The role of small regulatory RNAs in Mycobacterium tuberculosis pathogenesis

Mycobacterium tuberculosis, remains a major threat to global health. It is responsible for more deaths than any other bacterium and claims around 1.5 million lives every year in a deadly synergy with Human Immunodeficiency Virus (HIV), leaving ten million children orphaned by this disease. Rational design of drugs and vaccines to combat tuberculosis is currently hampered by limitations in basic understanding of the biology of the bacterium. M. tuberculosis persists in the human host as multiple subpopulations, an adaptation that requires significant and appropriate changes in gene expression. Gene expression is a multi-step process, but research in M. tuberculosis has until recently focused on only the first step, which is largely regulated by protein factors. However, it has become evident that novel and quite different non-protein regulators, the so-called small RNAs (sRNAs), play a major role in stress responses in all bacteria. In the case of disease causing bacteria (i.e. pathogens), these sRNAs play a crucial role in the adaptation to hostile host environments. sRNAs are able to modify the final outcome of gene expression after the initial step has taken place, by either promoting or inhibiting the next step in gene expression, that is, the production of bacterial proteins. These proteins include the building blocks of the cell as well as various effector molecules such as regulators and factors that are secreted in order to interfere with the host's defence against the invading pathogen. This project aims at investigating selected M. tuberculosis sRNAs that are highly expressed during infection and therefore might be of importance in host adaptation and development of the disease (pathogenesis). By artificially manipulating the levels of individual sRNAs we will be able to detect which genes and molecules may be affected by these regulators and hence what cellular mechanisms are involved. However, we will not only be able to determine what role the sRNAs play in pathogenesis. This project will also have broader implications in elucidating basic molecular mechanisms such as regulation of M. tuberculosis gene expression and metabolism, which will eventually provide us with a better understanding of the bacterium, and lead to the development of novel intervention strategies for tuberculosis.

The proposed project aims at investigating various aspects associated with M. tuberculosis small RNAs (Mtb sRNAs). I propose to characterize stimuli and regulators of expression, the targets that the sRNAs regulate, the role of one of the main RNases and whether the sRNAs require chaperones to facilitate interactions with their targets. Finally, I want to investigate the role that these sRNAs play in Mtb pathogenesis.
In order to investigate stimuli and regulators of expression I will subject cultures of Mtb to infection related stresses (e.g. oxidative stress, NO stress, hypoxia, iron depletion) and quantify the expression of each sRNA using northern blotting and qRT-PCR. In order to define regulons of the sRNAs, I will employ a panel of Mtb strains in which the expression of each sRNA can be manipulated (deletion mutants/complemented/over-expression strains). The strains will be subjected to different growth conditions in vitro and used to infect macrophages and mice via low-dose aerosol. RNA will be extracted and differential gene expression will be characterized using microarrays.
To further identify targets I have devised a strategy for isolating proteins and (m)RNAs that interact with the sRNAs. Aptamer-tagged versions of each sRNA will expressed in vitro and used to isolate interacting molecules from Mtb cell extracts using commercially available streptavidin beads. The tagged sRNAs will subsequently be introduced into their cognate knockout background, cultures will be grown under appropriate conditions and sRNA bound to interaction partners isolated from cell extracts. Weak interactions can be stabilized with an optional UV irradiation step before cell disruption. Isolated proteins will be identified by mass spectroscopy. Isolated RNA will be characterized by RNAseq.

Who will benefit from this research?

The immediate beneficiaries of this research will be scientists working in the fields of 1) M. tuberculosis (Mtb) basic and clinical research; 2) regulation of bacterial gene expression; 3) structure, function and evolution of bacterial small regulatory RNAs (sRNAs); 4) other, pro- and eukaryotic non-coding RNAs

Additional beneficiaries include clinicians working with tuberculosis (TB) patients and pharmaceutical companies in the development of novel diagnostic tools, drugs and vaccines. Ultimately, although more long-term, our research will benefit TB patients, the general public and the public sector, particularly in communities with a high prevalence of TB.

How will they benefit?

The TB research community will benefit from the increased understanding on how Mtb adapts to and survives in a normally hostile host environment. Previous efforts in characterising Mtb gene regulation have been dominated by a protein-centric view. However, at present there can be no doubt that sRNAs play an important role in stress responses and pathogenesis, and it is essential to include this hitherto uncharacterised reservoir of regulators in our efforts to understand Mtb.

sRNAs are deeply integrated into the regulatory networks of bacterial gene expression. Novel findings from non-model organism such as Mtb will contribute to the general knowledge about stress responses and adaptation and hence expand our understanding of basic regulatory mechanisms.

Scientists working on small regulatory RNAs in other more tractable organisms will benefit from the added knowledge about how GC-rich sRNAs interact with their targets without the aid of the otherwise widespread RNA chaperone Hfq. This knowledge will aid our understanding of the evolution of structure-function relationships in all types of non-coding RNAs.

The added knowledge of RNA-RNA interactions and the possible identification of novel RNA binding proteins may offer inspiration for more distantly related systems, i.e. eukaryotic miRNAs, piRNAs and siRNA.

Due to the extreme accumulation of some sRNAs during infection, clinicians working with TB patients may be able to phenotypically characterise Mtb isolated from patients where only a small number of bacteria can be isolated.

Although RNA is currently not a drugable target, the proposed research on sRNAs may provide novel insights into known targets by virtue of their association with these. Pharmaceutical companies will be able to tap into the increased knowledgebase concerning regulation of gene expression and metabolism in their efforts to develop novel drugs and vaccines. They may also be able to exploit the infection-induced accumulation of sRNAs in the development of diagnostic tools. Finally, deeper insights into RNA biology may improve methods in RNA interference/gene silencing technology, which in future will be applicable to a plethora of human disease.

The ultimate goal for research into any pathogen is eradication of the disease it causes. The long-term beneficiaries of our research are the patients who will profit from improved diagnostics and therapies. Currently the standard drug regimen for active TB extends over six months with a combination of four different antibiotics. Accumulation of sRNAs linked to different stress regulons may act as sensitive markers of the physiological status of the small numbers of bacteria present in different parts of human and non-human lesions, providing crucial information to support rational targeting of improved therapies

The general public will benefit from the improved interventions in the form of prevention (vaccines and diagnostics) and cure (diagnostics and drugs). This will have particular impact in communities with a high prevalence of TB, i.e. mostly developing countries, but also in more local (i.e. London) communities. Finally, improved health in the public will have a financial impact on the public sector.