MOLECULAR BASIS OF PKNB ESSENTIALITY IN MYCOBACTERIA

Mycobacteria are a group of versatile microorganisms which include medically important pathogens and environmental bacteria. Mycobacteria have developed distinct mechanisms enabling their prolonged survival in hostile conditions and adaptation to a wide range of environmental niches. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium with a complex lipid-rich cell wall that uses multiple signaling mechanisms to coordinate division with the biosynthesis of cellular components. Eleven serine/threonine protein kinases are particularly important for regulation of cellular processes in Mtb. The serine/threonine protein kinase B (PknB) is essential for mycobacterial growth, although the reasons for this are unknown. Previous attempts to deplete or over-produce PknB resulted in rapid death of mycobacteria. We have recently discovered special conditions, which support growth of mycobacteria that do not produce PknB, and compared phosphorylated proteins in mycobacteria producing and missing PknB. This novel approach has aided the identification of proteins that are phosphorylated by PknB, which is the first step in establishing the molecular mechanisms of PknB essentiality. Our findings and previously published results suggest that PknB phosphorylates enzymes involved in biosynthesis and remodelling of peptidoglycan, the major component of bacterial cell wall, however the precise role of phosphorylation of these proteins remains elusive. Within this project we propose to investigate the mechanisms of PknB-mediated regulation of peptidoglycan biosynthesis by measuring the activities of phosphorylated and non-phosphorylated enzymes, their localization in cells and export onto the cell surface, and the role of phosphorylation in the interaction of enzymes with other proteins. We will work on two genetically related organisms, Mtb and Mycobacterium smegmatis, and generate mutants missing the PknB substrates or expressing their altered forms which cannot be phosphorylated by PknB. The growth of these mutants in various media will be assessed. We will also use methods established by us to identify partners interacting with PknB-phosphorylated proteins and to elucidate the mechanistic details of peptidoglycan biosynthesis in mycobacteria and its regulation by PknB. In separate experiments we will study how PknB phosphorylation contributes to the formation of dormant mycobacteria and their resuscitation. The expected outcomes of this project will improve our understanding of fundamental cellular processes in mycobacteria and will stimulate the development of novel approaches to target Mtb in different physiological states. Our results will also contribute to deciphering the function of serine/threonine protein kinases in prokaryotes.

Tuberculosis is a global infectious disease which affected over 9 million people and caused 1.5 million deaths in 2014. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has evolved mechanisms to evade the host immune system allowing it to persist in vivo without causing an active disease. Eleven serine/threonine protein kinases control Mtb growth, persistence and cell wall biosynthesis. The protein kinase PknB is essential for mycobacterial growth, but the molecular basis of its essentiality is unknown. We have established that PknB-depleted Mtb can multiply and grow in a special osmoprotective medium. Comparative phosphoproteomic analyses of PknB-depleted and PknB-producing strains revealed several PknB substrates, including proteins involved in peptidoglycan biosynthesis, protein secretion and membrane protein assembly. Based on our data we hypothesise that PknB is dispensable for growth and division itself but plays a critical role in coordinating cell wall biosynthesis with cell growth. The proposed project is aimed to establish (i) the effect of PknB-depletion on peptidoglycan biosynthesis and cell wall structure; (ii) the role of PknB-mediated phosphorylation in regulation of the activities and interactions of the cell wall amidase Rv3915 and synthases PonA1, PbpA and MurG; (iii) the effect of phosphorylation on the cellular localisation of PknB substrates; and (iv) PknB-specific phosphoproteomic signatures of non-replicating and resuscitating mycobacteria. The experimental approaches will include phosphoproteomics and proteomics, genetic manipulations and the generation of mycobacterial mutants, peptidoglycan analysis, assays for measuring the activities of peptidoglycan synthases and hydrolases, and protein/protein interaction techniques. The project is expected to reveal the molecular basis for PknB-mediated signaling pathways and to stimulate the development of drugs against Mtb.

The proposed research will have impact on scientists working on 1) bacterial growth, dormancy and division mechanisms; 2) cell wall biosynthesis and peptidoglycan remodeling; 3) post-translational modifications and signaling mechanisms; 4) protein stabilization and improvement of protein activity; 5) tuberculosis diagnosis, treatment and prevention. Scientists will benefit from expanding of knowledge and understanding of molecular mechanisms. Advance in genetic manipulations and omics technologies will be also highly beneficial for fundamental and applied research. Findings of the project may also influence the current concepts of bacterial division and growth and therefore impact teaching of students.
Advance in our analysis of bacterial proteins and phosphoproteins will be also beneficial in development of research tools and improvement of existing kits for molecular research.
The project will be focused on investigation of mycobacteria, many of which are clinically important human and animal pathogens. One third of the entire world population are latently infected with tuberculosis and around 1.5 million people die every year from tuberculosis. Furthermore according to DEFRA "bovine tuberculosis (bTB) is the most pressing animal health problem in the UK". Despite the recent efforts to control the spread of bTB the number of new bTB incidents in the UK has not been reduced. Thus, the expected project outcomes on controlling mycobacterial growth and cell wall biosynthesis will be beneficial for clinicians and veterinarians.
The results of this project may be of interest for the pharmaceutical industry and drug design companies. Firstly, by providing important knowledge on essential proteins as important drug targets. Mycobacteria are particularly difficult organisms to treat as they have a highly hydrophobic cell wall. The project will be directed on study of secreted and cell surface proteins, which can be accessed by inhibitors and drugs without the necessity of penetrating the membrane to reach the targets inside the cell. Secondly, hydrolytic enzymes studied in this project can potentially be used for increasing of potency of known and novel drugs. The dysregulation of these cell wall hydrolases or their delivery by phages may be used as an alternative means of killing bacteria. These approaches would be particularly useful for the development of environment-friendly tools for the eradication of tuberculosis in cattle and badgers, which are believed to be the major contributing factor to the spread of bovine tuberculosis.
The proposed research may also impact biotechnological industry by expanding the expertise on stabilisation of enzymes and improving their activity by post-transcriptional modifications.