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Tuberculosis (TB) is a top infectious killer worldwide, and is responsible for more deaths than human immunodeficiency virus (HIV) and malaria, resulting in 400,000 deaths, annually. In 2016, the world health organization (WHO) estimated that 580 000 people worldwide develop Multidrug-Resistant (MDR) TB. India, China, and the Russian Federation accounted for 45% of the 580 000 cases. Although isoniazid (INH) is an essential element of all first-line treatment regimens for TB (1), INH resistance is an obstacle to the treatment of TB (2, 3). In order to achieve the ambitious target set in the End TB Strategy, these highest TB burden countries need to depend on advances in TB prevention and care.
According to broad analyses, mutations in the katG gene and inhA gene are the cause of the majority of INH resistance in clinical isolates (4), yet other mutations carry the explanation fora subset of INH resistance cases that lack the 2 most common canonical mutations (5, 6). These results contribute to making a broader search in the mechanisms of INH resistance.
In 2008, the first bacterial protein modifier, prokaryotic ubiquitin-like protein (Pup) was identified in Mycobacterium tuberculosis (MTB) (7). Functionally analogous to ubiquitin, conjugation with Pup serves as a signal for degradation by the mycobacterial proteasome (8). Protein-proteasome system (PPS) is a posttranslational regulatory mechanism (9). In the system, Pup could tag a variety of functional proteins and mediate these proteins degraded by proteasome, with the help of Dop (deamidase of Pup), PafA (Pup ligase), and Mpa. These proteins involve intermediary metabolism, lipid metabolism, and detoxification/virulence (10). However, whether PPS influences drug resistance of isoniazid mono-resistant M. tuberculosis (INH-MTB) is still unknown.
The purpose of this study was to clarify whether PPS effects drug resistance of INH-MTB strain.
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The current study showed that the MIC of INH in rINH -MTB: Dop did not change when compared with that in INH-MTB strain. This result indicated that Dop-over-expression had no effect on the drug resistance of the INH-MTB strain. The researchers speculated that Dop protein was partly degraded in order to maintain the functional stability of PPS.
Elharar et al. (9) found that exponential phase cells harboring Dop over-expression presented a 7-fold increase at the Dop mRNA level, compared with cells containing an empty vector. However, a barely detectable increase was noted when the Dop protein level was assessed, suggesting that Dop over-expression had been counterbalanced, potentially at the proteolytic level (9). Meanwhile, this study also found that low Dop protein levels promoted efficient pupylation, yet high Dop protein levels shifted the balance between the 2 activities of Dop to facilitate the process of de-pupylation (9). High Dop levels would lead to futile cycle between pupylation and de-pupylation.
The deletion of Dop gene and pafA gene could reduce the MIC of INH. The deletion of Dop or PafA interdicted the process of pupylation of substrate protein, leading to the accumulation of FabD (10, 18). The FabD involved in the synthesis of fatty acids and it was a crucial constituent of Mycobacterial cell wall. However, these abnormal FabD might hinder the synthesis of normal Mycobacterial cell wall, increasing the permeability of anti-tuberculosis drugs.
Delley et al. (19) found that in M. smegmatis (Msm) with a Dop deletion mutant, pupylation was severely impaired and proteasomal substrates were increased. Festa et al. (10) found that ∆PafA strain could cause the accumulation of substrate proteins in MTB. These results suggest that PafA or Dop are necessary to finish the degradation of substrate proteins. The MIC of INH in INH-MTB strain did not change when compared with that in rINH-MTB: Mpa strain and rINH-MTB: PafA strain. The Mpa protein could be pupylated and then degraded by proteasome. However, when PafA was knocked out, Mpa would accumulate in bacterial body, suggesting that the pupylation of Mpa could make itself be degraded (20).
Posttranslational modification of target proteins with the help of Pup, not only made a substrate be degraded by the proteasome but also regulated the enzyme activity of PPS itself (19). It has been reported that PafA could be poly-pupylated by itself and PafA accumulated in the strain with the deletion of Mpa or prcBA (21). The results suggest that the pupylation of Mpa or PafA intends to maintain the stability of PPS.
The current study showed that the MIC of INH in INH-MTB strain was lower than that in the INH-MTB∆Mpa strain and INH-MTB∆Pup strain. The Mpa contributed to the survival of MTB in a host; the virulence of ∆Mpa strain was reduced in the infection model of mice (22). The Pup made the target proteins to attach an unordered label to facilitate the degradation of target proteins. The researchers speculated that the deletion of Mpa or Pup would lead to the accumulation of target proteins, which decreased the tolerance of bacteria to all kinds of stress.
This study showed that the MIC of INH was lower in the INH-MTB strain than that in the rINH-MTB: Pup strain. They speculated that the increase of Pup protein accelerated the speed of degradation of harmful proteins in MTB, enhancing the adaptability of MTB. Ino1 could not be detected in Pup-over-expression Msm, speculating that the speed of elimination of Ino1 was expedited (23).
In summary, the experiments of this study clarify that MTB PPS affect drug resistance of the INH-MTB strain. This study provides new information for the treatment of drug-resistant TB.
