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. 2025 Jun 16;13(6):1401.
doi: 10.3390/microorganisms13061401.

The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates

H M Adnan Hameed  1   2   3   4 , Cuiting Fang  1   2   3   4 , Zhiyong Liu  1   5 , Yamin Gao  1   2   3   4 , Shuai Wang  1   2   3   4 , Xinwen Chen  5 , Nanshan Zhong  5   6 , Htin Lin Aung  2   7 , Jinxing Hu  1 , Tianyu Zhang  1   2   3   4   5
Affiliations

The Identification of Novel Mutations in ATP-Dependent Protease ClpC1 Assists in the Molecular Diagnosis of Obscured Pyrazinamide-Resistant Tuberculosis Clinical Isolates

H M Adnan Hameed et al. Microorganisms. .

Abstract

Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZAR) primarily related to pncA mutations. However, discordance between phenotypic resistance and conventional pncA-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among Mycobacterium tuberculosis (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZAR strains were genetically assessed by Sanger sequencing of PZAR-associated customary genes. Five pncA-wild-type PZAR strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZAR strains harbored mutations in pncA and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZAR strains lacked pncA mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating clpC1 mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (pncA alone) to 100%. This is the first report from southern China that identifies novel clpC1 mutations in wild-type pncA PZAR Mtb isolates. Our findings underscore the limitations of pncA-targeted diagnostics and support the integration of WGS and clpC1 analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.

Keywords: Mycobacterium tuberculosis; clinical isolates; clpC1; drug resistance; molecular diagnosis; pyrazinamide.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
PZase activity assay of Mtb clinical isolates. The tubes in the figure present the results of the PZase activity test. In these tubes, brownish liquid indicates positive PZase activity, reflecting the hydrolysis of PZA to POA, while colorless liquid (no change in color) indicates negative PZase activity, suggesting the absence of this enzymatic function.
Figure 2
Figure 2
Structural changes resulting from amino acid substitutions via in silico mutagenesis of ClpC1 of Mtb.
Figure 3
Figure 3
Proteolytic activity of the ClpC1P1P2 complex in response to POA treatment. **** p < 0.00001; ns, no significant difference, p > 0.05. The D-value, representing the change in fluorescence intensity, was calculated by subtracting the initial fluorescence intensity from the fluorescence intensities of subsequent detections.
Figure 4
Figure 4
Novel mutations across ClpC1 domains of Mtb clinical isolates.
See this image and copyright information in PMC

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