Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Abstract

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.

eLife digest

Tuberculosis (TB) is an infectious disease that affects over ten million people every year. The Mycobacterium tuberculosis bacteria that cause the disease spread through the air from one person to another and mainly infect the lungs. Although curable, TB is difficult to eradicate because it is remarkably widespread, with one third of the world’s population estimated to carry the bacteria.

Treatment for TB involves a mix of antibiotics that should be taken for several months to a year. The number of multidrug-resistant TB cases, where the infection is not treatable by the common cocktail of antibiotics, is rapidly increasing. There is therefore a need to discover new drugs that can kill the M. tuberculosis bacteria.

An antibiotic called fidaxomicin is used to treat intestinal infections. Although it can kill Mycobacterium tuberculosis cells in culture, it is not absorbed from the intestines to the blood and thus cannot reach the lungs to kill the bacteria. It may be possible to change the structure of the drug so that it can enter the bloodstream. Before this can be done, researchers need to understand exactly how fidaxomicin kills the bacteria so that they know which parts of the drug they can alter without making it less effective.

Fidaxomicin kills bacterial cells by binding to an enzyme called RNA polymerase. The antibiotic prevents the enzyme from reading and ‘transcribing’ DNA to form molecules that are essential for life. To learn more about how fidaxomicin has this effect, Boyaci, Chen et al. used cryo-electron microscopy to look at structures of the M. tuberculosis RNA polymerase in different states, including when it was bound to fidaxomicin.

The structures reveal the chemical details of the interactions between the RNA polymerase and the antibiotic. The two molecules bind to each other through a region of the RNA polymerase that is unique to M. tuberculosis and closely related bacteria. Fidaxomicin acts like a doorstop to jam the RNA polymerase in an open state that cannot bind to DNA and transcribe genes.

Medicinal chemists could now build on these findings to develop new drugs that might treat TB, either by modifying fidaxomicin or designing new antibiotics that bind to the same region of the RNA polymerase. Because the fidaxomicin-binding region of the RNA polymerase is specific to M. tuberculosis new antibiotics could be tailored towards the bacteria that have a minimal effect on a patient’s normal gut bacteria.