Late in 2016, the DST/NRF Centre of Excellence for Biomedical TB research (CBTBR) at Wits, under the leadership of Prof. Bavesh Kana published two landmark studies, one as a collaborative initiative with a group at Tsinghua University, Beijing China and the second as a multidisciplinary collaboration with several Wits researchers.
The first study, published in Nature Microbiology, a new Nature Publishing Group journal, focused on translational fidelity in mycobacteria. Modulation of translational fidelity is expected to result in metabolic plasticity that could lead to drug tolerance and the ultimate emerge of drug resistant variants. During protein biosynthesis, translation fidelity is maintained by ensuring the correct charging of tRNAs with the codon-specific cognate amino acids. For the case of glutamine and/or asparagine, this process entails a two-step mechanism that requires the glutamine amidotransferase, GatCAB. This study demonstrates that modulation of GatCAB activity results in mistranslation in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), thus leading to drug tolerance with first line TB antibiotics. A forward genetic screen was designed based on two complementary gain-of-function reporters that directly measured mistranslation to identify high mistranslator mutants. Initially, a reporter strain carrying a point mutation in the gene conferring kanamycin resistance was used to identify high mistranslator mutants of mycobacteria. The phenotypically resistant mutants were subsequently screened using a dual-luciferase assay to identify mistranslator strains specific for asparagine to aspartate. Following whole genome sequencing, SNPs were identified in the gatA gene that correlated with these observations. Moreover, clinical isolates of M. tuberculosis containing gatA SNPs were specifically associated with high mistranslation rates and rifampicin-specific phenotypic resistance. These mutations cause partial loss of the GatCAB holoenzyme, resulting in an increase in mistranslation rates. Using single-cell transcription dynamics it was demonstrated that reduced gatCAB expression results in elevated mistranslation rates. Collectively, these observations highlight a novel adaptation strategy that the tubercle bacillus uses to establish drug tolerant populations, which are required for the evolution of genetically stable drug resistance. CBTBR members involved in this study include Dr. Christopher Ealand and Professor Bavesh Kana
Su HW, Zhu JH, Li H, Cai RJ, Ealand C, Wang X, Chen YX, Kayani MU, Zhu TF, Moradigaravand D, Huang H, Kana BD, Javid B. The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity. Nat Microbiol. 2016 Aug 26;1(11):16147. doi: 10.1038/nmicrobiol.2016.147. PMID: 27564922
In their second landmark study, researchers at the CBTBR interrogated the presence of non-replicating, differentially culturable M. tuberculosis in the sputum of TB patients. This work was published in the American Journal of Respiratory and Critical Care Medicine (Impact Factor: 13.1, published by the American Thoracic Society). Anecdotal evidence pointed to the presence, in sputum, of differentially culturable tubercle bacteria (DCTB) that are unable to grow on solid media but can be recovered in liquid media supplemented with resuscitation promoting factors, a group of bacterial growth stimulatory enzymes secreted by M. tuberculosis. This intriguing observation was interrogated in a cross-sectional observational cohort of patients infected with TB or TB-HIV from various clinics in Soweto. The CBTBR first established the methodology to detect and quantify DCTB and thereafter, the combination of carefully collected sputum samples and a refined methodology, allowed for the detection and definition of five operationally distinct sub-classes of tubercle bacteria in the sputum of treatment naïve TB patients. These sub-populations are expected to respond differentially to TB therapy and most likely form the microbiological basis for the protracted treatment required for achieving functional cure in TB patients. Moreover, enhanced recovery of DCTB improved bacterial detection in sputum smear negative TB patients that are generally difficult to identify using standard diagnostics. Sputum from TB-HIV-1 infected individuals, with CD4 counts >200 cells/mm3, displayed higher levels of culture filtrate-responsive organisms than sputum from TB-HIV-1 infected individuals with CD4 counts <200 cells/mm3. This study represents the most comprehensive analysis of differentially culturable tubercle bacteria to date, with important implications for diagnosis of TB particularly in individuals with paucibacillary disease. Moreover, the quantitation of differentially culturable organisms now provides a novel biomarker to assess treatment response and risk of disease recurrence. Our data provide preliminary microbiological evidence to validate the long-standing hypothesis that the host immune response to TB infection drives bacteria into phenotypically distinct, drug tolerant states. Given the importance of these findings, this publication was selected by the Journal for an editorial feature. Current CBTBR members involved in the study include, Melissa Chengalroyen, Bhavna Gordhan and Neil Martinson.
Chengalroyen MD, Beukes GM, Gordhan BG, Streicher EM, Churchyard G, Hafner R, Warren R, Otwombe K, Martinson N, Kana BD. Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis. Am J Respir Crit Care Med. 2016 Dec 15;194(12):1532-1540. PMID: 27387272
Dartois V, Saito K, Warrier T, Nathan C. New Evidence for the Complexity of the Population Structure of Mycobacterium tuberculosis Increases the Diagnostic and Biologic Challenges. Am J Respir Crit Care Med. 2016 Dec 15;194(12):1448-1451. PMID: 27976945
||Christopher Ealand||Bhavna Gordhan||Neil Martinson||Bavesh Kana|