Protein O-glycosylation enzymes as targets for novel antimicrobials against mycobacteria

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Background

Mycobacterium tuberculosis (Mtb) infects around 2 billion people worldwide annually. The organism is a member of the Actinobacteria, a group of Gram-positive bacteria that includes corynebacteria and the streptomycetes. Actinobacteria have a protein-O-glycosylation system that is also conserved in eukaryotes, including man. Mtb mutants defective in protein glycosylation are dead whilst Streptomyces and Corynebacterium mutants are hypersensitive to certain antibiotics. Two enzymes required for protein glycosylation are a polyprenol phosphate mannose synthase (Ppm1) and a protein O-mannosyltransferase (Pmt). Ppm1 is a membrane associated enzyme that transfers mannose from GDP-mannose to a lipid carrier in the membrane, to form polyprenol phosphate mannose, PPM. Pmt is an integral membrane protein that transfers the mannose from PPM to periplasmic proteins. In mycobacteria PPM is also required for the synthesis of the essential cell envelope lipoglycans.

Objectives

Our goal is to understand the structure and mechanism of the protein glycosylation enzymes with a view to identifying novel inhibitors. This project will focus on Ppm1 to determine protein structure and enzyme properties. Ppm1 from Streptomyces is 57% identical to the Mtb homologue and has been overexpressed in E. coli to give approximately 8 mg/liter of soluble protein. We will use a novel assay to characterize the enzyme and attempt to determine its 3D structure or that of a close homologue. The structure will be used for fragment bases screening in collaboration with Prof Hubbard. In an independent line of study we will investigate the structure/function of Ppm1 by mutation and phenotypic analysis.

Novelty & Timeliness

Glycosyltransferases are extremely diverse and the 3D structures of only 7 out of >45000 CAZy GT2 enzymes are known. If solved the Ppm1 structure would be novel and provide insights into the eukaryotic homologue, Dpm. The project is timely as we have unpublished genetic and biochemical evidence for the importance of this enzyme in cell envelope homeostasis in bacteria.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 01/10/2015 30/09/2023
1643229 Studentship BB/M011151/1 01/10/2015 30/09/2019 Nathanial Holman
 
Title York Research Showcase Exhibition 
Description To showcase my research on Streptomyces bacteria. I presented agar plates of Streptomyces producing various antibiotics. These antibiotics are often brightly coloured and so are very attractive to the eye. The audience could also see the "fuzzy" spores made by these bacteria by eye and they could use a microscope to see these bacteria in more detail. I also presented antibiotic disc diffusion plates to illustrate to my audience how we test for antibiotic sensitivity in hospitals, research labs etc. Finally, I presented an accessible poster outlining the problem of antibiotic research and how my PhD research project aims to address this. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2016 
Impact The audience was a combination of University of York students and members of public. Regarding members of the public and non-science York students, this was the first time that many of them had seen an agar plate of bacteria, where they could make observations both by eye and with a microscope. The showcase was well received and I raised awareness of the emerging problem of antibiotic resistance, and the contribution I hope my research will make to this area. The accessible poster also acted as an anchor, allowing my audience to quickly understand critical statistics about antibiotic resistance and consequently appreciate why it is such a huge problem. 
 
Description Please see previous submission, no changes
Exploitation Route Please see previous submission, no changes
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC DTP representative for BBSRC funded PhD students at York University
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact I represented ~40 York University BBSRC funded PhD students at committee meetings with senior management, negotiating changes to training and deadlines requested by students/supervisors. some of these changes would also affect BBSRC funded PhD students at the other White Rose network universities (Sheffield and Leeds).
 
Description S. coelicolor Ppm1 Warwick Collaboration 
Organisation University of Warwick
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution I took a leading role in setting up and maintaining a collaboration with researchers from Warwick University. The collaboration involves myself, my PhD supervisor Prof Maggie Smith, Warwick PhD student Pamela Banana Dube and her supervisor Prof Maneula Tosin. We wanted to study the in vitro and in vivo consequences of S. coelicolor Ppm1 mutation. My contributions in the collaboration were focussed on the in vivo work. We received S. coelicolor Ppm1 mutant genes from Warwick, we cloned them into integration vectors, introduced them into Ppm1- strains of S. coelicolor (via ETZ conjugation), selected for and harvested the spores and finally were ready to carry out the in vivo work (antibiotic and phage sensitivity assays). We showed that the majority of Ppm1 mutants introduced into the Ppm1- S. coelicolor strain could not restore the wild type phenotype and were hypersensitive to multiple cell wall targeting antibiotics and resistant to phage infection. Our conclusions were that these mutants were inactive due to their failure to alleviate the antibiotic sensitivity phenotypes and the absence of phage infection (this is based on previously published data from Prof Smith). Of the mutants screened, 2 were partially active, 2 were active and 7 were inactive.
Collaborator Contribution Pamela Dube made the mutants genes and expressed and purified the mutant proteins for biochemical screening. She carried out a Malachite Green biochemical assay to test for activity. Her in vitro biochemical data was in very good agreement with our own in vivo molecular microbiology assays. Biochemical assays involved carrying out kinetic profiling where the Km, vmax and Kcat were determined. In addition to the biochemical assays, she also chemically synthesised multiple donor and acceptor substrates to be used in the assays.
Impact In vitro biochemical and in vivo molecular microbiology assays data is in very good agreement. Experimental work has been concluded and we are in the process of writing a manuscript for publication. I have written the draft of the York portion of this work.
Start Year 2017
 
Description Gradshare Manager 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Gradshare is an event were internal and external guest speakers who have forged successful careers both inside and outside academia are recruited to talk to an audience of mostly PhD students about career opportunities post PhD. During my time as Gradshare manager I:
1) Arranged five speakers from diverse research backgrounds to give careers talks to students
2) Publicised the talks by word of mouth, email and social media to roughly double attendance
3) Negotiated a departmental budget allowing me to source refreshments that further increased attendance
Year(s) Of Engagement Activity 2015,2016
 
Description Pint of Science 2019 York Event Manager for Tech Me Out 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I have arranged several speakers for the 2019 Pint of Science event Tech Me Out. I am now in the process of trying to publicise the event, we anticipate that across all the Tech Me Out speaker events in May, we can hopefully reach several hundred people. Three pubs have been booked already to serve as host venues.
Year(s) Of Engagement Activity 2019
 
Description RSC Chemistry at Work (Antibiotics Awareness Workshop) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I organised and ran my own antibiotics awareness workshop in the University of York Chemistry Department as part of the RSC outreach event last July: Chemistry at Work. The purpose was to inspire a greater understanding of the importance of antibiotics in the field of medicine and how we can combine our understanding of chemistry and biology to not only better understand how antibiotics work, but also identify and make new/better ones. As the workshop was focussed mostly on chemistry, I had the school students build antibiotic structures using molymod kits and then had them relate structure to function. A mini lecture was also given on the history of antibiotics, their successes and limitations and alternatives such as the use of phage therapy . In total, I engaged with between 50-70 students. Feedback from teachers and students was very positive, the students especially enjoyed the use of the molymod kits as they could build and see the antibiotics for themselves, rather than seeing a 2D drawing on a board, this practical approach allowed the students to appreciate how the structure related to function. The mini lecture was also well received and there were many questions especially regarding phages e.g. what they are, and why we in in the West have focussed preferentially on antibiotics, and continue to do so. Given the success of this event, there are plans to organise more workshops for the next RSC Chemistry at Work event and I have been asked to take part again.
Year(s) Of Engagement Activity 2017,2018,2019
 
Description School visits (York) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Every term we get local and regional students who come to the University of York Biology Department. These students will spend the day carrying out some practicals to gain an understanding of what studying for a science degree would be like in University, practicals run in the past include DNA fingerprinting and Locust jumping. I assist the Teaching Fellows with these sessions every term and have done so for the past two years. As well as giving the students exposure to University practicals and helping to build their confidence with lab work generally, I also answer any questions they may have regarding the pursuit of a scientific degree and career. The students are often very enthusiastic and eager to ask questions. We often get repeat visit requests from schools who have previously participated in the visits, consequently these visit days are often oversubscribed, demonstrating the impact they have on the students and teachers who attend.
Year(s) Of Engagement Activity 2016,2017