Xuelian Zhang

Background: Endophytic fungi are important sources of structurally diverse antimicrobial natural products, but their potential to yield antitubercular scaffolds remains insufficiently explored. This study aimed to uncover new decalin polyketides from the endophytic fungus, Trichoderma longicollum 17007, and to evaluate their biological activity. Methods: Feature-based molecular networking (FBMN) analysis of the ethyl acetate extract from rice-based cultures was performed using trichoharzin as a seed compound to guide targeted isolation. Structures were established by HRMS, 1D and 2D NMR spectroscopy, NOESY analysis, and quantum chemical ECD calculations. Cytotoxicity and activity against the avirulent Mycobacterium tuberculosis H37Ra strain were evaluated, and genome mining was performed to identify a candidate biosynthetic gene cluster. Results: Six new decalin polyketides, tandyukisins K–P (1–6), and two known analogues, trichoharzin (7) and tandyukisin J (8), were isolated. Compounds 1–6 are C1-O-acetylated analogues bearing a 3-methylpentenedioate-type acyl side chain. Comparative ¹³C NMR analysis revealed empirical features useful for assigning side-chain attachment site, double-bond position and geometry. Tandyukisins K (1) and P (6) showed moderate activity against the avirulent M. tuberculosis H37Ra strain, with a MIC value of 12.5 μg/mL. Conclusions: These findings expand the structural diversity of decalin polyketides and provide the first evidence of antitubercular activity by this compound family.

The caseinolytic protease (Clp) system, which is highly conserved and essential for protein homeostasis in mycobacteria, comprises the ClpP1P2 proteolytic core and the essential ATPases ClpC1 and ClpX. While ClpX has a defined role in DNA replication, and some differential substrates have been identified, the physiological functions distinguishing ClpC1 from ClpX remain incompletely understood. Here, using Clustered Regularly Interspaced Short Palindromic Repeats-mediated knockdown in Mycolicibacterium smegmatis and the attenuated Mycobacterium tuberculosis H37Ra strain, we showed that ClpX impairment caused complete growth arrest, cell filamentation, and dramatically increased intracellular reactive oxygen species (ROS). In contrast, clpC1 knockdown resulted in only moderate growth defects but induced profound hypersensitivity to high oxygen and catalase. Mechanistically, ClpC1 maintained iron homeostasis; its loss elevated free Fe² ⁺ levels, driving Fenton chemistry, catastrophic ROS bursts, and redox collapse upon oxygen exposure. This iron-dependent survival defect was confirmed in a murine model. We thus establish ClpC1 as a central regulator of iron metabolism and oxidative defense, implicating its roles in bacterial reactivation from dormancy and persistence under fluctuating oxygen conditions. Furthermore, knockdown of either clpC1 or clpX increased mycobacterial susceptibility to multiple antibiotics, highlighting the Clp system as an attractive anti-tuberculosis drug target. Together, our findings reveal overlapping yet non-redundant roles for ClpC1 and ClpX, delineating their distinct regulatory networks. These results advance our understanding of the Clp system. Importantly, although these conclusions are derived from studies in surrogate mycobacterial species, they raise the possibility that similar mechanisms may underlie adaptation, dormancy, and resuscitation in pathogenic Mycobacterium tuberculosis . IMPORTANCE: Tuberculosis remains a major global health burden. The ClpC1/ClpX-ClpP1P2 proteolytic complex has emerged as a highly attractive drug target. This study investigates the role of ClpX in maintaining fundamental bacterial biological processes and suggests a specific function in regulating iron homeostasis and oxidative defense. This proposed mechanism may contribute to the bacterium’s ability to survive environmental shifts, such as resuscitation from latency. By demonstrating an involvement of caseinolytic proteases (Clps) in iron metabolism, this research provides additional insights into mycobacterial adaptive strategies within the host. Importantly, the results highlight a putative vulnerability during latent infection and reactivation: mycobacteria appear to depend on ClpX and ClpC1 for dormancy regulation, while ClpC1 is likely important for mitigating reoxygenation-induced oxidative stress. This discovery may inform future therapeutic approaches specifically targeting the reactivation phase of latent tuberculosis.

A series of nitrile derivatives of Petromurin C were synthesized and assessed for their cytotoxic effects on tumor cell lines and inhibitory activity against Mycobacterium tuberculosis (M.tb H37Ra). Bromoalkyl nitriles (Br(CH2)nCN; n=1-5) and (o-, m-, p-) bromomethyl benzonitriles were used as alkylating reagents. The majority of the syntheses consisted of O-alkylated derivatives in which the two NH group were not alkylated, as expected from the treatment of petromurin C with K2CO3 in DMF followed by treatment with alkylating reagents. Similarly, treatment of Petromurin C with NaH in DMF and the treatment with alkylating reagents yielded both OR and bis-NR alkylated structures. In the first method, 7 different O-alkyl Petromurin C derivatives were obtained. The second method yielded 7 different Petromurin-C derivatives containing both O-alkyl and bis-N-alkyl groups. Notably, compounds 1-4 and 7-10 demonstrated selective inhibitory activity against the acute myeloid leukemia cell line MV4-11, with IC50 values ranging from 12.96 to 20.00 μM. Compound 15 exhibited a minimum inhibitory concentration (MIC) of 6.25 μM against M.tb H37Ra. Importantly, all synthesized compounds showed negligible inhibition (below 50%) against human normal cell lines L-02 and 293T at a concentration of 100 μM. These findings suggest that the compounds possess high efficacy and low toxicity, indicating their potential as novel therapeutic agents for the treatment of leukemia and tuberculosis.

Ribonucleotide reductase (RNR) catalyzes the synthesis of four deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication. Although dNTP imbalances reduce replication fidelity and elevate mutation rates, the impact of RNR dysfunction on Mycobacterium tuberculosis (Mtb) physiology and drug resistance remains unknown. Here, we constructed inducible knockdown strains for the RNR R1 subunit NrdE in Mtb and Mycolicibacterium smegmatis (Msm). NrdE knockdown significantly impaired growth and metabolic imbalances, indirectly disrupting oxidative homeostasis and mycolic acid synthesis, while increasing levels of intracellular ROS accumulation and enhancing cell wall permeability. Additionally, we developed genomic mutant strains, Msm-Y252A and Msm-Q255A, featuring targeted point mutations in the substrate-specific site (S-site) of the RNR loop domain, which determines NDP reduction specificity. The Msm-Y252A displayed a 1.9-fold decrease in dATP and increases in dGTP (1.6-fold), dTTP (9.0-fold), and dCTP (1.3-fold). In contrast, Msm-Q255A exhibited elevated intracellular levels of dGTP (1.6-fold), dTTP (6.1-fold), and dATP (1.5-fold), while dCTP levels remained unchanged. Neither the NrdE knockdown strain nor the S-site mutants exhibited direct resistance development; however, they both showed genomic instability, enhancing the emergence of rifampicin-resistant mutants, even with a 70-fold and a 25-fold increase in mutation frequency for Msm-Y252A and Msm-Q255A, respectively. This study demonstrates that NrdE is integral to Mycobacterium survival and genomic stability and that its RNR dysfunction creates a mutagenic environment under selective pressure, indirectly contributes to the development of drug resistance, positioning NrdE as an effective target for therapeutic strategies and a valuable molecular marker for early detection of drug-resistant Mtb.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge, primarily due to the increasing prevalence of drug resistance. Consequently, the development of drugs with novel modes of action (MOAs) is urgently required. In this study, we discovered and characterized two potent inhibitors, Pi-1-58 and Pi-2-26, targeting the prokaryotic ubiquitin-like protein (Pup) ligase PafA of Mtb. Pi-1-58 was identified through computer-aided drug screening. The binding mode of Pi-1-58 and PafA was investigated through molecular docking, followed by experimental validations. Based on the core structure of Pi-1-58, we developed a more potent inhibitor, Pi-2-26, through structure-guided drug design. Both Pi-1-58 and Pi-2-26 exhibited selective and specific inhibition of PafA according to biochemical and cell-based assays. Importantly, the inhibitors demonstrated significant inhibition on Mtb survival in the presence of nitric oxide, mimicking the in vivo nitrogen limited environment that Mtb encountered in macrophage. Our findings provide a comprehensive understanding of the structural and functional aspects of these PafA inhibitors and establish a solid foundation for the development of novel therapeutics against tuberculosis.

Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the β-CASP and the β-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid β-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis, and the ability of Mtb to survive harsh host conditions has been the subject of intensive research. As a result, we explored the molecular mechanisms guiding downstream target genes of CRP when Mtb responds to its environment.

Summary: As the FAIR (Findable, Accessible, Interoperable, Reusable) principles have become widely accepted in the proteomics field, under the guidance of ProteomeXchange and The Human Proteome Organization Proteomics Standards Initiative, proteomics public databases have been providing Application Programming Interfaces for programmatic access. Based on generating logic from proteomics data, we present Patpat, an extensible framework for searching public datasets, merging results from multiple databases to help researchers find their proteins of interest in the vast mass spectrometry. Patpat’s 2D strategy of combining results from multiple databases allows users to provide only protein identifiers to obtain metadata for relevant datasets, improving the ‘Findable’ of proteomics data.Availability and implementation: The Patpat framework is released under the Apache 2.0 license open source, and the source code is stored on GitHub (https://github.com/henry-leo/Patpat) and is freely available.Supplementary information: Supplementary data are available at Bioinformatics online.

The PE/PPE protein family commonly exists in pathogenic species, such as Mycobacterium tuberculosis, suggesting a role in virulence and its maintenance. However, the exact role of most PE/PPE proteins in host–pathogen interactions remains unknown. Here, we constructed a recombinant Mycobacterium smegmatis expressing M. tuberculosis PE_PGRS19 (Ms_PE_PGRS19) and found that PE_PGRS19 overexpression resulted in accelerated bacterial growth in vitro, increased bacterial survival in macrophages, and enhanced cell damage capacity. Ms_PE_PGRS19 also induced the expression of pro-inflammatory cytokines, such as IL-6, TNF-α, IL-1β, and IL-18. Furthermore, we demonstrated that Ms_PE_PGRS19 induced cell pyroptosis by cleaving caspase-11 via a non-classical pathway rather than caspase-1 activation and further inducing the cleavage of gasdermin D, which led to the release of IL-1β and IL-18. To the best of our current knowledge, this is the first report of a PE/PPE family protein activating cell pyroptosis via a non-classical pathway, which expands the knowledge on PE/PPE protein functions, and these pathogenic factors involved in bacterial survival and spread could be potential drug targets for anti-tuberculosis therapy.