Novel molecular targets to combat antibiotic resistance: probing the assembly dynamics of a bacterial mitotic spindle

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Bacteria resistant to multiple antibiotics are a growing hazard to human health. When bacteria are not killed by a first antibiotic, the treatment has to be switched to a second or third alternative drug. Sometimes no alternative drug is available. Thus antibiotic resistance costs lives and money and puts a heavy burden on health care resources: it has been estimated that the costs for the NHS amount to around #1 billion a year. The emergence of superbugs among bacterial populations results from naturally occurring phenomena in the genetic patrimony of bacteria, which consists of a single circular (more rarely linear) chromosome of DNA and sometimes smaller circles of DNA, called plasmids. Resistance can develop in a bacterium as a consequence of a change or mutation in its chromosomal DNA or by acquisition of a plasmid carrying resistance genes from another bacterium. Plasmids are mobile genetic elements that are able to transfer from one bacterium to another of the same or different species. This mobility is dangerous as it foments the dissemination of antibiotic resistance genes. Multidrug resistance plasmids harbour their own survival kit, a partition cassette, consisting of two genes and a stretch of noncoding DNA. This cassette ensures accurate segregation of the plasmids from one generation to the next at cell division. When this system malfunctions, the plasmid is not stably inherited and is ultimately lost. In our laboratory, we have been investigating the molecular mechanisms involved in the inheritance and maintenance of the multidrug resistance plasmid TP228, which contains genes responsible for the resistance to six different antibiotics. The partition cassette of TP228 contains two genes, designated parF and parG, and a region of noncoding DNA. The protein specified by the parF gene is very peculiar as it forms cable-like filaments that can be seen by using an electron microscope. The protein specified by the parG gene is a DNA-binding factor contacting the noncoding DNA region on the plasmid. The aims of this project are: to explore the dynamics of the interaction of the ParF and ParG proteins in the cell; to investigate the mechanism whereby ParG helps the assembly of ParF into cable-like structures; and to identify other bacterial proteins that associate with the ParF-ParG complex. These studies will allow us to learn more about the mechanism whereby plasmids are maintained in bacterial cells and will identify novel targets for the development of new antimicrobial agents.

Technical Summary

The emergence of multidrug-resistant strains among bacterial populations results either from mutations within the bacterial genome or from the horizontal transfer of resistance genes often present on mobile genetic elements such as plasmids, transposons and pathogenicity islands. Large, low copy number plasmids, such as those implicated in antibiotic resistance, have evolved sophisticated strategies to ensure their faithful distribution at cell division. These plasmids harbour a partition locus, which ensures an accurate and equitable segregation of the plasmids from one generation to the next. Our model system is the partition cassette harboured by the multidrug resistance plasmid TP228, that replicates at low copy number in Escherichia coli. This mobile element specifies resistance to a range of antibiotics, including tetracycline, spectinomycin and sulphonamides. The segregation locus of TP228 consists of the parF and parG genes and upstream parH centromere, which comprises four direct repeats. ParF is an ATPase that polymerizes into multistranded filaments; ParG is a DNA-binding protein that contacts the centromeric DNA and recruits ParF into the nucleoprotein partition complex. The resulting segrosome is a positioning apparatus that localizes the attached plasmids to specific subcellular addresses. The overall aim of this project is to continue the dissection of the TP228 model system by elucidating the molecular mechanisms underpinning its segregational stability at cell division.
The first objective (months 1-12) is to analyze the subcellular distribution of the two trans-acting factors ParF and ParG and to study their assembly dynamics in vivo by immunofluorescence and live fluorescence microscopy. Preliminary results on this aspect of the project appear very promising and exciting.
The second objective (months 1-24) of the work is a detailed exploration of the mechanism/s whereby the ParG flexible N-terminus nucleates and bundles ParF filaments. This will involve site-directed mutagenesis of the region of parG encoding for its unfolded N-terminus and in vivo and in vitro characterization of the mutants.
The third objective (months 18-30) is to investigate the topology of the arginine finger-like motif in ParG in relation to the stimulation of ParF ATPase activity, which is essential for plasmid partition.
The fourth objective (months 18-36) will consist in a detailed investigation of newly emerging connections between cell division and plasmid segregation and in the identification and characterization of other potential E. coli host factors interacting with the TP228 partition complex. These studies will contribute novel insights into various facets of segregation process of multidrug resistance plasmids.

Publications

10 25 50
 
Title A collection of ParG mutants 
Description This collection includes more than 20 mutants of our protein of interest, ParG. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The mutants constructed have helped and will further our knowledge on how our plasmid segregation system operates. 
 
Title E. coli DNA library 
Description The library contains DNA fragments covering the entire E. coli genome. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We are using the library to extend the objective of the grant project with the aim to discover new factors interacting with our multidrug resistance plasmid segregation system. 
 
Title Fluorescent fusion proteins 
Description DNA partition proteins fused to different fluorescent proteins (mCherry, Emerald and Blue fluorescent proteins). These tools have allowed us to determine the cellular localization of the DNA segregation proteins under investigation in this project. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact These research materials have allowed us to determine the in vivo localization of the proteins that are the focus of this research project. In addition, they have allowed us to build up fluorescent miscroscopy expertise in our laboratory. 
 
Description Finbarr Hayes - Genome segregation mechanisms 
Organisation University of Manchester
Department Manchester Interdisciplinary Biocentre
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing crucial reagents (DNA clones), providing expertise on specialised techniques, intellectual input to design projects and training of staff.
Collaborator Contribution The research sinergy betweeen my collaborator's group and my group resulted in the publication of a number of high profile papers.
Impact A number of significant publications: 16415929 16584885 17261809 17920627 18245388
Start Year 2006
 
Description Joe Pogliano - Super resolution microscopy 
Organisation University of California, San Diego (UCSD)
Country United States 
Sector Academic/University 
PI Contribution Providing DNA clones and bacterial strains.
Collaborator Contribution Provided state-of-the-art microscopy facilities and expertise.
Impact We are currently writing a manuscript.
Start Year 2011
 
Description Maria Schumacher - Structural studies of the TP228 segrosome 
Organisation Duke University
Department Department of Biochemistry
Country United States 
Sector Academic/University 
PI Contribution My team provided DNA constructs of wild type and mutant alleles to the collaborator at Duke. We also performed biochemical experiments like fluorescence anisotropy, dynamic light scattering and ATPase assays to characterize mutant proteins.
Collaborator Contribution Our collaborator solved the 3D structure of the ParF protein.
Impact *Schumacher, M.A., Ye, Q., Barge, M.T., Zampini, M., *Barillà, D., *Hayes, F. (2012) Structural mechanism of ATP induced polymerization of the partition factor ParF: implications for DNA segregation. J Biol Chem 287, 26146-26154.
Start Year 2009
 
Description ASM conference on Prokaryotic Cell Biology and Development (Montreal, Canada) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The talk generated quite a bit of discussion and allowed me to promote my research to an international audience.

It triggered invited talk at other international meetings.
Year(s) Of Engagement Activity 2012
 
Description International Plasmid Biology Conference 2010. Bariloche, Argentina, 2010. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact One postdoc presented a poster on our multidrug resistance plasmid segregation project (MRC).
Another postdoc gave a talk about our archaea project (BBRSC).

Promotion of the research ongoing in our group.
Year(s) Of Engagement Activity 2010
 
Description Invited talk, Department of Biology and Biotechnologies, University of Pavia, Italy, 2012. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I talked about the progress made on the work on plasmid segregation funded by the MRC.

Promotion of my research to an international audience.
Year(s) Of Engagement Activity 2012
 
Description Plasmid Biology Conference 2012 (Santander) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The talk sparked questions.

I guess that the talk increased the visibility of my research team.
Year(s) Of Engagement Activity 2012
 
Description UCAS days for undergraduate recruitment 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Talk to perspective undergraduate students

Trying to enthuse the next generation of microbiologits.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011