Deep eutectic solvents (DESs) were evaluated for their role in enhancing green tea extract (GTE)-based biosynthesis of silver nanoparticles (Ag NPs). Four types of DESs with varying water contents (10-50 %) and hydrogen bond acceptor (HBA) mole fractions (0.17-0.83) were evaluated. While HBA composition had little effect on catechin and phenolic compound extraction, higher water content usually improved yields by reducing DES viscosity. Compared with water-based GTE, all DES-based GTEs produced Ag NPs with higher surface plasmon resonance intensities, smaller particle sizes, and narrower distributions. In GTEs derived from non-urea-based DESs, these enhancements were consistent regardless of the water content and HBA mole fraction, suggesting inherent beneficial effects of the DESs. However, urea-based DESs that contained ammonia as a byproduct during DES preparation, exhibited different characteristics: although the geometric sizes of the Ag NPs remained unchanged, increasing water content led to larger hydrodynamic sizes, indicating intensified surface modification of the Ag NPs. The surface modification correlated positively with antioxidant activity, negatively with catalytic activity, and exhibited no significant association with antibacterial activity. Overall, DESs generally enhance the GTE-based biosynthesis of Ag NPs without the need for precise condition optimizations. However, ammonia-generating DESs may require tailored conditions to balance synthesis and activities.

Cyclic di-AMP (c-di-AMP) is an essential bacterial second messenger that coordinates multiple cellular processes and is closely linked to central physiology in many bacteria. In Staphylococcus aureus, both depletion and excessive accumulation of c-di-AMP impair bacterial fitness, indicating that this signaling pathway must be tightly regulated. Recent studies have identified c-di-AMP receptor proteins in S. aureus, revealing how c-di-AMP maintains osmotic balance through ion transport and influences cell envelope function and stress adaptation. These regulatory effects also influence bacterial growth and cell size, and are linked to biofilm formation and β-lactam resistance. Elevated c-di-AMP levels can alter peptidoglycan architecture and reduce autolysis, thereby increasing β-lactam resistance, whereas reduced c-di-AMP levels can increase β-lactam susceptibility. This review summarizes current understanding of c-di-AMP synthesis, degradation, and receptor-mediated signaling in S. aureus. We also discuss emerging approaches to perturbing intracellular c-di-AMP balance as potential antimicrobial strategies.

Type I interferons (IFNs) are central regulators of innate immunity, coordinating antiviral defense and tumor immune surveillance. However, therapeutic strategies that selectively engage IFN signaling while maintaining controlled outputs remain limited. Here, we identify a synthetic small molecule that induces robust type I IFN responses. Mechanistic analyses demonstrate that the compound directly engages TANK-binding kinase 1 (TBK1), leading to IRF3 phosphorylation and selective activation of IFN-stimulated gene programs. In contrast to nucleotide-based innate immune agonists that signal through upstream pattern-recognition receptors, this molecule activates TBK1 through direct chemical engagement. TBK1 activation confers host-directed antiviral activity against coronaviruses, including SARS-CoV-2, through induction of interferon-stimulated genes. The compound also modulates the tumor immune microenvironment toward an immunologically active state, characterized by enhanced antigen presentation and increased infiltration of cytotoxic T cells, resulting in augmented antitumor immune responses in vivo. These immunomodulatory effects reflect coordinated activation of innate and adaptive immune pathways downstream of IFN signaling. Chemical activation of the TBK1-IRF3-IFN axis underscores the druggability of this signaling pathway for selective innate immune modulation in viral infection and cancer.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major global health threat because of its ability to adapt. In North America, the USA300 lineage ST8 has become the predominant MRSA clone, whereas the ST72 lineage has emerged as an important MRSA in East Asia. Here, we compare USA300 and SAWL001 from the ST72 at the phenotypic, genomic, and transcriptomic levels. Phenotypic assays assessed antibiotic susceptibility, intracellular invasion, oxidative stress survival, biofilm formation under β-lactam exposure, blood-induced cell clumping, persister formation, and virulence in a mouse sepsis model. For genomic analyses, we compared the SAWL001 genome against USA300 and other major S. aureus strains. SAWL001 showed modestly higher resistance to rifampicin, gentamicin, and linezolid compared with USA300. We also found that SAWL001 mecA is inducible only under oxacillin, whereas USA300 mecA is constitutively expressed. Consistent with these differences, SAWL001 invaded human epithelial cells far less efficiently and survived H2O2 exposure at a significantly lower rate than USA300. Furthermore, our genome analysis revealed that SAWL001 has features different from USA300, such as the beta-lactamase gene locus. Finally, our transcriptomic profiling shows that USA300 maintains virulence features such as PVL, while SAWL001 shows adaptation toward greater horizontal gene transfer and antibiotic resistance. Together, our findings highlight that MRSA lineages can branch toward different evolutionary trajectories, such as becoming more antibiotic resistant or more invasive, underscoring the need for lineage-specific analysis to identify competence determinants and to tailor treatment strategies to each clone's strengths and weaknesses.IMPORTANCEMethicillin-resistant Staphylococcus aureus remains a leading cause of antibiotic-resistant infections worldwide, and its lineages can differ widely in antibiotic resistance and virulence. In this study, we compared the North American USA300 lineage (ST8) with an emerging East Asian ST72 strain, SAWL001. SAWL001 showed higher resistance to several antibiotics than USA300, although the overall resistance levels were moderate. Also, SAWL001 exhibits an inducible mecA-mediated methicillin resistance, whereas USA300 expresses mecA constitutively. Conversely, USA300 invades host epithelial cells more effectively and survives oxidative stress better than SAWL001. Genome and transcriptome analyses show that USA300 retains classical virulence factors, while SAWL001 is primed for horizontal gene acquisition. Our findings underscore distinct evolutionary strategies: USA300 appears to favor aggressive virulence, whereas SAWL001 shows greater metabolic and genomic flexibility, suggesting the need for lineage-specific control strategies.

Elevated oxygen levels in the intestine during antibiotic exposure and disease perturb gut communities, yet the metabolites underlying bacterial adaptation remain unclear. Here, we show that oxygen stress is associated with increased production of a family of antibacterial imidazolium metabolites in the facultative anaerobe Enterococcus faecalis KCTC 5191T. Comparative LC-MS profiling under aerobic versus anaerobic culture revealed increased accumulation of metabolites, including nine previously undescribed imidazoliums, enterozolium A-I. Uniform 13C/15N tracer feeding (l-phenylalanine and l-tyrosine) established their origins as biogenic amines, and biomimetic syntheses corroborated the proposed structures and nonenzymatic steps. A tdc locus encoding tyrosine decarboxylase (tyrDC) was required for production of the amine precursors: a Δtdc mutant of E. faecalis v583 showed markedly reduced metabolite levels and tyrDC transcripts increased ∼4-fold under aerobic conditions. Several metabolites were active against methicillin-resistant Staphylococcus aureus (MRSA); 10 and 17 displayed minimum inhibitory concentrations (MICs) of 5.2 and 3.1 μg/mL, respectively, and 17 showed efficacy in a Caenorhabditis elegans model of MRSA infection. Mechanistic assays indicate dissipation of the membrane proton motive force (PMF) as the antibacterial mode of action. These findings uncover an oxygen-responsive metabolic shift that yields PMF-targeting imidazolium antibiotics and highlight the imidazolium scaffold as a promising lead for antibacterial discovery.

Antibiotic resistance in bacteria can be shaped by environmental factors, including heavy metal contamination. Cadmium (Cd) can enhance bacterial tolerance against other toxic agents, including antibiotics. In this study, we investigated how Cd exposure influences antibiotic resistance across environmental and host-associated contexts. We demonstrate that chronic Cd contamination in soil (5.8 mg Cd/kg) reshapes bacterial communities, enriching Proteobacteria (32.9-37.1 %) that show increased resistance to both Cd (16 mg/L) and four major antibiotics targeting cell wall synthesis (fosfomycin, D-cycloserine), DNA synthesis (ciprofloxacin), and protein synthesis (gentamicin). Extending these findings to host-associated contexts, we found that over 70 % of the Cd-resistant lactic acid bacteria (82 Cd-resistant strains out of 145 isolates) exhibited resistance to at least one antibiotic. To reinforce these findings, we screened Cd-resistant isolates (31.2 μg/mL Cd) from human skin commensals and identified Dermacoccus nishinomiyaensis. D. nishinomiyaensis showed strong resistance to multiple antibiotics, with enhanced thiol-based detoxification that mitigates Cd-induced oxidative damage and contributes to antibiotic resistance. Finally, using Staphylococcus aureus as a model, we demonstrate that Cd exposure primes bacteria for antibiotic tolerance through the bacillithiol pathway via bshC. Bacillithiol reduces oxidative stress and drives a 10-100-fold increase in persister cell formation and antibiotic tolerance. Collectively, our results reveal mechanisms by which heavy metal exposure drives antibiotic resistance, underscoring the importance of controlling environmental Cd contamination to limit the spread of antibiotic resistant bacteria.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.

Elevated oxygen levels in the intestine during antibiotic exposure and disease perturb gut communities, yet the metabolites underlying bacterial adaptation remain unclear. Here, we show that oxygen stress is associated with increased production of a family of antibacterial imidazolium metabolites in the facultative anaerobe KCTC 5191T. Comparative LC-MS profiling under aerobic versus anaerobic culture revealed increased accumulation of metabolites, including nine previously undescribed imidazoliums, enterozolium A-I. Uniform 13C/15N tracer feeding (l-phenylalanine and l-tyrosine) established their origins as biogenic amines, and biomimetic syntheses corroborated the proposed structures and nonenzymatic steps. A tdc locus encoding tyrosine decarboxylase (tyrDC) was required for production of the amine precursors: a Delta tdc mutant of v583 showed markedly reduced metabolite levels and tyrDC transcripts increased similar to 4-fold under aerobic conditions. Several metabolites were active against methicillin-resistant (MRSA); 10 and 17 displayed minimum inhibitory concentrations (MICs) of 5.2 and 3.1 mu g/mL, respectively, and 17 showed efficacy in a model of MRSA infection. Mechanistic assays indicate dissipation of the membrane proton motive force (PMF) as the antibacterial mode of action. These findings uncover an oxygen-responsive metabolic shift that yields PMF-targeting imidazolium antibiotics and highlight the imidazolium scaffold as a promising lead for antibacterial discovery.

Antibiotic-resistant bacterial infections continue to increase globally, creating an urgent need for new antibacterials with novel mechanisms of action. Early stages of antibiotic discovery are often limited by the difficulty of identifying compounds that act through previously unrecognized pathways and by challenges in determining their mechanisms. Machine learning (ML) integrated with high-throughput profiling now provides systematic approaches to overcome these barriers. Beyond initial hit discovery, multilayer profiling using morphological phenotyping, transposon sequencing, transcriptomics, proteomics, and metabolomics captures cellular responses that reflect the mechanisms of action. Because profiling data sets are typically high-dimensional and contain defined features and variables, ML can extract complex patterns associated with pathway-level responses and predict mechanisms for unknown compounds. In this review, we summarize current progress in high-throughput profiling and describe how ML applied to each data set can accelerate the identification of antibacterials with new mechanisms. These approaches accelerate the transition from large-scale compound screening to mechanistic validation and enable effective prioritization of lead compounds in early-stage antibiotic discovery.

The efficacy of cancer immunotherapy is often limited by the immunosuppressive tumor microenvironment (TME) and insufficient immune activation in tumor-draining lymph nodes (TDLN). Since the TME and TDLN form a dynamic axis crucial for tumor metastasis and resistance to immune checkpoint blockade, strategies that effectively modulate both sites are critical. Here, we present a dissolving microneedle (MN) system that generates nanomicelles (NMCs) for localized delivery of a newly identified dual-functional macrocyclic trichothecene, Roridin E (R.E). R.E induces cancer cell-autonomous secretion of IFN-β and immunogenic cancer cell death (ICD). Direct delivery of R.E to the TDLN via the MN platform reshapes the local immune landscape to suppress cancer while minimizing off-target toxicity. In a B16F10 melanoma model, MN-guided R.E. delivery significantly improved tumor control, reduced lung metastases, and extended overall survival. This approach provides a minimally invasive and effective strategy for integrating natural-product-based therapies with advanced drug delivery systems to target the TME-TDLN axis, thereby improving outcomes in metastatic cancer.

The ability of Staphylococcus aureus to adapt and thrive in diverse host niches adds to the challenge in combating this ubiquitous pathogen. While extensive research has been pursued on the adaptive mechanisms of methicillin-resistant S. aureus (MRSA) in various infection models, a comprehensive analysis of its fitness across different host niches is lacking. In this study, we employ transposon sequencing to analyze the adaptive strategies of MRSA in various infection niches. Our analysis encompasses a cell model that mimics an intracellular niche, human blood, which represents a major extracellular environment as well as a major intermediary route encountered by bacteria during systemic infection, and a male murine sepsis model that recapitulates intra-organ environments. Our findings reveal substantial differences in the genetic determinants essential for bacterial survival in intracellular and blood environments. Moreover, we show that each organ imposes unique growth constraints, thus fostering heterogeneity within the mutant population that can enter and survive in each organ of the mouse. By comparing genes important for survival across all examined host environments, we identify 27 core genes that represent potential therapeutic targets for treating S. aureus infections. Additionally, our findings aid in understanding how bacteria adapt to diverse host environments.

Infections associated with bacterial persisters are challenging to cure because they can evade antibiotics and regrow, often resulting in relapse. Current antibiotics are not optimized to target persisters, highlighting the urgent need for new therapeutics. Here, we report that bakuchiol, a plant-derived natural product, exhibits anti-persister and adjuvant properties. Bakuchiol eradicates persisters formed by the gram-positive bacterium Staphylococcus aureus at 8 μg/mL and, in combination with 1 μg/mL colistin, completely eliminates persisters formed by the gram-negative bacterium Acinetobacter baumannii. Mechanistic analyses revealed that bakuchiol selectively disrupted bacterial membrane phospholipids while sparing mammalian membranes and exhibited low cytotoxicity. In Acinetobacter baumannii persisters, bakuchiol likely damages phospholipid patches in the outer membrane, causing nominal lethality but facilitating membrane permeabilization. This activity synergizes with colistin, which targets the lipooligosaccharide layer, resulting in the mutual reinforcement of their bactericidal effects. These findings highlight the potential of dual glycolipid–phospholipid targeting as a strategy to combat gram-negative persisters and highlight natural products as valuable sources for anti-persister therapeutics with membrane selectivity.

Multidrug-resistant (MDR) Acinetobacter baumannii is a major clinical threat with limited treatment options, as current therapies rely on polymyxins such as colistin. Targeting the lipopolysaccharide (LPS) biosynthetic pathway offers a new target for new antibiotic discovery, yet most efforts have focused on enzyme-based assays that do not reflect cell level physiology. Here, we developed a cell-based screening strategy that links colistin resistance to inhibition of lipooligosaccharide (LOS) biogenesis in A. baumannii. Using colistin, we established a phenotypic platform in which compounds that inhibit LOS synthesis rescue bacterial growth from colistin's mode of action. This approach allows direct identification of inhibitors acting on essential LPS enzymes, including LpxC. Screening a library of about 7000 small molecules discovered non-hydroxamate compounds that restored growth under colistin stress. Hit compounds were validated through LpxC enzyme assays and protein-compound binding assays. Furthermore, our molecular docking study suggests that the hit compounds bind to the LpxC catalytic pocket similarly to CHIR-090. Together, our work introduces a novel phenotypic screening strategy for discovering LPS targeted inhibitors and provides new chemical scaffolds for developing antibiotics against A. baumannii and other Gram-negative pathogens.

Staphylococcus aureus, a leading cause of serious infections, produces various factors important for intrinsic resistance to antibiotics. Understanding what intrinsic resistance factors do may enable strategies to potentiate existing antibiotics. The membrane protein AuxB is an intrinsic resistance factor that helps S. aureus withstand diverse compounds that target the cell envelope, but its cellular functions are unknown. We show here that AuxB is a four-pass transmembrane protein with an intracellular C-terminus that interacts directly with the cytosolic cell cycle regulator GpsB. We also show AuxB's membrane domain forms a homodimer that exists in equilibrium with a heterodimer of AuxB and PknB, a eukaryotic-like serine/threonine kinase that has been implicated in cell envelope processes. Shifting the equilibrium to favor AuxB-bound PknB impairs growth on tunicamycin, a condition where PknB is essential, which suggests that AuxB binding antagonizes a PknB function. To link PknB's domains to compound susceptibility phenotypes, we assessed the fitness of PknB variants under several conditions. We find that PknB's extracellular and kinase domains are not functionally interdependent but instead play distinct roles in withstanding cell envelope stress. AuxB evidently antagonizes functions of PknB's extracellular PASTA (penicillin-binding protein and Ser/Thr kinase-associated) domain, the presence of which is beneficial under tunicamycin treatment regardless of whether the kinase domain is active. On compounds where the PASTA domain is deleterious, increasing the amount of AuxB-bound PknB can also ameliorate sensitivity. Collectively, our data suggest that AuxB, as a homodimer and through its interactions with GpsB and PknB, modulates cell envelope processes during cell growth and division.IMPORTANCEStaphylococcus aureus is a leading cause of fatal infections worldwide. It encodes diverse genes that contribute to the organism's high intrinsic resistance to antibiotics. Understanding the biological roles of these genes and how their features contribute to intrinsic resistance may enable better antibiotic therapies. Here, we investigate AuxB, an intrinsic resistance factor to compounds that target the cell envelope. We find that AuxB interacts directly with the cell cycle regulator GpsB and the eukaryotic-like serine/threonine kinase PknB, another intrinsic resistance factor that is proposed to sense and respond to cell wall status. Based on our findings, we propose that AuxB impacts cell physiology through three mechanisms: (i) by antagonizing PknB's penicillin-binding protein and Ser/Thr kinase-associated domain function; (ii) by coordinating the phosphorylation of cell division proteins; and (iii) by forming a homodimer that interacts with GpsB hexamers to enable the formation of extended GpsB interaction networks.

The interplay among antibiotics, gut microbiota, and disease pathogenesis remains poorly understood, particularly in the context of rare gut bacteria. This study identifies a novel correlation between erythromycin-induced stress and the production of antiangiogenic metabolites in Aneurinibacillus aneurinilyticus, a human gut bacterium. We report the isolation and structural characterization of aneuristatin (1), a metabolite featuring a unique pyrrolo[1,2-a]pyrazine scaffold, along with seven structurally related metabolites (2-8) from A. aneurinilyticus ATCC 12856T. These metabolites were upregulated via the erythromycin-induced activation of the arnA biosynthetic gene. Aneuristatin (1) enhanced prolyl hydroxylase activity, promoting hypoxia-inducible factor-1α (HIF-1α) degradation and reducing downstream targets, including VEGF and EPO. It also exhibited antioxidant effects by reducing ROS levels under hypoxia. Additionally, it inhibited angiogenesis in HUVECs and zebrafish and effectively reduced inflammation, fibrosis, and angiogenesis in a mouse corneal injury model. Our study establishes a molecular basis for the potential of erythromycin-induced aneuristatin (1) to prevent or treat angiogenesis-related diseases such as cancer.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major infectious disease with high mortality globally. Polyhexamethylene guanidine (PHMG), a cationic polymer and major ingredient of humidifier disinfectants, was implicated in an outbreak of severe pulmonary diseases including TB in the Republic of Korea. During the period when humidifier disinfectants were widely used, the incidence of TB exhibited a rising trend. In this study, we aimed to investigate whether PHMG aggravates TB pathogenesis and elucidate the underlying mechanisms by which PHMG exposure modulates TB progression. In a murine model of Mtb infection, PHMG exposure accelerated TB progression, characterized by increased Mtb burden, alveolar macrophage (AM) depletion, excessive neutrophil accumulation-mediated severe pulmonary inflammation, and impaired Th1 immunity. Transcriptomic profiling of PHMG-exposed Mtb-infected AMs revealed induction of type I interferon (IFN) signatures, inflammatory cytokines including Il1a, Tnf, and Il6, and chemokines for neutrophil recruitment such as Cxcl2 and Cxcl3, indicating a pathway associated with aggravated TB outcomes. Consistently, blockade of type I IFN receptor signaling or depletion of neutrophils by relevant antibodies significantly reduced inflammation and Mtb burden in the lungs. Our study demonstrated that PHMG exacerbated TB via the elevated type I IFN signaling and neutrophilic inflammation and uncovered how environmental toxicants such as PHMG would influence host defense system and act as risk factors for TB progression.

Preterm infants are frequently administered antibiotics to prevent infections, yet their impact on the developing gut microbiota and metabolome remains complex and clinically significant. To systematically assess these effects, we analyzed longitudinal stool samples from 54 extremely- and very-low-birthweight infants by integrating clinical data, 16S rRNA-based microbiome profiling, targeted metabolomics, and community-scale metabolic modeling. Antibiotic exposure disrupted microbial diversity, depleted beneficial taxa, and altered metabolites such as short-chain fatty acids (SCFAs) and bile acids. Class-specific antibiotic effects were observed, with cephalosporins promoting Staphylococcus dominance and potentially reducing bile acid diversity. Necrotizing enterocolitis (NEC) samples showed SCFAs depletion and enrichment of antibiotic-resistant genera. In silico models further identified microbial contributors to SCFAs production and recapitulated metabolite trends. These findings demonstrate how antibiotic regimens can perturb the neonatal gut ecosystem and highlight the need for precision antibiotic stewardship to preserve microbiome-derived metabolic functions and reduce disease risk in preterm infants.

Bacterial persisters are dormant phenotypic variants that are tolerant to antibiotics, contributing to treatment failure and the emergence of antimicrobial resistance. Although the formation of persisters has been extensively studied in regards to bacterial infections and treatment, such as antibiotic exposure or intracellular survival within macrophages, the role of environmental stressors in persister formation remains largely unexplored. In this study, we investigate the role of environmental heavy metals, specifically arsenic (As), cadmium (Cd), and mercury (Hg), in promoting persister cell formation in Staphylococcus aureus and Escherichia coli. Log-phase cultures were exposed to heavy metals (5 mM As, 1.25 mM Cd, 4 µM Hg for S. aureus; 12.5 mM As, 2 mM Cd, and 15 µM Hg for E. coli) for 0.5 h to induce persister cells. We observed that exposure to these metals induced persister cell formation, confirmed by intracellular ATP levels through microscopy and luciferase assays, as well as by reactive oxygen species (ROS) levels using carboxy-H2DCFDA. Short-term heavy metal exposure strongly depleted intracellular ATP while generating ROS. Moreover, we observed enhanced expression of genes involved in the SOS response, including recA, umuC, dinB, rexA, rexB, sulA, rpoS, and soxR, as measured by qPCR. This response was likely induced by elevated ROS levels following heavy metal exposure. Furthermore, we demonstrate that heavy metal-induced bacterial persisters exhibited a substantially increased emergence of antibiotic resistance, as shown by ciprofloxacin resistance developing in the presence of heavy metals. Therefore, our results clearly demonstrate that heavy metals can induce persister cells by depleting cellular ATP and generating ROS, and these bacterial responses to heavy metals substantially contribute to antibiotic resistance. These findings highlight the intricate relationship between environmental heavy metals, bacterial persister formation, and antibiotic resistance, emphasizing the need for a “One Health” strategy to address the growing antibiotic resistance crisis.

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

ABSTRACTAntibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment.IMPORTANCEThe significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.