| More people are killed each year by Mycobacterium tuberculosis (MTb) than any other bacterial pathogen, due to the long and often ineffective treatment regimen required to eradicate the infection. This treatment regimen is becoming increasingly fruitless as multi-drug resistant (MDR) and extensively-drug resistant (XDR) MTb infections become more prevalent. MDR and XDR MTb pose a grave threat to global health, and it is imperative that new drugs are developed to fight these infections. We conducted two screens in order to identify new drug targets and compounds to combat MTb. First, transposon mutagenesis and sequencing, or Tn-seq, was conducted. Tn-seq is a whole genome method of identifying genes required for survival in a given environment. We challenged a library of transposon mutants with a high dose of rifampicin and then identified, through sequencing of the transposon junctions, which genes were under-represented in the surviving population. We found that genes involved in the plasma membrane and cofactor metabolic processes were likely to be required for survival during rifampicin treatment. Second, we conducted a natural products screen to identify compounds with activity against MTb. One compound, initially called Novo23 and later renamed "lassomycin", was identified. That in vitro study had determined that lassomycin binds to the ClpC1 ATPase subunit of the ClpCP proteolytic complex and both inhibits proteolysis and increases ATP hydrolysis. In order to determine lassomycin's mechanism of action, we conducted a proteomic analysis of MTb cells with and without lassomycin treatment, and found that lassomycin treatment causes a shift in MTb's proteome. We also measured ATP concentration in cultures treated with lassomycin, and found that lassomycin significantly decreased ATP concentration in comparison to untreated or rifampicin controls, similar to bedaquiline, a known ATP synthase inhibitor. Lassomycin is an entirely new class of drug and reveals a heretofore unknown mechanism of antibiotic action. |
