Characterisation of the effector ligand recognised by VanS, a key protein triggering resistance to vancomycin

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

Bacteria possess a wall that forms a barrier around their cells, helping to protect them. Antibiotics that disrupt bacterial cell wall construction, such as vancomycin, are clinically important in the treatment of infectious diseases. However, bacteria can adapt by developing resistance to these antibiotics making them ineffective for treating infections.

Despite some recent progress, relatively little is understood about exactly how bacteria sense an attack on their cell wall by an antibiotic, and how this can then be turned into responses that allow them to survive. We are using a harmless group of bacteria that live in the soil, streptomycetes, as a model system to investigate how this is achieved. Streptomycetes make the majority of antibiotics that we use to treat infectious diseases, and so are the ultimate source of most antibiotic resistance systems. This is because they have to be able to avoid the harmful effects of the antibiotics that they produce.

If we can understand better how these resistance systems work, it may eventually be possible to develop new drugs which are still active but which do not trigger the bacterial resistance mechanisms, consequently outsmarting antibiotic resistance in clinical infections.

Technical Summary

Glycopeptides, including vancomycin, are clinically important in the treatment of infectious diseases, but resistance systems in pathogens have evolved that reduce their effectiveness. Despite significant effort studying the mechanism of vancomycin and other glycopeptide antibiotics resistance over many years, the exact nature of the ligand(s) that activate(s) VanS, a key sensor protein that triggers the expression of genes conferring resistance, is not known. The aim of the proposed work is to use non-pathogenic Streptomyces strains as models to understand the nature of the specific ligand(s) of VanS through genetic and biochemical approaches.
Streptomyces coelicolor is a non-pathogenic, non-glycopeptide producing strain but carries an inducible novel vancomycin resistance system. This strain is an attractive model for studying the mechanisms of sensing and responding to vancomycin and glycopeptide antibiotics as it is genetically the best characterised streptomycete, and many useful biochemical and genetic tools have already been developed for the study of this organism. Streptomyces toyocaensis, a glycopeptide antibiotic A47934 producer, is another attractive model to study. The A47934 resistance genes are associated with the antibiotic biosynthetic cluster, and the DNA sequence of the cluster has been published. Both Streptomyces strains will be used as host systems to help characterise the ligand(s) that activate(s) the VanR/VanS signal transduction systems derived from other glycopeptide resistance clusters from a range of organisms, including pathogenic enterococci. The outcome of this project may provide information which enables us to design new glycopeptide antibiotics that are still active but which are not recognised by the VanS sensor and therefore do not trigger the resistance system, consequently outsmarting glycopeptide resistance in clinical infection.
The work will initially focus on genetic approaches using gene swap experiments, and eventually will extend to structural studies to identify the exact nature of the VanS inducer(s). The objectives of the proposed work are: (i) Characterisation of the inducer(s) for glycopeptide resistance using gene swap experiments: a. Swapping the complete VanR/VanS two-component system; b. Swapping only the VanS sensor domain; c. Individually swapping only VanR or VanS. (ii) Screening vancomycin derivatives for the ability to induce resistance systems. (iii) Construction of a strain constitutively producing cell wall precursors terminating in D-Ala-D-Lac, bypassing the activity of the van genes. (iv) Biochemical and structural characterisation of VanS-ligand interaction.

Publications

10 25 50
 
Description The Royal Soceity University Research Fellowship Renewal
Amount £301,287 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Title screening system for wide range glycopeptide antibiotics 
Description based on our experience working on molecular mechanism of vancomycin resistance particularlly controlled by two-component system VanR/S, we have constructed various different type of bioassay systems for broad range of glycopeptide antibiotics via creaing a glycopeptide dependent cell lines. 
Type Of Material Cell line 
Year Produced 2007 
Provided To Others? Yes  
Impact This simple bioassay system analogous to what we have patented previously will enable us to sceen and discover novel glycopeptide antibiotics. In collaboration with a Korean group who provided us a library of natural compounds extracted from about 10000 different actinomycete species, we were able to isolate one which may be a novel glycopeptide antibiotic so far. We are on the process of analysing the compound as well as the producer strain and we are screening for more. 
 
Description VanS-ligand Gerry Wright 
Organisation McMaster University
Country Canada 
Sector Academic/University 
PI Contribution My group has provided expertise and results from molecular genetic analysis.
Collaborator Contribution Professor Gerry Wright group provided an important biochemical result for the work which complemented the genetic results provided from my group. We have just submitted the outcome of this collaboration to Nature Chemical Biology.
Impact The first result from this collaboration has recently been submitted for publication in Nature Chemical Biology (although not yet accepted for the publication). It is envisaged that this work will lead to further collaboration between the groups because the study so far has thrown up many interesting new questions.
Start Year 2007
 
Description screening for new glycopeptide antibiotics 
Organisation Myongji University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution My group has developed a simple bioassay tool which is able to screen glycopeptide antibiotics specifically.
Collaborator Contribution Professor Suh's group in Myongji University has establisehd a natural product extract library from over 10000 different actinomycetes so we screened them using the bioassay tool that we develped in collaboration and finally managed to identified a new actinomycete isolate producing a glycopeptide antibiotic.
Impact - the collaboration produced 2 publications and is like to produce 1-2 more publication within next year. - the collaboration is still on going and we are discussing in a stage where we may apply for an internatioanl collaboration grant proposal in near future.
Start Year 2007
 
Description 'The Magic of DNA' on 2009 Cambridge Sicence Day and School Visit Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact about 500 people within age 8-60 attended for hands-on activity, presentation and talks

also 50 schools pupils attended the same activity but organised in a different day in the Department

I've got loads positive feedbacks by emails.
Year(s) Of Engagement Activity 2009,2013
 
Description Antibiotic Unearthed school project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I have started to be involved in a school project called the 'Antibiotic Unearthed' since 2016 summer with attending a summer school at Reading University in July 2016. This project is sponsored by Microbiology Society UK which was a good opportunity for outreach experience. The project involves visiting Oxfordshire UTO in Didcot, Oxfordshire every Wednesday morning since January 2017 till Easter term to lecture and demonstrate our usual lab work particularly to do with discovering antibiotics from soil samples to school students. We repeated this again from September 2017 till January 2018 and are willing to continue this activity.
Year(s) Of Engagement Activity 2016,2017,2018
URL https://microbiologysociety.org/publication/past-issues/microbiology-in-popular-culture/article/scho...