Program: Molecular Microbiology and Microbial Pathogenesis
Current advisor: Christina L. Stallings, PhD
Undergraduate university: Washington University
I recently rotated in the lab of Christina Stallings, participating in studies of Mycobacterium tuberculosis (Mtb), the leading infectious cause of death worldwide. During my rotation I investigated a molecular compound, termed EC309, found to have inhibitory effects in Mycobacterium spp. My goal was to determine if EC309 has a similar mechanism of action to C10, an analogous compound that inhibits Mtb respiration and biofilm production and sensitizes it to the antibiotic isoniazid. To assess this, I supplemented EC309-treated M. smegmatis cultures with intermediates of the TCA cycle and associated methylcitrate cycle (MCC), and measured respiration inhibition. Such supplementation has been shown to alter C10’s inhibitory function in Mtb. C10 has also been shown to alter transcription of genes encoding MCC enzymes, so I additionally collected RNA from EC309-treated M. smegmatis cultures and performed rtPCR for several MCC genes. While the results of these experiments were strong, they were unfortunately negative and suggested that EC309 has a different mechanism of action than C10, at least in M. smegmatis. Further investigation will be performed to see if this phenotype is conserved in Mtb, or if it is a species-specific result. Additionally, I investigated CarD, a Mycobacterium RNA polymerase-binding transcription factor that stabilizes the RNAP-promoter complex, previously shown by the group to influence genome-wide expression. Particularly, I was interested to see how changes in CarD abundance influenced gene regulation in times of nutrient starvation, when Mycobacterium must adapt in order to persist. Western blot of lysate from wild type and CarD mutant strains grown in both nutrient-supplied and nutrient-starved conditions suggested that CarD protein abundance was lower under starved conditions. Furthermore, rtPCR of starvation-associated genes in these strains suggested that expression of genes associated with translation, transcription, and energy metabolism are altered in starvation conditions, largely promoting an efficient, energy-conserving state. However, the integrity of the housekeeping genes used for these rtPCR analysis is a point of ongoing investigation and the role of the specific CarD mutations on protein function requires further clarification.