Inhibition of Riboflavin Biosynthesis Combined with First-Line Drugs to Combat Mycobacterium tuberculosis
The biosynthesis of riboflavin is essential for the survival of Mycobacterium tuberculosis (M. tb), creating a promising target for novel therapeutic treatments. This study aims to evaluate whether the inhibition of riboflavin biosynthesis enhances the efficiency of anti-TB drugs to kill M. tb. Essential riboflavin biosynthesis genes, ribC and ribG, were silenced using CRISPRi. The CRISPRi system was placed under the control of an inducible promoter, anhydrotetracycline (ATc). Growth curves were performed, (+/-) ATc to identify the sgRNAs targeting ribC and ribG capable of inducing growth inhibition in riboflavin deficient M. tb. Quantitative RT-PCR was used to test for transcriptional repression in CRISPRi strains. To assess potential synergistic effects of inhibiting riboflavin biosynthesis with anti-TB drugs, Dose-Centering Assays were performed. The assays allowed the determination of the IC50 and IC90 of the first-line M. tb drugs (rifampicin, isoniazid, and ethambutol) on the CRISPRi M. tb strains. The results revealed that there was significant growth inhibition when RibC and RibG were grown with ATc on day 7 of growth, but not over the entire two week growth period. Only RibG revealed significant repression in transcription when qRT-PCR was performed. Dose-centering assays revealed that the CRISPRi strains and the control both had their growth inhibited when exposed to ATc. These findings suggest that there was another factor inhibiting growth and the CRISPRi strains would not be effective enhancers of anti-TB drugs.