Defining the mechanisms-of-action of nitrofuranyl piperazines against Mycobacterium abscessus
Defining the functions of the small molecule to inhibit M. abscessus growth and comparitive studies examining the mechanisms of action in M. tuberculosis vs. M. abscessus. Overall design: Mab cultures were treated in two biological replicates with 10 µM of HC2210 and an equivalent volume of DMSO for 24 hours at T25 standing flasks in 37°C incubator (without 5% CO2). The same is done for Mtb cultures except that 2 µM of HC2210 is used and cultures were incubated in T25 standing flasks at 37°C, 5% CO2 for 24 hours. After treatment, the pellets were harvested, and the bacterial RNA was extracted using the TRIzol-based protocol as previously described(56). Samples were DNAse treated with Invitrogen DNAse (RNAse free). Library preparation was performed using Illumina's Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10bp unique dual indices (UDI). Sequencing was done on a NovaSeq X Plus, producing paired end 150bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v4.1.5). The sequencing reads were analyzed using the commercially licensed CLC Genomics Suite and are presented in Dataset 3.1 (for Mab) and Dataset 3.2 (for Mtb).