Mechanisms of fitness cost reduction for rifampicin-resistant strains with deletion or duplication mutations in rpoB

Gerrit Brandis,Diarmaid Hughes

doi:10.1038/s41598-018-36005-y

Abstract

Rifampicin resistance (RifR) is caused by mutations in rpoB, encoding the β-subunit of RNA polymerase. RifR mutations generally incur a fitness cost and in resistant isolates are frequently accompanied by compensatory mutations in rpoA, rpoB or rpoC. Previous studies of fitness compensation focused on RifR caused by amino acid substitutions within rpoB. RifR is also caused by deletion and duplication mutations in rpoB but it is not known whether or how such mutants can ameliorate their fitness costs. Using experimental evolution of Salmonella carrying RifR deletion or duplication mutations we identified compensatory amino acid substitution mutations within rpoA, rpoB or rpoC in 16 of 21 evolved lineages. Additionally, we found one lineage where a large deletion was compensated by duplication of adjacent amino acids (possibly to fill the gap within the protein structure), two lineages where mutations occurred outside of rpoABC, and two lineages where a duplication mutant reverted to the wild-type sequence. All but the two revertant mutants maintained the RifR phenotype. These data suggest that amino acid substitution mutations are the major compensatory mechanism regardless of the nature of the primary RifR mutation.

Highlights

Rifampicin is a bactericidal antibiotic that is part of the short-course anti-tuberculosis treatment[1]
Three independent lineages of each of the seven Rifampicin resistance (RifR) isolates were evolved by serial passage in Luria broth (LB) medium with selection for increased growth rates and after 100 generations, diluted cultures were plated on LA agar
The fourth isolate had acquired a mutation outside the RNAP genes that has previously been shown to be adaptive to growth in LB medium[13]. These results indicate that adaptation to growth under laboratory condition is only a minor contributing factor in those lineages where compensatory mutations to the fitness costs of RifR mutations were selected within the RNA polymerase genes, at least during the timespan of the evolutionary experiment (100 generations)

Summary

Introduction

Rifampicin is a bactericidal antibiotic that is part of the short-course anti-tuberculosis treatment[1]. Resistance to rifampicin can be caused by any of over 100 distinct changes in the β-subunit of the bacterial RNA polymerase (RNAP)[2] where rifampicin binds and exerts its function by inhibiting transcription[3] The majority of these mutations cause a significant fitness cost to the bacteria[2] and recent studies using Salmonella as a model organism and/ or genome sequences of clinical MTB isolates have shown the importance of fitness-compensatory evolution in the development of clinical rifampicin resistance[2,4,5,6,7]. Our aims were: (i) to identify growth-compensatory mutations, and (ii) to compare the compensatory evolution of RifR deletions and duplications to the compensatory evolution of RifR amino acid substitutions

Objectives

Methods

Results

Conclusion