusive. I propose to characterize the biochemical function of mycobacterial BrkB and resolve its role in virulence by completing the following three specific aims: (1) Identify mycobacterial BrkB channel substrate(s) and test for protein interactions, (2) identify mechanism of BrkB extracellular growth requirement, and (3) Measure mycobacterial BrkB growth under late-stage infection conditions. " - excerpted from the project abstract. For further reading, visit here.

Presently, students are purifying recombinant tuberculosis BrkB from insect cell culture and are working to crystallize this purified protein to evaluate its structure. As his thesis research, Piotr Pawlowicz purified the m. smegmatis BrkB-Dendra2-FLAG using immunoprecipitation, and identified coprecipitants by protein/peptide mass spectrometry. Fiona Oberhofer is presently developing a protein-protein interaction platform to test top candidates for interaction in e. coli, m. smegmatis, and m. marinum as a means of identifying the function of BrkB.

Mycosin protease inhibitors

Proteases are enzymes that cut proteins and at least three different proteases exist in Mycobacterium tuberculosis that exist outside the cell and are required for tuberculosis to survive inside its host. Furthermore, proteases are one of the most druggable enzymes known. We use protease inhibitors against viruses (paxlovid for COVID and kaletra for HIV) and against heart disease and stroke (lotensin for high blood pressure and apixaban as a blood thinner). Therefore, we think purifying and exploring tuberculosis protease inhibitors presents an important avenue for future therapies. In my lab, students have purified mycosin protease 1 that is recombinantly expressed in the model organism e. coli. Students have developed protease kinetics assays and inhibitor assays. Students have modeled binding of inhibitors to the active site of mycosin protease using swissdock. Beginning in the spring of 2026 students will study mycosin protease 1 and 3 as part of a course-based undergraduate research experience and lecture (BMB305: Advanced Biochemistry) modeled after the ASBMB BASIL curriculum.  

TB Prophylaxis project

"Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC–MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells." - excerpted from Robbins et al. 2023.

For this project students students engaged in problems of genetic engineering, biochemistry, and bioethics. They considered and published the ethical framework for genetic engineering as a therapy regarding informed consent, safety and efficacy testing, and how genetic engineering approaches can be measured against each other. They reviewed all available antibiotics against tuberculosis to identify d-cycloserine as the most viable antibiotic based on the presence of precursors in the macrophage metabolome and the short overall biosynthetic pathway. They cloned all six d-cycloserine biosynthetic enzymes from Streptomyces lavendulae into a custom flag-GFP expression construct. They optimized expression of all six enzymes and purified one of the enzymes to determine that human cells express active enzyme. They made progress towards measuring efficacy of the genetic chemoprophylaxis approach. They optimized transfection of human lung cells, developed chromatography methods to detect enzyme product formation by mass spectrometry, and connected their work at the molecular level to effects on the cellular level. They concluded that prophylaxis requires a high safety threshold, and engineered plasmids to be excisable, to express functional enzyme only in the presence of tuberculosis, and to localize expression to the site of infection.

Moving forward, motivated students will need to select antibiotic enzymes, peptides, and pore forming toxins from mycobacteriophages and other sources to identify a simpler genetic prophylaxis that draws on the engineering problems solved by their predecessors.