The group of Babak Javid recently published a paper titled ‘The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity’ in Nature Microbiology in August, 2016. This work showed for the first time how GatCAB – an essential enzyme complex involved in the synthesis of glutamine and asparagine tRNAs regulates and modulates the fidelity of translation of glutamine and asparagine codons. Using a forward genetic selection and screen strategy, coupled with whole-genome sequencing they identified that mutations in gatA led to increased rates of mistranslation (specific errors in gene translation) of glutamine to glutamate and asparagine to aspartate.  These mutant strains also showed increased phenotypic resistance to rifampicin – the most important antibiotic used for the treatment of tuberculosis.

Model for how cellular heterogeneity in RpoB and GatCAB expression predicts rifampicin tolerance.

(A) GatCAB functions to amidate the physiologically misacylated Asp-tRNAAsn and Glu-tRNAGln aminoacyl tRNAs to their cognate Asn-tRNAAsn and Gln-tRNAGln. Limiting GatCAB increases the abundance of misacylated Asp-tRNAAsn and Glu-tRNAGln, which in turn cause generalized mistranslation, including specific, critical residues of RpoB, rendering it resistant to rifampicin. (B) The abundance of mistranslated, rifampicin-resistant RpoB is a product of the total expression of RpoB and the cellular mistranslation rate (represented by degree of shading). Cells with higher mistranslation rates due to limited GatCAB and hence increased concentrations of rifampicin-resistant RpoB variants will be able to continue transcription at doses of rifampicin sufficient to inhibit and kill the majority population (a). Ongoing transcription supports essential cellular activities allowing not only survival but growth in the presence of otherwise lethal concentrations of rifampicin (b and c).

Tuberculosis (TB) caused by Mycobacterium tuberculosis is the world’s most deadly infectious disease, and the leading cause of death from a preventable and curable cause.  More than 1.8 million people died from TB in 2015 according to the World Health Organisation, and each year there are approximately 1 million cases of active TB in China, making it a key pathogen in the nation. A major roadblock for the eradication of TB is that treatment of even drug-sensitive strains take at least 6 months continuous treatment with antibiotics – drug resistant strains need 20 months or more of treatment. The long treatment time is due to “tolerance” or “phenotypic resistance” of drug-sensitive bacterial populations to the antibiotics – and this study for the first time determined the molecular mechanism of antibiotic tolerance in clinical isolates of Mycobacterium tuberculosis.

Since China in general and Tsinghua in particular do not have appropriate facilities for the study of pathogenic Mycobacterium tuberculosis, study co-first author, Hongwei Su, a graduate student of Javid from the PTN programme, spent 12 months in the laboratory of Bavesh Kana, HHMI, in South Africa to perform the necessary bacterial genetics experiments to verify that mutations in gatA identified from clinical isolates do indeed cause increased mistranslation and tolerance to rifampicin.  After verifying this, in collaboration with the Beijing TB hospital, the group identified a clinical isolate with a mutation in gatA and showed that this strain was highly tolerant to rifampicin specifically.  The isolate was from a patient who had failed conventional TB treatment.

Study senior author Babak Javid said: “We had previously shown that mycobacteria had very high specific error rates of mistranslation, but it wasn’t clear what the mechanism was.  Here, we have shown that GatCAB, which had previously been assumed to be a highly efficient enzyme, can actually modulate the error rate in mycobacteria according to environmental conditions.  Our work has very important implications for strategies to shorten the duration of TB therapy.  China still has a huge TB problem, which the Government is committed to tackling – but Chinese universities, even Tsinghua, still lack the basic infrastructure needed to perform world-class TB research.  Our current study would have been impossible to perform solely in China, but thanks to our collaborators in South Africa, which has far more advanced facilities, we were able to elegantly demonstrate the essential role of mycobacterial GatCAB in antibiotic tolerance.  I look forward to when Tsinghua is able to be self-sufficient in these cutting edge studies.”

Babak Javid from Tsinghua University School of Medicine is the sole corresponding author of this work, Su Hongwei from Tsinghua University 2012 PTN program and Zhu Junhao from Peking University 2011 PTN program are the co-first authors. This work was funded in part by the Bill&Melinda Gates Foundation (OPP1109789) and start-up funds from Tsinghua University to Babak Javid. We also acknowledge technical assistance from the microbial sorting facility at Peking University and the Clinical Database and Sample Bank of Tuberculosis of Beijing (D131100005313012) of the National Clinical Lab on Tuberculsis, Beijing Chest Hospital for access to their strain collection.