Objective: Pyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA⁺ upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis. Methods: Drug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA (Rv2044c-pncA-Rv2042c) and PZase assay were performed for 448 M. tuberculosis clinical isolates. Results: Our data showed that among 448 M. tuberculosis clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drug-resistance. A total of 60.04% (269/448) M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZA^R isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZA^R. Compared with phenotypic data, 84.38% (227/269) PZA^R strains with mutations in pncA⁺ upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C− 114del) in upstream of pncA^wt nullified the effect of A− 11G mutation and induced positive PZase activity, divergent from five PZase negative A− 11G PZA^R mutants. Twenty-six PZA^R strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms. Conclusion: Our study revealed that PZA^R rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA⁺ UFR, rpsA, panD, Rv2783c, and Rv2044c provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.

Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF. Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay. Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to > 64 μg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426– 452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284– 1289 CCAGCT), (1295– 1303 AATTCATGG), and insertion at (1300– 1302 TTC) were detected within RRDR of three RIF^R strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIF^R strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively. Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIF^R clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.

The caseinolytic protease (Clp) system has recently emerged as a promising anti-tuberculosis target. The anti-cancer drug bortezomib exhibits potent anti-mycobacterial activity and binds to Mycobacterium tuberculosis (Mtb) Clp protease complexes. We determine cryo-EM structures of Mtb ClpP1P2, ClpC1P1P2 and ClpXP1P2 complexes bound to bortezomib in different conformations. Structural and biochemical data indicate that sub-stoichiometric binding by bortezomib to the protease active sites orthosterically activates the MtbClpP1P2 complex. Bortezomib activation of MtbClpP1P2 induces structural changes promoting the recruitment of the chaperone-unfoldases, MtbClpC1 or MtbClpX, facilitating holoenzyme formation. The structures of the MtbClpC1P1P2 holoenzyme indicate that MtbClpC1 motion, induced by ATP rebinding at the MtbClpC1 spiral seam, translocates the substrate. In the MtbClpXP1P2 holoenzyme structure, we identify a specialized substrate channel gating mechanism involving the MtbClpX pore-2 loop and MtbClpP2 N-terminal domains. Our results provide insights into the intricate regulation of the Mtb Clp system and suggest that bortezomib can disrupt this regulation by sub-stoichiometric binding at the Mtb Clp protease sites.

A series of 3-amino-5-benzylphenol derivatives were designed and synthesized. Among them, (3-benzyl-5-hydroxyphenyl)carbamates were found to exert good inhibitory activity against M. tuberculosis H37Ra, H37Rv and clinically isolated multidrug-resistant M. tuberculosis strains (MIC = 0.625–6.25 μg/mL). The privileged compounds 3i and 3l showed moderate cytotoxicity against cell line A549. Compound 3l also exhibited potent in vivo inhibitory activity on a mouse infection model via the oral administration. The results demonstrated 3-hydroxyphenylcarbamates as a class of new antitubercular agents with good potential.

The escalating threat of mycobacterial infectious diseases, particularly those caused by nontuberculous mycobacteria (NTM), poses a serious challenge to public health. Linezolid (LZD), an oxazolidinone antimicrobial, exhibits potent activity against Mycobacterium tuberculosis and NTM. Generally, mutations in the rrl and rplC genes are widely associated with resistance to LZD. However, in this study, we screened Mycobacterium marinum strains lacking such mutations, indicating the presence of an alternative resistance mechanism. Notably, through whole-genome sequencing, we identified a novel mutation C395T in the MMAR_0911 ( grcC1 ) gene that has never been linked to drug resistance. This mutation leads to an A132V substitution in the encoded protein, a polyprenyl diphosphate synthase potentially involved in the synthesis of cell wall components and menaquinones. We found that the overexpression of grcC1 caused resistance to multiple drugs including LZD, clarithromycin (CLR), vancomycin (VAN), clofazimine (CFZ), rifampicin (RIF), cefoxitin (CEF), levofloxacin (LEV), and moxifloxacin (MXF) and reduced cell wall permeability, while the silence and knockout of grcC1 showed increased cell wall permeability and susceptibility to these drugs. Using CRISPR/Cpf1-assisted gene editing, we confirmed that the A132V mutation conferred low-level resistance to the aforementioned drugs in Mycobacterium abscessus and Mycobacterium smegmatis . Furthermore, thin-layer chromatography analysis indicated reduced glycolipid polarity in the grcC1 mutant strains, suggesting an impact on the cell envelope integrity. Our findings suggest that GrcC1 contributes to low-level drug resistance in mycobacteria by potentially reducing cell wall permeability, highlighting its potential as a novel target for antimicrobial agents and as a diagnostic marker. IMPORTANCE: Our study uncovers a novel drug resistance mechanism in mycobacteria, focusing on the previously uncharacterized grcC1 gene. We identified a new mutation, A132V, in GrcC1, which is involved in cell wall component synthesis and menaquinone production. This mutation contributes to low-level resistance not only to linezolid but also to a broad range of drugs, including clarithromycin, vancomycin, and rifampicin. Through advanced techniques like CRISPR interference and gene editing, we demonstrated that GrcC1 plays a critical role in drug susceptibility and cell wall permeability across multiple Mycobacterium species. These findings represent the first connection between GrcC1 and drug resistance, offering new insights into combating infections caused by nontuberculous mycobacteria (NTM). Our work highlights the potential of GrcC1 as a target for novel therapeutic approaches and as a diagnostic marker for drug-resistant NTM infections.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains poses serious challenges to global tuberculosis control, highlighting the urgent need to elucidate the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant Mycobacterium smegmatis (Msm) mutants and identified a strain, Msm-R1-2, exhibiting 16- and 64-fold increases in minimum inhibitory concentrations (MICs) to BTZ and linezolid (LZD), respectively, compared to the parental strain. Whole-genome sequencing revealed resistance-associated mutations in two functionally distinct genes: MSMEG_1380, encoding a transcriptional regulator involved in efflux pump expression, and MSMEG_0965, encoding a porin protein. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that single mutations in either MSMEG_1380 or MSMEG_0965 caused low-level resistance (4-fold MIC increase) to BTZ and LZD, while dual mutations conferred resistance levels comparable to Msm-R1-2, with 16- and 64-fold increases in MICs for BTZ and LZD, respectively. An ethidium bromide accumulation assay demonstrated that mutations in MSMEG_0965 reduce cell wall permeability, contributing to multidrug resistance. Furthermore, quantitative real-time PCR showed that mutations in MSMEG_1380 upregulate the mmpS5-mmpL5 efflux system. Together, these dual mechanisms function synergistically: restricted drug entry combined with enhanced drug efflux confers robust multidrug resistance. These findings provide novel insights into the evolutionary mechanisms of resistance in mycobacteria.