William Jacobs, by initiating work with mycobacteriophages, has developed novel genetic approaches to make mutations and transfer genes in Mycobacterium tuberculosis. With these tools, he has identified drug targets and novel virulence factors of M. tuberculosis, many of which are enzymes or products of complex lipid metabolism. These complex lipids are unique among bacterial pathogens and likely to contribute significantly to the pathogenic property of mycobacteria. His lab uses this knowledge to develop novel chemotherapies, vaccines, and diagnostic tests to treat tuberculosis.
Trigger Factor: A Chaperonin Essential for Mycobacterium Tuberculosis Virulence is Exploited by Mycobacteriophage DS6A for Successful Propagation
Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that continues to be a global health burden primarily effecting people in the developing world. M. tuberculosis is part of a family of mycobacteria that contain both slow-growing and fast-growing species. M. tuberculosis is slow-growing with an average generation time of 24 hours, while the very similar M. smegmatis has a generation time of only 3 hours. The molecular basis for this slow growth remains a mystery. For the last 30 years, my laboratory has used mycobacteriophages (viruses that infect mycobacteria) to develop genetic tools and investigate the properties of the mycobacterial species. Mycobacteriophages can be readily found in most soil samples along with mycobacteria. Amongst the 6000+ known mycobacteriophages, DS6A is unique in that it plaques on members of the M. tuberculosis complex but not M. smegmatis. Thus, we reasoned that understanding the genetic basis of this specificity might shed light on unique properties of M. tuberculosis.
Using a transposon mutagenesis screen, Trigger Factor (Rv2462c) was found to play an essential role in DS6A propagation in M. tuberculosis. Trigger Factor is a ubiquitous chaperonin that binds nascent peptides as they leave the ribosomal channel. Bound peptides are protected from misfolding and aggregation as they’re either combined with additional peptides to form complexes or are transported to target cellular compartments. No host range mutants able to overcome a tig null deletion have arisen; interestingly however, no other tested phages able to infect M. tuberculosis were affected by this tig deletion. We have generated two independent DS6A fluorescent reporter phages and demonstrated that in M. tuberculosis, ∆tig mutants, infection, DNA entry, and transcription/translation occur if the DS6A phage occurs. We are currently using transmission electron microscopy, small molecule binding, and co-immunoprecipitation assays to determine the role Tig is playing in the assembly or escape of the DS6A phage.
We have demonstrated that Trigger Factor plays an important role in virulence of M. tuberculosis. Mutants with a tig deletion took about twice as long to kill SCID mice as the wild type when given as an IV injection. In healthy mice exposed to wild type and tig deletion mutants in a low dose aerosol study, tig deletion mutants had 2-log fewer cells at their peak numbers compared to the wild type in the lungs, while they took longer to grow up in the spleens than the wild type, and were very quickly cleared. The tig deletion may be stimulating the adaptive immune system to more aggressively clear bacteria from the lungs and spleen. Current efforts will be focused on not only elucidating the mechanism of M. tuberculosis specificity, but also understanding why this mutant is highly attenuated in mice. Further studies on how the bacteria are initially cleared as well as what immune factors it is eliciting are required.