Genomic and functional analyses of Mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance

Abstract

A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.

Figure 1

Significance of associations between genotypic variants and drug resistance phenotypes. Each circle represents a genotypic feature that is plotted at the intersection of the negative log p-values from Fisher’s exact and correlated evolution tests for each drug. P-values were corrected for multiple comparisons using the Benjamini-Hochberg method. Genotypic variants known to confer resistance are colored according to the drug to which they confer resistance, and genotypes with no known effect on drug resistance are grey. Genotypes scoring well in both tests appear in the upper right quadrants.

Figure 2

Convergent evolution of loss-of-function mutations in ald in MDR and XDR M. tuberculosis. A) Positions of insertions, deletions, and nonsense mutations in ald. Insertions are marked as blue triangles while deletions and nonsense mutations are marked as red triangles. Size of the indel or details of nonsense mutation are shown above or below each triangle. The location of the frameshift in BCG is also included. B) Phylogeny of lineage 4 strains. Inner tick marks denote MDR (gray) and XDR (black) M. tuberculosis. Outer tick marks denote insertions (blue) and deletions or nonsense mutations (red).

Figure 3

Alanine metabolism pathway in M. tuberculosis. L-alanine-dehydrogenase catalyzes the NAD-dependent interconversion between L-alanine and pyruvate. Under aerobic conditions this reaction allows for the utilization of L-alanine as a nitrogen source, whereas under hypoxic conditions this reaction allows for NADH recycling to NAD⁺. Separately, the initial step of peptidoglycan synthesis involves conversion of L-alanine to D-alanine by alanine racemase. Two D-alanine molecules are joined by D-ala-D-ala ligase to produce the dipeptide D-alanine-D-alanine, which is subsequently incorporated into the pentapeptide chain of the peptidoglycan cell wall. Both alanine racemase and D-ala-D-ala ligase are competitively inhibited by D-cycloserine, an analog of D-alanine.

Figure 4

A single gene knockout of ald confers a growth advantage relative to wild type M. tuberculosis when cultured in the presence of D-cycloserine. Four laboratory strains—wild type M. tuberculosis (WT), ald knockout (Δald ), ald complemented back into Δald (ald-comp) and BCG—were cultured in triplicate in 0, 7.5 and 15 μg/mL of D-cycloserine. The resulting time to positivity (ttp) in MGIT was recorded as days since inoculation, and the resulting ttp was normalized to the no drug control for each strain to calculate the growth inhibition index for each drug concentration. The mean growth inhibition index is plotted with error bars to represent the SEM. P-values were calculated using a two-way ANOVA.

Figure 5

Clinical strains with mutations in L-alanine dehydrogenase (ald) and alanine racemace (alr) exhibit increased resistance to D-cycloserine. A set of 44 clinical strains of M. tuberculosis with differing genotypes with respect to ald and alr were selected, representing wild type alleles (WT), loss-of-function mutations (LOF), nonsynonymous mutations (NSY) and promoter mutations (PRO). The minimum inhibitory concentration (MIC) to D-cycloserine was determined for each strain on Löwenstein–Jensen media. For wild type strains and strains with ald loss-of-function mutations, the median is shown as an open circle and quartiles are shown as connected bars. When compared to WT isolates, strains with ald loss-of-function had significantly increased resistance to D-cycloserine (p < 0.0002, Mann-Whitney U test).