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

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.

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.