MOLECULAR BASIS OF PKNB ESSENTIALITY IN MYCOBACTERIA

Lead Research Organisation: University of Leicester
Department Name: Infection Immunity and Inflammation

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50

 
Description We found that serine/threoinine protein kinase B (PknB) is not essential for mycobacterial division but required for synthesis of peptidoglycan. We established CwlM as a major PknB substrate and determined functions of phosphorylated and non-phosphorylated forms of CwlM. We established that CwlM interact with the key protein involved in peptidoglycan biosynthesis (MurJ and FhaA) and characterised a post-transcriptional regulation of CwlM by aconitase. Furthermore we found that PknB phosphorylates Lsr2, a global transcriptional regulator, to adjust gene expression according to bacterial growth. We have also shown that serine/threonine protein phosphatase may directly influence PknB activity and investigated phosphoproteomics of PstP depleted mycobacteria. These findings have been published.
Exploitation Route We developed methodology for growing PknB-depleted mycobacteria; this can be used by others for research. We conducted phosphoproteomics analysis of PknB-depleted mycobacteria; data are available to researchers. We generated several bacterial strains that can be used for investigation of mycobacteria and expression of mycobacterial proteins.
Sectors Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Determining the persister populations in sputum during tuberculosis therapy. A supplementary study to the RIFASHORT trial (Co-Investigator)
Amount £472,512 (GBP)
Funding ID MR/P011357/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 12/2020
 
Description MIBTP iCASE
Amount £21,400 (GBP)
Organisation Public Health England 
Sector Public
Country United Kingdom
Start 08/2018 
End 09/2022
 
Description Newton Mobility Grants 2020 Round "Identification and evaluation of novel PknB inhibitors as anti-tuberculosis agents), applicant Dr Pharit Kamsri
Amount £12,000 (GBP)
Funding ID Royal Society-Newton Mobility Grant NMG\R1\201061 
Organisation Nakhon Phanom University 
Sector Academic/University
Country Thailand
Start 12/2020 
End 12/2022
 
Description PhD studentship
Amount £43,525 (GBP)
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom
Start 08/2017 
End 09/2020
 
Title Development of media for growing PknB-depleted Mtb 
Description We developed a special medium which enables cultivation of PknB-depleted Mtb and application of comics approaches. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact This is a novel approach that can be used for investigation of conditional mutants. 
URL https://www.cell.com/cell-reports/pdf/S2211-1247(18)31419-0.pdf
 
Title NMR solution structure of LSR2 binding domain. 
Description Linked to a publication doi: 10.1111/mmi.14398. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact No direct impact. However, this finding aid our understanding of phosphorylation impact on protein dynamics. 
URL https://bmrb.io/data_library/summary/?bmrbId=34358
 
Title NMR solution structure of LSR2-T112D binding domain. 
Description It is described in publication doi: 10.1111/mmi.14398. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact This structure revealed how phosphorylation changes dynamics of protein domains. 
URL https://bmrb.io/data_library/summary/?bmrbId=34359
 
Title Vaccines Sulman et al 2021 
Description This is the core data for the paper Balance between Protection and Pathogenic Response to Aerosol Challenge with Mycobacterium tuberculosis (Mtb) in Mice Vaccinated with TriFu64, a Fusion Consisting of Three Mtb Antigens 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact No direct impact. 
URL https://leicester.figshare.com/articles/dataset/Vaccines_Sulman_et_al_2021/14607879
 
Description Collaboration with Dr Pharit Kamsri and Dr Pornpan Pungpo to design novel PknB inhibitors 
Organisation Nakhon Phanom University
Country Thailand 
Sector Academic/University 
PI Contribution We initiated design of novel inhibitors of PknB as drugs to target mycobacteria.
Collaborator Contribution My partners will design and synthesise PknB inhibitors.
Impact No outputs yet. Our collaboration resulted in successful grant application for Royal Society-Newton Mobility Grant.
Start Year 2020
 
Description Collaboration with TB centre (Georgia, Tbilisi) 
Organisation National TB Reference Laboratory, Tbilisi, Georgia
Country Georgia 
Sector Private 
PI Contribution Transferring expertise to handle Rpf-dependent mycobacteria.
Collaborator Contribution Access to clinical Mycobacterium tuberculosis isolates and samples.
Impact No current outputs.
Start Year 2017
 
Description DSTL 2017 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution Studentship jointly funded by the University of Leicester and DSTL. Leicester provides expertise in macrophage infection, activity assays and resuscitation experiments.
Collaborator Contribution DSTL provides expertise in Burkholderia pseudomallei biology.
Impact Currently no outputs.
Start Year 2017
 
Description Newcastle PknB essentiality 
Organisation Newcastle University
Department Institute of Cellular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution My group focuses on mycobacterial dormancy, resuscitation and growth and provides expertise in serine/threonine protein kinase signalling, genetic manipulations and phenotypic characterisation of mycobacteria. We generated strains for production of several recombinant proteins and provided them to the partners for assessment of peptidoglycan-hydrolysing activity.
Collaborator Contribution Prof Vollmer is a world leading expert in peptidoglycan biosynthesis and remodelling. His group provides expertise in analysis of cell wall enzymes, novel activity assays and identification of protein=protein interactions.
Impact No specific outcome generated at this stage.
Start Year 2016
 
Description Establishment of Leicester Infectious Disease Centre 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Organise LeMID seminars, worshops and grant development sessions.
Year(s) Of Engagement Activity 2019
 
Description Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Open day activities and discussions with potential candidates about research and study in Leicester.
Year(s) Of Engagement Activity 2016,2017,2018,2019
 
Description World tuberculosis day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact 24th March is the World Tuberculosis day. We organised a special event to mark this day and increase public awareness on tuberculosis.
Year(s) Of Engagement Activity 2019
URL http://le.ac.uk/news/2019/march/22-world-tb-day