Mycobacterium tuberculosis is an ancient pathogen that causes tuberculosis (TB), an infectious disease that results in over a million deaths every year. The increased prevalence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) TB is imposing an even greater threat to public health.  

Isonicotinic acid hydrazide (INH) is an important first-line anti-mycobacterial antibiotic that has been widely used in the treatment of active and latent TB. M. tuberculosis can enter latency after it has sensed a stringent environment via a complex regulatory system. Rv1996 belongs to the universal stress protein (USP) family and is regulated by the dormancy  survival regulator (DosR）. It is located in a deletion hot spot region of clinical strains, suggesting that rv1996 may be related to drug resistance.

Dr. MI’s team at the Institute of Microbiology, Chinese Academy of Sciences has obtained evidence that the latency protein, USP-Rv1996, is associated with INH resistance in mycobacteria.

Firstly, the latency protein Rv1996 was overexpressed in M. bovis BCG (BCG-Rv1996) to mimic the physiological latency status. Under these conditions, susceptibility to INH was increased compared to the control strain (BCG-pMV261), as shown by minimum inhibitory concentration (Fig. 1) and killing curves assays (Fig. 1).

To explore the effect of Rv1996 on INH susceptibility further, MI and her colleagues compared protein expression levels between BCG-pMV261 and BCG-Rv1996 in the early log phase (OD₆₀₀ = 0.3). In collaboration with Dr. DENG Haiteng’s lab (Tsinghua University), they identified 50 up-regulated proteins and 26 down-regulated proteins using TMT-labeled proteomic analysis. Specifically, the amount of the KatG protein, which is known to be essential for INH activity, was show to be higher in BCG-Rv1996 compared with BCG-pMV261.

Interestingly, it was also shown that overexpression of Rv1996 increased the activity of KatG activity in vivo. Additionally, the protein level of MprA (Mycobacterium persistence regulator) was decreased in BCG-Rv1996 compared with BCG-pMV261.

The authors further set up a Wayne model of hypoxia-induced Mycobacterial dormancy and observed an increase in the mRNA levels of both katG and rv1996 and a decrease in the mRNA level of MprA in hypoxia bacillary compared with aerobic ones. 

Their study first shows a direct relationship between latency regulatory gene and drug susceptibility. The results provide an explanation for the epidemiological data that have identified the presence of Rv1996 in a deletion hot spot of clinical strains. During latency status, the latency USP-Rv1996 is overexpressed which leads to a decrease in MprA expression, followed by up-regulation of KatG and, consequently increased INH susceptibility.

Figure 1 : Overexpression of latency protein Rv1996 increase susceptibility to INH (Image from Dr. Mi's

Key words: Universal stress protein Rv1996, isoniazid, mycobacteria

Funding: This work was supported by the National Basic Research Program of China (2012CB518700 and 2014CB744400), the National Natural Science Foundation of China (31270178 and 31070118), the Key Program of the Chinese Academy of Sciences (KJZD-EW-L02); and the Chinese Academy of Sciences Visiting Professorship (2010T1S18 to John Chan).

Article Website:

http://pubs.acs.org/doi/abs/10.1021/pr5011058

Contact:
Dr. MI Kaixia

CAS Key Laboratory of Pathogenic Microbiology and Immunology ，Institute Of Microbiology，Chinese Academy of Sciences，100101，Beijing, China
E-mail: mik@im.ac.cn