Fitness costs of rifampicin resistance in Mycobacterium tuberculosis are amplified under conditions of nutrient starvation and compensated by mutation in the β' subunit of RNA polymerase

Abstract

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the β subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi β-barrel domain of the β' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.

Trial registration: ClinicalTrials.gov NCT00341601.

Conflict of interest statement

The authors of this study declare that they have no conflicts of interest with respect to any aspect of this research.

© 2014 John Wiley & Sons Ltd.

Figures

Figure 1

Relative risk for mutation in proteins from drug-resistant as compared to drug-susceptible clinical isolates from South Korea. We calculated the relative risk of mutation from 15 drug-sensitive and 18 drug-resistant South Korean isolates in each protein in the drug-resistant strains. Known genes associated with drug-resistance alleles are red, while genes associated with potential compensatory suppressor alleles are shown in blue.

Figure 2

RpoB and RpoC protein sequence variation amongst 41 drug-susceptible and 35 drug-resistant isolates based on combining new data from South Korean strains with available published data. Amino acid substitutions relative to the consensus are displayed according to the key in the figure. Strains in green represent sequential evolution of drug-resistance within a single South Korean subject, strains in gold represent sequential evolution of drug resistance within previously reported sequenced strains from three individuals in the Western Cape from South Africa. The strains in red represent the likely clonal expansion of an XDR lineage from KwaZulu Natal, South Africa. The two pairs with black brackets represent sequences from sequential isolates in the same individual. More details concerning the source of the isolates can be found in Tables S1 and S2, and source and description of the previously sequenced isolates is in Table S3. The K at the start of a sequence name indicates a new sequence from South Korea. Sequences were obtained from 2 different laboratories in this study, and a few isolates shown here were sequenced twice, once in each laboratory. Those sequences obtained at the NIH are labled with an asterisk, the others were sequenced by Novartis. The country of origin for the other strains are indicated at the beginning of the name using the International Standard 2 letter designations: United States, US; South Africa, ZA; and Canada, CA. The year of ioslation is indicated when reported, and the region of origin for South African set is also indicated (Western Cape, WeC; Durban, Dur; and Tugela Ferry TUF). The two letter code at the end of each isolate name indicates the reported drug profile as defined and detailed in Table S2. DS indicates drug sensitive; XR, XDR; MR, MDR; PR, pre-XDR; IR, INH resistant; and DR, drug resistant with a specfic profile that is not readily categorized. Fig. S1 is a companion to this figure, showing the same mutational patterns shown highlighted here, but presented in the context of a SNP-based phylogeny using all available sequence data to illustrate the genetic relatedness of the strains.

Figure 3

Compensatory mutation of the S531L allele is driven by both transmission and convergent evolution. A) Sequencing of 170 RIF-resistant strains from South Korea showing linkage between rpoB and rpoC alleles. Distinct amino acids substitutions are indicated by colored tickmarks as detailed in the inset. The mutations are summarized by strain in Table S4 and shown relative to the protein sequences in Fig. S2. (B) A Neighbor-joining tree of the strains based on 15-locus MIRU-VNTR patterns showing that specific combinations of alleles occurred in clusters, while others appeared to have evolved convergently. Strains carrying the rpoB S531L mutation are marked with symbols, while being colored for the alleles that occurred in multiple strains. Gray circles indicate strains with all other additional mutations in rpoB outside of the RRDR and rpoC.

Figure 4

Fitness defect of the rpoB S531L mutant and compensation by rpoC alleles. (A) The growth of M. smegmatis strains in which the wild type rpoB and rpoC have been replaced by the corresponding rpoB and rpoC genes from Mtb on 7H10 plates. The leftmost panel shows the growth phenotype of the wild type Mtb alleles, the second panel from the left shows the diminished growth by the rpoB S531L allele and the two right panels show compensation of this defect by the corresponding rpoC alterations at F452L and V483G. (B) The fitness impact is muted in 7H9 media supplemented with 0.2% glucose (open bars) and 7H9 with reduced iron and biotin levels (light gray bars) but this defect is amplified under conditions of carbon source limitation (0.002% glucose, black bars) and under conditions of restriction of carbon source, biotin and iron deprivation (dark gray bars). (C) Competitive fitness of the rpoB S531L mutant and two compensated strains by either rpoC F452L or rpoC V483G when co-cultured with the strain carrying wild type Mtb rpoB. Culture conditions indicated as bars are same as in (A).

Figure 5

Restoration of RNA polymerase activity by rpoC F452L and rpoC V483G alleles. M. smegmatis strains carrying the respective Mtb rpoB and rpoC genes were induced for amiE gene expression, and the slope of the line plotting the proportional change in 2ȒΔΔC† per minute of the induced amiE transcript relative to a constitutive recA transcript was represented as transcription efficiency.

Figure 6

Compensatory mutations in RpoC might reduce impedance of RNA exit from RIF-resistant mutant RNA polymerase. (A) A model of the RNAP from Mtb (based on the structure from T. thermophiles PDB: 2O5J) is shown with the DNA in blue and RNA in orange. The β subunit is shown in space filling representation, while the β’ subunit is shown in magenta ribbon for contrast (except the three helix loop in cyan which is the focus of many of the potential compensatory mutations). RIF resistance inducing mutations are shown in red and noted, and RIF is shown in green. (B) The exit tunnel for RNA is highlighted, while in (C) the critical contacts of this three-α-helix loop at the exit of the tunnel is shown compared to Ser531 of β subunit. (D) The position of the F452L compensatory mutation in the β’ subunit is shown, as well as the proximity of the I1187 residue in the β subunit.

Figure 7

Three-dimensional representation of the RIF-resistance conferring and compensatory mutations on the crystal structure of the E. coli RNAP-RIF complex (PDB: 4JK1, (Mechold et al., 2013)). (A) The RIF-resistance conferring rpoB S531L mutation and its compensatory mutations. The RNAP core enzyme is depicted as an α-carbon backbone trace (α subunit, white; β subunit, cyan; β‘ subunit, pink; ω subunit, gray). The Ser531 residue of the β subunit is shown as yellow sphere. Compensatory mutations found on αI, αIIβ and β‘ subunits are highlighted as gray, black, blue and dark pink, respectively. Two compensatory mutations, rpoC F452L and rpoC V483G, characterized in the in vitro transcription assay were shown as red. The ppGpp binding site is indicated as green sphere and the DPBB domain of the β‘ subunit containing the RNAP active site (magenta sphere) is highlighted in yellow. (B) RIF-resistance associated mutations other than rpoB S531L and their compensatory mutations. Rifampicin resistant mutations are shown as yellow spheres, and compensatory mutations found on αI, αIIβ and β‘ subunits are highlighted as gray, black, blue and dark pink, respectively.