Oligosaccharides from Scorzonera yildirimlii and S. zorkunensis, and their potential antimicrobial, enzyme inhibition, cytotoxic effect, and in silico study.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt bd-II from Mycobacterium smegmatis, a member of a hitherto uncharacterized evolutionary group (qOR-2) of bd oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the appCB operon in its native host led to production of a highly active bd-II (kobs = 30 e- s-1) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

Biological nanopores enable single-molecule sensing but often lack specificity when analytes produce similar current signatures. Covalent tethering of recognition elements improves selectivity, yet site-specific functionalization of symmetric, multimeric pores like Mycobacterium smegmatis porin A (MspA) remains challenging. Existing gel-based strategies for isolating heteromeric pores are labor-intensive and low-throughput and can compromise protein functionality. Here, we report a modular strategy for producing hetero-octameric MspA nanopores with a single site-specific modification. By coexpressing MspA and a D56C MspA mutant bearing a C-terminal D16 tag in Escherichia coli (E. coli), we directed the assembly of asymmetric pores with defined subunit composition. To isolate the modified pores, we developed a rapid, nondenaturing purification method that integrates magnetic bead capture with toehold-mediated DNA strand displacement, demonstrating selective enrichment of functionalized pores in under 3.5 h while preserving structural integrity. Using this platform, we generated MspA nanopores functionalized with single DNA probes targeting dopamine, microRNA, and thrombin. Single-channel recordings demonstrated distinct current signatures upon target recognition, enabling label-free and selective detection. This approach offers a robust and scalable framework for engineering functionalized nanopores for diverse molecular sensing.

Novel routes for bioproduction of delta lactone aroma compounds.

In silico identification of quinoline-pyridine hybrids binding to Mycobacterium protein kinase B, assessment by molecular dynamics simulation and quantum mechanics calculation, and in vitro validation of antimicrobial activity.

Bacterial adhesion on glyco-hydrogels: impact of glycan and hydrogel stiffness.

Background Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), is the leading cause of death due to a single infectious pathogen globally. The increasing prevalence of drug-resistant Mtb strains underscores the pressing need for the development of new antimycobacterial drugs with novel mechanisms of action. Targeting pathogen drug efflux, a key antimycobacterial drug resistance mechanism, is an attractive, viable strategy for the development of new TB therapeutics. Methods In this study, we utilised Mycobacterium smegmatis ( Msm ), a non-pathogenic Mtb surrogate, to delineate the ability of natural-product based compounds to augment the efficacy of bedaquiline (BDQ), clofazimine (CFZ) and doxycycline (DOX), probably via efflux inhibition (EI). Literature reporting the plant sources of the known efflux inhibitors (EIs) reserpine (RES), berberine (BER) and piperine (PIP) was scoped and additional compounds, (+)-lyoniresinol-3-Alpha-O-Beta-D-glucopyranoside (LYO-3) and lyoniresinol (LYO), isolated from the same plant species were chosen for testing. In vitro screening of the selected compounds was performed using the two-dimensional (2-D) checkerboard assay in which each likely efflux disruptor was tested in combination with BDQ, CFZ and DOX against Msm and the effect of the combinations ascertained. Thereafter, compounds that exhibited probable EI activity were docked onto potential targets namely MSMEG_5187, a Msm homologue of Mtb Rv1258c efflux pump (EP) and MmpL5. Results Molecular docking revealed that the EIs avidly bound to the EPs with docking scores of <-7 kcal/mol while the checkerboard combination assays demonstrated strong synergistic interactions for BDQ plus BER, LYO-3 and LYO. Conclusion These results point to a probable disruption of the Msm efflux system by Berberis species derivatives.

Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB_L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aurum, and Escherichia coli. The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB_L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB_L378R mutation.

Bacteriophages represent a vast reservoir of genetic diversity however, functional annotation remains a major challenge, as most predicted gene products lack detectable similarity to characterized gene families. Experimental approaches such as systematic overexpression screens provide an avenue for identifying phage genes that influence bacterial physiology. Here, we report an overexpression screen of all 123 predicted protein-coding genes from Cluster L1 mycobacteriophage LeBron, the first representative of this cluster to undergo genome-wide functional analysis. Expression assays in Mycobacterium smegmatis revealed that 39 genes (32%) impaired host growth, with nineteen of these toxic genes (49%) assigned no known function. The proportion of cytotoxic genes observed in LeBron is comparable to findings from Clusters K and F, despite minimal sequence conservation across clusters. Interestingly, a subset of LeBron's toxic genes appear to be functionally analogous to previously identified toxic genes in other clusters, suggesting conserved biological activities carried out by non-homologous proteins. Additionally, this analysis uncovered several novel gene families that elicit strong cytotoxic effects, expanding the known catalog of phage-derived bacterial growth inhibitors. These results provide new insights into phage gene functions and demonstrate the value of genome-wide expression screening for uncovering conserved and cluster-specific interactions between bacteriophages and their hosts.

Mycolic acids (MAs) are the major component of the mycobacterial outer membrane, a key contributor to the intrinsic resistance of mycobacteria to external insults including multiple antibiotics. After being synthesized in the cytoplasm and before reaching the outer membrane, MAs are transported across the inner membrane in the form of an acylated sugar, generally believed to be trehalose monomycolates (TMMs). Whether trehalose is the only mycolate carrier during transport is under debate, and why this highly abundant disaccharide is essential for mycobacterial growth is unclear. To address these questions, we leveraged a trehalose auxotrophic Mycobacterium smegmatis strain to investigate the biosynthetic steps affording TMMs. We show that in addition to TMMs, mature MAs are also not produced in the absence of intracellular trehalose. This is likely due to a product inhibition mechanism where unreduced MA precursors accumulate on Pks13, the protein catalyzing the ligation of mycolic acids and the sugar head group. We establish that the unreduced mycolates could only be released by trehalose, revealing exquisite Pks13 specificity, and subsequently reduced by CmrA in vitro. Furthermore, only trehalose and its analogs can reactivate MA biosynthesis in cells. Finally, by replacing trehalose with a 6-deoxy analog in cells, we demonstrate that the cord factor trehalose dimycolate is dispensable for M. smegmatis growth in vitro. Our work gives a clear depiction of how TMMs are formed and provides a compelling reason for the essentiality of trehalose, shedding light on the development of future antimycobacterial strategies.

The rise of drug-resistant microbes has made antimicrobial therapy increasingly challenging, and despite several reports on peptide-functionalized silver nanoparticles, their efficacy against Mycobacterium species remains largely unexplored. In this study, we synthesized short peptide functionalized silver nanoparticles to develop an effective antimycobacterial agent, where peptides acted as both reducing and stabilizing agents for the one-pot synthesis of silver nanoparticles (AgNPs). The developed nanoparticles were characterized by high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), and UV-visible spectroscopy (UV-vis). The positively charged peptide-capped silver nanoparticles exhibited significant antimycobacterial activity against acid-fast mycobacterial strains, including Mycobacterium smegmatis, Mycobacterium bovis, and Mycobacterium marinum, compared to peptides alone, which could be due to the integrated effect of the peptide-functionalized AgNPs. Among the synthesized nanoparticles, linear peptide 2 (LP 2) functionalized AgNP exhibited the highest antimycobacterial efficacy against the Mycobacterium strains, with the lowest MIC (5 μM). AgNP LP 2 was found to be efficient to penetrate the mycobacterial cell wall, inducing membranolytic activity, triggering oxidative stress and degrading DNA, which led to the death of mycobacterial cells. Molecular docking and molecular dynamics (MD) simulations of the peptides with key enzyme FadD32 (MsmFadD32), Mycobacterium smegmatis, demonstrated strong interactions near the active site cleft, indicating potential inhibition of the mycolic acid biosynthesis pathway by the LP 2 peptide. This disruption likely challenges the organism's pathogenicity and supports the peptides' role in contributing to membranolytic activity. Additionally, AgNP LP 2 demonstrated the ability to inhibit biofilm formation and effectively disrupt preformed mycobacterial biofilms while exhibiting negligible cytotoxicity toward human embryonic kidney (HEK293) cells. In summary, our results suggest that newly developed AgNPs exhibit antimycobacterial activity without compromising the cell viability of normal cells, making them highly potent as prospective antimycobacterial agents.

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). l,d-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of β-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

Background/Objectives: The increase in incidences of multidrug resistance exacerbates tuberculosis-related global health challenges and underscores a call for more efforts for development of new antitubercular drugs, including the use of medicinal plants, especially those that have been used for generations by traditional healers. Despite reports of antimicrobial activity and chemical profiling of Kirkia wilmsii (K. wilmsii) extracts, chemical structures of the bioactive agents have not been elucidated. Here, we used a combination of bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance to purify and elucidate the chemical structure of antimycobacterial agents contained in leaf and twig extracts for K. wilmsii. Results: After overnight extraction with acetone and 90 g of dry twigs and leaves produced 5.38 g (6%) and 4.56 g (5%) of product, which displayed moderate antimycobacterial activity of 0.5 and 1 mg/mL, respectively. The antimycobacterial activity was increased six- and three-fold, respectively, after the crude extracts were subjected to solvent-solvent partitioning. Due to many bioactive fractions being obtained after silica gel chromatography purification, fraction 5 of twig extract was prioritized for further purification due to its low minimum inhibitory concentration (MIC) (0.25 mg/mL) and cytotoxicity (20%, in THP-1 cells). Sequential purification of the fraction 5 (twig extract) extracts through the C18 cartridge and high-performance liquid chromatography (HPLC) produced four fractions, which were subjected to structural elucidation. The high-resolution mass spectrometric analyses revealed that the first two eluting peaks had the same mass ion of 441.0822 m/z (M - H-), which corresponded to catechin monogallate, and so were the last two eluting peaks, which had a mass ion of 539.0932 m/z (M - H-), corresponding to catechin digallate. Further analyses by 1H, 13C, and 2D NMR confirmed the chemical structures of compounds eluting in the first two peaks on HPLC as structural isomers of catechin 3'-monogallate and catechin 4'-monogallate (MIC not determined). Similarly, compounds eluting in the last two peaks were identified as structural isomers catechin 3'-digallate and catechin 4'-digallate, with an MIC of 250 µg/mL against Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv and an MBC of 500 μg/mL against M. smegmatis. Conclusions: To the best of our knowledge, this study is the first to report the structure of catechin 3'- and 4'-digallate, their antimycobacterial activity, and the existence of acyl migration involving galloyl 3' and 4'-hydroxyl groups of catechin ring B.

Porphyrin secretion does not alter heme biosynthesis in the nontuberculous mycobacteria.

Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens.

Multidrug-resistant (MDR) mycobacteria are considered a major challenge in tuberculosis treatment, creating an urgent need for novel antimycobacterial drugs. Actinobacteria, known for their ability to produce bioactive compounds, are considered promising sources for new drug discovery. In this study, 87 actinobacteria isolates were successfully obtained from five samples collected in a karst cave on Sumba Island, Indonesia. The isolates were screened for antimycobacterial activity against Mycobacterium smegmatis wild-type (WT-M.smeg), rifampicin-resistant (RIFR-M.smeg), isoniazid-resistant (INHR-M.smeg), and multidrug-resistant (MDR-M.smeg) strains. Sixteen extracts were found to inhibit WT-M.smeg, with three extracts from isolates KRST 02-20, KRST 03-10, and KRST 05-08 showing potent activity against all resistant strains (≥95% growth inhibition). The extract of isolate KRST 03-10 was observed to exhibit the most significant inhibition, with IC50 values of 9.63 µg/mL (WT-M.smeg), 29.64 µg/mL (RIFR-M.smeg), 10.89 µg/mL (INHR-M.smeg), and 27.76 µg/mL (MDR-M.smeg). Molecular identification showed that this isolate has the highest similarity (98.91%) with Streptomyces cinereoruber NBRC 12756. Gas chromatography-mass spectrometry (GC-MS) analysis identified 2,4-di-tert-butylphenol as a compound with potential antituberculosis activity, while liquid chromatography-high-resolution mass spectrometry (LC-HRMS) detected nocardamine, L-α-palmitin, erucamide, and 2-anisic acid, all known for their antimicrobial activity. An unidentified compound, NP-011220 (C11H18O2), was also detected in high relative abundance. Further research is needed to evaluate the activity of the most promising isolate, KRST 03-10, against Mycobacterium tuberculosis and to purify its active compounds.

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

Heme is an essential cofactor and dietary source of iron for the obligate human pathogen, Mycobacterium tuberculosis (Mtb). Consequently, heme is required for Mtb growth and pathogenicity, and strategies to limit heme represent a promising therapeutic approach. Although Mtb can both make and scavenge heme, it was previously found that de novo synthesized heme is substantially more bioavailable and metabolically active than exogenously scavenged heme. These findings provided a strong justification to target the terminal heme biosynthetic enzyme, coproheme decarboxylase (ChdC), in the development of antimycobacterial therapies. Herein, we sought to characterize heme homeostasis in a ΔchdC deletion mutant in Mtb. Surprisingly, we found that ablation of ChdC in Mtb and Mycobacterium smegmatis resulted in the enhanced accumulation and bioavailability of exogenously scavenged heme compared with WT or mutants lacking glutamyl tRNA reductase, the first enzyme in the heme synthetic pathway. Moreover, we found that Mtb has a preference for scavenging reduced ferrous heme and exhibits a heme reductase activity that is inhibited by ChdC. We further found that ChdC expression is downregulated when iron is limiting, which in turn increases both heme import and bioavailability. Such a mechanism may serve to protect cells from heme toxicity while trying to meet the nutritional demand for iron. Importantly, our results also suggest that caution must be taken if targeting ChdC because of feedback mechanisms that lead to enhanced heme scavenging in response to ChdC ablation.

In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR–Cas and restriction–modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance toward high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium–phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution.

The rise of multidrug-resistant tuberculosis (TB) necessitates alternative therapeutic sources. This study investigated the polyphenolic content and the antioxidant, antimycobacterial, and anti-virulence activities of selected medicinal plants traditionally used to treat TB and related symptoms. Total phenolics, tannins, and flavonoids were quantified using colorimetric assays. Antioxidant capacity was assessed via DPPH and ferric-reducing power assays. Antimycobacterial activity against Mycobacterium smegmatis was evaluated using broth microdilution, growth kinetics, cell constituent leakage, and respiratory chain dehydrogenase inhibition assays. Anti-virulence effects were examined using crystal violet biofilm and swarming motility assays. Tarchonanthus camphoratus showed the highest polyphenolic levels and, together with Combretum hereroense, strong antioxidant activity. Extracts of Senecio macroglossus, Nerium oleander, and Tetradenia riparia displayed potent antimycobacterial activity (MIC = 0.16 mg/mL), characterized by delayed exponential growth, membrane damage, and metabolic inhibition. Tabernaemontana elegans exhibited the weakest activity (MIC > 2.5 mg/mL). Most extracts also significantly impaired motility (12-100%) and early-stage biofilm formation. Polyphenolic-rich plant extracts demonstrated promising antimycobacterial and anti-virulence properties against M. smegmatis, highlighting their potential as leads for developing novel anti-TB agents.