Fuzhen Zhang, 1, 2,* Shanshan Li, 2,* Shuan Wen, 2 Tingting Zhang, 2 Yuanyuan Shang, 2 Fengmin Huo, 2 Yi Xue, 2 Ling Li, 1, 3 Yu Pang 2 1Biosafety Level 3 Laboratory, School of Public Health, Southern Medical University, Guangzhou, People’s Republic of China; 2National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People’s Republic of China; 3Experimental Teaching Center, School of Basic Medical Science, Southern Medical University, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yu PangBeijing Chest Hospital, Capital Medical University, No. 97, Machang, Tongzhou District, Beijing 101149, People’s Republic of ChinaTel/Fax +86 10 8950 9359Email pangyupound@163.comLing LiSouthern Medical University, No. 1023-1063, Shatai South Road, Baiyun District, Guangzhou 510515, People’s Republic of ChinaTel +86 18680280210Email liling@smu.edu.cnObjective: PA-824 (Pretomanid), a bicyclic nitroimidazole drug, exhibits significant bactericidal activity toward Mycobacterium tuberculosis (MTB) in vitro and in vivo, but not against Mycobacterium smegmatis. Through catalytic bioreduction, deazaflavin-dependent nitroreductase (Ddn) within MTB directly converts PA-824 to potent bactericidal products. This study aimed to identify key MTB Ddn residues involved in PA-824 conversion toward development of in vitro surrogate markers for detection of mycobacterial resistance to PA-824.Methods: We evaluated in vitro activity of PA-824 toward MTB and nontuberculous mycobacterial species using antimicrobial susceptibility testing. Ddn amino acid sequence alignments and phylogenetic analysis revealed putative key enzyme active site residues. Candidate MTB Ddn residues required for PA-824 conversion activity were evaluated for loss-of-function using recombinantly cloned Ddn mutant proteins expressed in Mycobacterium smegmatis.Results: PA-824 minimum inhibitory concentrations of 90% of bacterial growth (MIC 90s) against MTB and Mycobacterium kansasii were 0.12 mg/L and 8 mg/L, respectively, but > 32 mg/L for Mycobacterium spp. M. avium, M. intracellulare, M. abscessus and M. fortuitum. MTB Ddn and M. kansasii Ddn homologous sequences shared the greatest similarity (89.3% amino acid identity). M. smegmatis expressing Ddn proteins with Y65L, A76V or Y133F substitutions (but not V75L, Q125K or V148I) were resistant to PA-824.Conclusion: Our data demonstrated that PA-824 exhibited excellent and moderate levels of in vitro activity against MTB and M. kansasii, respectively. Substitutions of Ddn residues Y65, A76 or Y133 conferred mycobacterial resistance to PA-824.Keywords: mycobacteria, PA-824, susceptibility, Ddn
