## 4 Discussion
Although several antibiotics share ClpC1 as a macromolecular target, the differences in their binding patterns can lead to varying effects on ClpC1 proteolytic and ATPase activities [17; 50]. Moreover, the N-terminal domain of ClpC connects with adaptor proteins, either by serving as a binding site or by assisting in substrate recognition [52], suggesting that mutations in this region could interfere with substrate processing or drug binding, contributing to PZA resistance. Besides the N-terminal mutations, we also identified mutations in the D2 domain (Ala567 Val) and C-terminal domain (Pro796 Leu) of ClpC1 (Figure 4). The C-terminal domain has been found essential for the stability of ClpC1 hexamer and plays a critical role in protein oligomerization, which is required for its chaperone activity; however, alterations in the C-terminus lead to defects in oligomerization [47]. Owing to its crucial role in Mtb viability, ClpC1 has emerged as an attractive target for novel anti-TB agents, including rufomycin [50] and four other cyclic peptides: cyclomarin A [53], lassomycin [49], ilamycin [54], and ecumicin [51]. The anti-persister activity of these cyclic peptides through the disruption of ClpC1-mediated protein degradation is mechanistically analogous to PZA's interference with the protein degradation system via the inhibition of trans-translation [14]. Using FITC-casein as a substrate, we confirmed that PZA's active form, pyrazinoic acid (POA), does not directly inhibit ClpC1P1P2 proteolytic activity, unlike inhibitors such as ILE, which significantly reduced protease function [17; 50; 55; 56]. Our data suggest that POA likely exerts its effect indirectly by promoting the degradation of PanD rather than by binding ClpC1 directly. Future studies employing detailed metabolomic and proteomic analyses are crucial to elucidate the functional consequences of ClpC1 mutations on PZA resistance mechanisms. The increasing emergence of non-canonical resistance mechanisms that evade molecular diagnostics poses a growing challenge [57]. Mutations in clpC1 and insertions/deletions in flanking regions of pncA are often missed by conventional diagnostic assays, thereby compromising the accuracy of PZA resistance detection. According to the WHO, new molecular assays for Mtb should achieve a diagnostic sensitivity of >90% and specificity of >95% [58]. In our study, the cumulative assessment of resistance-conferring mutations demonstrated a marked improvement in diagnostic performance. The sensitivity and accuracy of PZA resistance detection increased from 48.3% and 69.8% when targeting _pncA_ alone to 100% when the analysis expanded by including novel resistance markers and flanking regions. This performance surpassed the sensitivities reported in previous studies: 95.9% in Hunan [32]; 90.1% in Hangzhou [59]; 89.5% in Henan [60]; 83.1% in Ningbo [33]; 91.3% in Ireland [30]; and 97.1% in London, UK [61]. In the referenced studies, which involved comprehensive molecular analyses of PZA resistance, the most commonly examined genes besides _pncA_ included _rpsA_ and _panD_, which are well-established candidates associated with PZA resistance and are often included in molecular diagnostic investigations. Therefore, these genes were referred to as 'customary genes' in our study. Some studies have also explored additional targets such as clpC1 [17; 62], Rv2783c [12], and others, albeit  
Figure 4: Novel mutations across ClpC1 domains of Mtb clinical isolates. less consistently; however, combined evaluations of these genes assisted in increasing the diagnostic sensitivity [12]. Among them, pncA was the primary resistance determinant, with the highest frequency of mutations, particularly among the isolates belonging to the Beijing genotype. In agreement with recent reports [12; 28], our findings strongly reinforce the necessity of broadening molecular diagnostic targets beyond pncA alone.