Time to Diagnosis and Persistence: The Two Major Determinants of Effective Tuberculosis Control

The greatest challenge confronting effective tuberculosis (TB) eradication is the time to diagnosis, and duration of treatment of chronically infected individuals which represent a pool of infection. In an attempt to help limit the spread of TB in New Zealand, a fast SNP based diagnostic test was developed, to quickly identify the highly transmissible and virulent endemic Rangipo strain. The role of VapBC toxin-antitoxin systems in M. tuberculosis has been the subject of great interest recently, due to their expanded number in the genome and links with virulence and the regulation of cell growth in response to environmental stress. Their ability to regulate growth under adverse conditions for presumed survival advantages possibly leading to dormancy or persistence, make them ideal candidates for the development of new M. tuberculosis treatments. To establish differential expression of vapC, and therefore identify possible pathways and functions of the VapBC proteins, RT-qPCR was used to assess the expression levels of vapB and vapC in M. smegmatis under conditions of stress. No consistant changes in vapC mRNA levels were observed, resulting in the hypothesis that it is not the transcriptional differences which are important in the regulation of VapC, but post-transcriptional factors. In order to investigate the function(s) of M. tuberculosis VapBCs, these VapBC proteins were expressed and purified in M. smegmatis, and the VapC toxin tested for RNase activity. The purification, expression, RNase testing and bioinformatic analysis of M. tuberculosis VapCs suggested that VapCRv2530c, VapCRv0065 and VapCRv0617 may all target the same recognition sequence, UA*GG. Bioinformatic analysis revealed an abundance of this target sequence in horizontal gene transfer and TA genes, raising the possibility that VapC toxins could be functioning as selfish elements, or initiating transcriptional regulation cascades when a rapid change in the proteomic response and metabolic state of the cell is required. It is intriguing that the three M. tuberculosis VapC proteins tested thus far appear to target the same recognition sequence, possibly suggesting that all 47 VapCs are RNases and are targeting the same sequence. Alternatively; VapCs may belong to sub-groups targeting different sequences, allowing M. tuberculosis to exude both gross and fine metabolic control; or, they may share the same target, but are regulated by different activators triggered in response to different environmental stimuli