NEW YORK (GenomeWeb) – By analyzing nearly 500 Mycobacterium tuberculosis genomes and performing functional studies, researchers from the Broad Institute and South Africa's KwaZulu Natal Research Institute for TB and HIV have identified a new mechanism by which the pathogen acquires drug resistance.
The team reported today in Nature Genetics that loss of function of the Ald gene confers resistance to second line treatment. In addition, the researchers also predicted that mutations to Ald and Alr genes are deleterious and lead to drug resistance.
To look for mechanisms of drug resistance, the team analyzed whole-genome sequence data from two clinical collections from South Africa and China, comprising 498 isolates. The two collections had diverse phenotypic drug resistance profiles.
One major goal was to look specifically for new resistance mechanisms for drugs with particularly toxic side effects and for which resistance screening is not routinely done. One such drug, D-cycloserine, has severe psychiatric and central nervous side effects and current molecular diagnostic guidelines do not suggest testing patients for resistance due to technical difficulties with the existing phenotypic assay.
In addition, the molecular basis of D-cycloserine resistance is also poorly understood. The researchers noted that a goal of identifying resistance mechanisms would be to "further restrict its use to patients harboring strains that are susceptible to D-cycloserine."
The group identified two studies from South Africa and China that had performed whole-genome sequencing and drug resistance profiling on a total of 498 tuberculosis strains. The strains had varying degrees of drug resistance and included a large number of strains with multi-drug resistance (MDR) and some that were designated extensively drug resistant (XDR).
They then used bioinformatics strategies to identify candidate drug resistance genes from the data. First, they wanted to get an idea of how much unexplained drug resistance there was in each strain. They compared the variants identified in each strain with a list of resistance-associated variants and found that per drug, there could be between eight and 21 strains without known resistance-conferring mutations.
Next, they looked specifically at loss-of-function variants and came up with 11,704 genotypic features for downstream analysis. To figure out which ones were associated with drug resistance, they assumed that both the resistance mutation and the drug would be positively associated, and that resistance and the mutations should evolve together. They then removed the known resistance mutations from further analysis, coming up with 21 genes with loss-of-function mechanisms that were also associated with drug resistance to at least one of nine different drugs.
Only one of the mutants demonstrated in a knock-out screen that it alone was sufficient to confer drug resistance — a loss-of-function mutation in the gene Ald. The researchers found 11 different Ald loss-of-function mutations in 25 South African strains. In addition, they found the mutations appeared "exclusively and repeatedly in MDR and XDR genetic backgrounds, suggesting selection for loss of function of this gene among patients receiving second-line drug regimens." They then confirmed that the mutation did indeed confer resistance to D-cycloserine in several assays.
The team also looked at a few other genes involved in the same D-alanine pathway as Ald: Alr, DdlA, and CycA — characterizing all promoter, non-synonymous, and loss-of-function mutations. Twenty-five strains had Ald mutations, 17 had Alr mutations, and seven had either DdlA or CycA mutations — none of which corresponded to previously known resistance-conferring mutations.
To compare the effect of Alr mutations on drug resistance, they looked at 22 clinical isolates from South Africa that were wild-type for both genes, 13 isolates with Ald mutations, seven with Alr mutations, and two with mutations in both genes.
Strains with Ald mutations were more resistant than wild-type strains, although the researchers noted that in some cases, Ald mutations conferred moderate resistance. By contrast, Alr mutations tended to elicit much higher drug resistance, with "high-level resistance" found in five of the seven strains with Alr mutations. The team hypothesized that this was because Alr encodes the "direct target" of D-cycloserine, while Ald is involved in the pathway, but not the direct target.
Going forward, the researchers wrote, it will be important to incorporate "resistance mechanisms to D-cycloserine and other highly toxic drugs into new molecular diagnostics" in order to "prevent these drugs from being prescribed in a treatment setting where they might be ineffective and needlessly toxic."