Date Published: March 21, 2017
Publisher: Public Library of Science
Author(s): Alexandra Moores, Ana B. Riesco, Stefan Schwenk, Kristine B. Arnvig, Tanya Parish.
Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.
In spite of significant endeavours in both drug discovery and vaccine development, tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global threat to human health. Drug treatment is lengthy and complex often with severe side effects, especially for patients with multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB). MDR-TB accounted for an estimated 480 000 incidents in 2014, with almost 10% of those being XDR-TB . Moreover, the Mycobacterium bovis BCG vaccine does not always offer the desired or even expected protection, and in most cases protection only lasts a decade or two . It is therefore essential that we investigate all aspects of M. tuberculosis gene regulation in order to understand the host-pathogen interactions important for establishing and maintaining infection. A substantial volume of literature describes stress induced transcriptional changes in the M. tuberculosis gene expression program [3–6]. Less well-known are the post-transcriptional changes mediated by regulatory RNAs such as riboswitches and small regulatory RNAs (sRNAs).
Within the last decade it has become increasingly evident that post-transcriptional regulation of gene expression, including sRNA expression and sRNA/mRNA stability, contributes significantly to pathogen adaptation and survival. Nevertheless, knowledge about M. tuberculosis RNA biology is limited, and in order to fully understand the potential of this pathogen’s ability to adjust to the host environment we need to study these molecular mechanisms involved in regulating gene expression. To shed more light on these processes we characterised the expression, maturation, stability and turnover of DrrS, a DosR regulated sRNA from M. tuberculosis. We found that in M. bovis BCG, the activity of the main DrrS core promoter was significantly activated between exponential and stationary phase, suggesting that the promoter may be recognised by an alternative sigma factor, either in addition to, or instead of SigA, which is mostly log-phase associated . However, scrutiny of the DrrS core promoter region did not reveal any obvious motifs for alternative sigma factors. Finally, we found that in M. smegmatis the DrrS core promoter was significantly activated (13–29 fold) in the presence of DosRtb, while the activity of the extended promoter region, which includes a known DBS, was only activated 5–8 fold and overall showed a decrease in promoter activity relative to the core promoter (Table 3). As this pattern was similar in two different strain backgrounds, i.e. mc2155 and ΔdosRsm we believe that the results are authentic, but based on our current results we cannot determine if this is due to a novel molecular mechanism of DosR or if there are other factors involved. The weaker activation by DosRtb in the presence of DosRsm may be due to competition between the two factors, which have been shown to functionally overlap , although we would expect the level of DosRtb to be significantly higher than DosRsm due to the expression from a strong, exponential-phase promoter of the former. There are no reports on sigma factor expression being induced by DosR and hence we assume that activation of the DrrS core promoter by DosR is via a hitherto unknown molecular mechanism that does not require DBS. Some regulators stimulate transcription without DNA-binding but rather by directly interacting with RNA polymerase , although in our case it remains unclear how the promoter specificity would be achieved. It is possible that only promoters with -10 regions that diverge from the SigA consensus in a specific fashion, either by sequence or by spacing to the TSS would be recognised. The increased expression from the empty vector + dosRtb suggests that the dosRtb insert may contain a cryptic antisense promoter that drives low level expression of the lacZ reporter. However, the activity of the core promoter is still more than ten-fold higher than the empty vector, and hence the presence of such a cryptic promoter does not explain the dramatic activity of the core promoter in the presence of DosRtb.