these particular strains had not
only
acquired a mutation that caused rifampicin resistance, but also acquired so-called compensatory mutations that restored the fitness deficit.” Gagneux’s more recent work, published in
a paper earlier this year, expanded on these findings by trying to identify the specific mutations that could cause this fitness compensation. Firstly, using genome sequencing techniques, they found a number of mutations in the clinical strains that could potentially be involved in the compensatory mechanism. They then went back to the lab-generated strains, all of which had a fitness cost, and sub cultured them repeatedly, attempting to select for compensatory mutations in the lab. They then repeated what they had done before; by genome sequencing the evolved forms along with their non-evolved ancestors, they were able to identify the mutations that may have happened during this period of in vitro evolution. “We then compared the evolved strains
from the labs with the natural isolates from the patients, and tried to look for genes that were hit lots of
times i.e. mutations that
were promising in terms of whether they might be involved in compensation or not,” explains Gagneux. The mechanism of rifampicin resistance is
a very well understood topic. Rifampicin binds with RNA polymerase, particularly to a sub-section known as the beta sub-unit encoded by the gene rpoB, and it is mutations in this area that mostly cause
resistance to the drug. However, the mutations found in the artificially evolved strains and in some of the clinical strains were found in the rpoA and rpoC genes which encode different sub-units of RNA polymerase and interact with rpoB. “We then thought, now that we had
identified putative compensatory mutations, could we go back to our in vitro testing to see whether, in the clinical paired isolates, the strains that had no fitness cost had these putative compensatory mutations,” says Gagneux. “This indeed was the case; the strains with fitness costs had none of these mutations, whereas three out of four of the strains with no fitness cost had these mutations. Although this was a small sample, the results were statistically significant, and thus a first indication that these mutations really do matter.” Multi-drug-resistant tuberculosis is non-
randomly distributed; many of the former Soviet Union republics such as Georgia, Kazakhstan and Uzbekistan are so-called hot-spots, whereas other areas have much less of a prevalence of these strains. The global proportion of multi-drug-resistant tuberculosis amongst all tuberculosis is between 3-5%, whereas in some of these high-burden countries it can be up to 30%. Thus, Gagneux and his colleagues predicted that the frequency of the putative compensatory mutations would be much higher in these countries. “We took a global collection of clinical
strains, some of which were from these high-burden countries, and screened all of them for the putative compensatory mutations,” says Gagneux. “We found that up to 30% of multi-drug-resistant strains in the hot-spot areas had at
least one of
these mutations, whereas only around 10% were seen with them in low-burden multi drug-resistant countries. This association suggests that these compensatory mutations do indeed contribute to the success of multi-drug-resistant strains in these high-burden countries.” With other teams of researchers now
researching what the mechanism of these mutations might be, Gagneux and his team are looking even further ahead. “What we would like to do is to carry out transmission studies in some of the high-burden countries, looking at particular multi- drug-resistant strains that have these particular combinations of drug-resistance mutations. We also plan to study the corresponding compensatory mutations, and discover whether they transmit more successfully.”
★
www.projectsmagazine.eu.com really do 39
Project Information AT A GLANCE
Project Title: The Evolution of Multidrug-resistant Tuberculosis
Project Objective: The objective of this project is to study the evolution and ecology of multidrug-resistant tuberculosis with a particular focus on the interactions between drug resistance-conferring mutations and compensatory mutations, and their effect on bacterial fitness.
Project Duration and Timing: 5 years, 2010 to 2015
Project Funding: Swiss National Science Foundation (SNF-professorship) and European Research Council (ERC Starting Grant)
Project Partners: MRC National Institute for Medical Research, London, UK & National Centre for Tuberculosis and Lung Diseases, Tbilisi, Georgia.
Main Contact:
Sébastien Gagneux Dr. Gagneux received his PhD from the University of Basel and worked as a postdoctoral fellow at Stanford University and at the Institute for Systems Biology in Seattle. Before joining Swiss TPH, he spent three years as a Program Leader at the MRC National Institute for Medical Research in London, UK.
Contact: Tel: +41 61 284 83 69 Email:
Sebastien.gagneux@
unibas.ch Web:
www.swisstph.ch/?id=1336
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