The efficient removal of pharmaceuticals and personal care products (PPCPs) in wastewater remains a significant challenge due to their limited degradability in conventional treatment processes and interference of humic acid (HA).Biological ion exchange (BIEX) has emerged as a promising alternative technology, integrating the adsorption capacity of resin with biodegradation capability of attached microorganisms. This work investigated the performance of BIEX system constructed with magnetic polyacrylic resin (NDMP) for removing ibuprofen (IBU) and diclofenac (DCF) under HA stress, benchmarked against systems constructed with commercial A860 resin and high-density polyethylene (HDPE). NDMP system exhibited superior steady-state removal of IBU (100%), dissolved organic carbon (DOC, 70%), and DCF (29%), outperforming A860 and HDPE. While initial removal was dominated by adsorption, long-term stability was sustained by biodegradation, with biological contributions exceeding 63% for DOC and 96% for IBU in NDMP system. NDMP supported greater attached biomass (9.5 vs. 3.3 mg/mL carrier on HDPE) and metabolic activity (2.2 vs. 0.6 μg ATP/mL carrier on HDPE). Crucially, NDMP selectively enriched fungal populations (7.2 vs. 1.1 × 108 copies/g microorganisms). Antifungal assays using amphotericin B quantitatively confirmed that fungi accounted for approximately 20% of IBU removal in NDMP system and 13% of IBU removal in HDPE system, consistent with their level of fungal abundance. Under HA stress, bacterial diversity declined, whereas fungal diversity increased. These findings underscore the advantage of NDMP as BIEX carrier and provide explicit evidence for the role of fungi in PPCPs removal, which has been largely overlooked in previous BIEX research.

Reproductive synchronization is suggested to enhance fitness in many insects, yet the mechanisms enabling such coordination remain unknown. In Locusta migratoria, we identify a microbiota-derived volatile, 2,4,6-trimethylpyridine (2,4,6-TMP) that coordinates communal oviposition in gregarious females while simultaneously deterring egg predators. Eggs laid by gregarious females emit 2,4,6-TMP, which attracts conspecifics by activating the olfactory receptors LmOR34 and LmOR36; disruption of either receptor abolishes attraction and alters oviposition behavior. 2,4,6-TMP is synthesized by the egg-associated bacterium Enterobacter roggenkampii through a lysine degradation pathway. Deletion of lysine decarboxylase abolishes 2,4,6-TMP production and associated behavioral responses. Beyond facilitating reproduction, 2,4,6-TMP repels soil-dwelling predators, including ants, beetles, and potworms. In the ant Solenopsis invicta, avoidance is mediated by the olfactory receptor SinOR43a-1; RNA interference (RNAi) knockdown of this receptor abolishes repellence. Semi-field assays confirm the ecological relevance of 2,4,6-TMP. These findings reveal an egg-associated bacterial signaling mechanism that couples reproductive coordination with predator defense in L. migratoria.

Interkingdom competition between bacteria and fungi in nutrient-limited environments drives microbial community dynamics, yet the molecular mechanisms enabling bacterial dominance during bacterial-fungal interaction remain poorly characterized. This study reveals a paradigm-shifting role for bacterial type IV secretion system (T4SS) by demonstrating that a specialized bacterial-killing T4SS (T4SSBK) in Lysobacter enzymogenes serves as a universal cross-kingdom antifungal weapon. Combining genetic, biochemical, and bioinformatical approaches, we show that T4SSBK mediates direct contact-dependent suppression of phylogenetically diverse fungi-including unicellular yeasts and filamentous species-through delivery of a conserved deoxyribonuclease effector, LtdD. Structural characterization reveals that LtdD homologs are evolutionarily conserved across T4SSBK-harboring bacterial lineages, suggesting widespread ecological deployment of this antifungal strategy. These findings overturn the classical dogma of T4SSBK as an antibacterial apparatus and establish it as a multifunctional microbial combat system capable of trans-kingdom interference. By elucidating a DNA-targeting mechanism of fungal growth inhibition, our work not only redefines the ecological role of T4SSBK in microbial warfare but also provides a molecular blueprint for engineering DNA-based antifungal therapies.

Bacterial diseases are a major cause of crop losses worldwide, threatening food security and the sustainability of agricultural production. Traditional management practices heavily rely on antibiotics, resistant strains and copper-based compounds but their continued efficiency is hampered by antimicrobial resistance, ecological issues and rapid evolution of pathogens. Nano-enabled anti-virulence strategies offer a novel approach that specifically targets bacterial virulence instead of the killing of the pathogens alone, which can potentially reduce the pressure for resistance development. This review rigorously investigates mechanistic basis of nano-facilitated interference in bacterial communication and pathogenicity in the plant pathosystems. We evaluated the impact of metallic, metal oxide, silica-based, carbon-based and polymeric nanomaterials on quorum sensing (QS), biofilm formation, extracellular polysaccharide synthesis, motility, secretion pathways, phytotoxin production and host colonization. Particular emphasis is placed on nanoparticle-pathogen interactions, nano-enabled delivery of quorum quenchers, RNA molecules and biological agents, microbiome compatibility, and the integration of these processes within a mechanistic anti-virulence framework. Current evidence indicates that nanomaterial efficacy is strongly influenced by physicochemical properties, target virulence mechanisms, delivery efficiency, and environmental conditions; therefore, no single nanoplatform is universally optimal across plant-pathogen systems. Although smart delivery systems provide opportunities to simultaneously interfere with interconnected virulence pathways, their practical application remains constrained by field stability, biosafety, non-target effects, scalability, and regulatory uncertainty. Overall, advancing nano-enabled anti-virulence technologies from laboratory efficacy toward sustainable crop protection will require mechanism-guided nanomaterial design, microbiome-compatible formulations, rigorous field validation, and comprehensive environmental risk assessment.

Chronic wounds have presented a significant clinical challenge due to persistent infection, uncontrolled inflammation, and impaired tissue regeneration. Existing therapeutic platforms have failed to integrate these critical aspects into a single, cohesive strategy. To address this, a novel multifaceted agent, zinc silicate flower-like microsphere (ZnSi FMPs), was designed with controlled hierarchical architecture to achieve chronic wound repair by mediating bacterial metabolic interference and microenvironment remodeling. The unique nanosheet structure with sustained Zn2+ release endows ZnSi FMPs with favorable pH-independent antibacterial activity. This activity is mechanistically rooted in metabolic interference against Staphylococcus aureus (S. aureus), including disruption of ABC transporters and inhibition of the biosynthesis of essential amino acids (valine, leucine, isoleucine) and ribosomes. Concurrently, ZnSi FMPs can promote microenvironment remodeling by modulating inflammatory responses and angiogenesis, evidenced by suppressed inflammation mediated by the complement and coagulation cascades pathway and robust activation of the Areg/HB-EGF-mediated ErbB signaling pathway. Hence, in both S. aureus-infected and diabetic wound models (mice and minipigs), ZnSi FMPs exhibited superior chronic wound healing capacity compared with commercial silver sulfadiazine® (AgSD) and 45S5 bioglass®. This improved healing was achieved through enhanced antibacterial activity, anti-inflammatory effects, and angiogenesis, accompanied by elevated expression of CD163, CD206, CD31 and α-SMA. By synergizing bacterial metabolic interference with microenvironment regulation, ZnSi FMPs effectively facilitate tissue healing and provide a promising candidate for clinical wound treatment.

Wound infections represent a major clinical concern due to their high incidence and subtle early manifestations, often resulting in prolonged healing and increased risk of complications. While wearable bioelectronics facilitate real-time diagnostics and therapeutic interventions, their widespread clinical translation remains constrained by the complexity, power demands, and high cost of current systems. Here, a wearable platform that integrates colorimetric sensors with a wireless optoelectronic microneedle patch was presented for in situ wound infection status detection and on-demand photodynamic therapy (PDT). This wireless colorimetric optoelectronic microneedle (COMN) patch enables rapid (15 min) and reliable detection of typical wound infection biomarkers with minor interference. By integrating microneedles and wireless light-emitting diodes (LEDs), this system supports concurrent delivery of photosensitizers and light into deeper wound tissues, achieving > 99.9% eradication of drug-resistant bacteria through penetration-enhanced PDT. In a murine full-thickness wound infection model, this system promptly assessed the infection status of the wound and then triggered PDT, resulting in > 3 log reduction in bacterial burden and accelerated healing, with > 80% wound closure observed by day 14. This work demonstrates a flexible and adaptive bioelectronic platform based on colorimetric analysis and optoelectronic microneedle therapy, offering an accessible strategy for precision wound care.

Angelica glauca Edgew., a high-altitude Himalayan medicinal plant, is an important source of bioactive essential oil rich in phthalide derivatives. The present study investigated the influence of environmental conditions and extraction parameters on essential oil yield and chemical composition, with particular emphasis on Z-ligustilide. Essential oils obtained from roots collected across different altitudes revealed marked variations in yield and phytochemical profile, with plants growing at 3500-3800 m producing the highest concentration of Z-ligustilide (75.2%). The optimised essential oil exhibited broad-spectrum antimicrobial activity against both Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Candida species. Molecular docking studies further supported its therapeutic potential by demonstrating strong interactions with microbial target proteins, with binding affinities reaching approximately -6.9 kcal/mol. The predicted mechanisms of antimicrobial action include membrane disruption, interference with ATP production, and inhibition of biofilm formation, indicating a multi-target mode of action. Overall, these findings demonstrate that altitude-driven phytochemical variation combined with optimised extraction significantly enhances the quality and biological efficacy of A. glauca root oil, highlighting its potential as a natural antimicrobial agent for pharmaceutical and nutraceutical applications.

The CRISPR-Cas12 family encompasses diverse RNA-guided nucleases with both DNA- and RNA-targeting subtypes. They can trigger antiviral activities through either direct elimination of invading nucleic acids or activating broad collateral cleavage to induce abortive infection. Here, we report a novel type V CRISPR effector BaCas12a3 that causes growth inhibition through a unique tRNA-cleavage mechanism. Plasmid interference and western blot assays showed that BaCas12a3 induces host growth arrest without DNA damage response, suggestive of the absence of double-strand DNA breaks. Indeed, biochemical characterization of the BaCas12a3-crRNA ribonucleoprotein unraveled that the effector is an RNA-activating nuclease that cleaves the 3' terminal CCA of tRNAs. Cryo-EM structures of BaCas12a3 reveal a conserved bilobed architecture featuring a unique tRNA-loading domain (tRLD) adjacent to the RuvC catalytic center. Structural and mutagenesis analyses show that the tRLD domain, together with a zinc ribbon domain, form a gated substrate groove. Target RNA binding induces conformational changes that open the groove and expose the RuvC active site, enabling specific tRNA 3' end cleavage while preventing other non-specific degradation. Our findings identify the tRLD domain aside the RuvC active site responsible for the tRNA recognition in BaCas12a3, expanding the functional diversity of CRISPR immunity.

Plant-derived exosome-like nanoparticles (P-DENs) have emerged as promising bio-nanocarriers with antimicrobial potential. In this study, we isolated and characterized exosome-like nanovesicles from Rosa damascena and evaluated their antibacterial efficacy against Salmonella enterica, a significant foodborne pathogen. Rosa damascena-derived exosomes-like nanovesicles (R-DENs) exhibited 8.9 × 10[9] particles/mL in nanoparticle tracking analysis. Growth assays revealed significant inhibition of bacterial proliferation (63.52%). Propidium iodide (PI) staining in flow cytometry demonstrated concentration-dependent bacterial cell death (65.78%), while scanning electron microscopy (SEM) visualized distinct ultrastructural damage to the bacterial envelope, confirming membrane disruption as a primary antibacterial mechanism. Consistent with these effects, R-DENs markedly reduced biofilm formation (69.88%) and suppressed exopolysaccharide (EPS) production (69.10%). Elevated intracellular reactive oxygen species (ROS) levels (up to 65.37%) further indicated oxidative stress as a major antibacterial contributor. Moreover, R-DENs treatment impaired bacterial adhesion (77.88%) and invasion (67.37%) in L929 fibroblasts, correlating with attenuation of pathogenicity. Gene expression analysis confirmed significant downregulation of biofilm- and virulence-associated genes (fimA, csgD, bcsA, rsmA, and rpoS), linking the phenotypic changes to molecular interference with pathogenic pathways. Collectively, these findings demonstrate that R-DENs exert multifaceted antibacterial activity through membrane disruption, biofilm and EPS inhibition, ROS generation, and suppression of virulence traits, underscoring their potential as natural, biocompatible nanotherapeutics against drug-resistant bacterial infections.

Epitranscriptomics has recently gained significant momentum due to technological advances and translational applications; however, studies on bacterial RNA modifications remain limited. Bacterial RNA is notoriously prone to degradation, and methodologies to investigate the epitranscriptome are challenging. Prior research has shown RNA modifications modulate antimicrobial resistance, virulence and pathogenicity. This research employed CRISPR interference to knock down five known Escherichia coli rRNA modification genes (rlmF, rlmJ, rluD, rsmF and rsmG) in three E. coli strains. These isolates were investigated for growth delays, changes in the proteome and the influence on mRNA modifications via native RNA sequencing. CRISPRi adequately silenced the majority of RNA modification genes in E. coli (> 80% reduction). Significant growth delays were associated with rlmF, rluD and rsmF repression. Unique protein pathways corresponding with RNA modification loss were found for rlmJ (TreB, XylF), rluD (CysH, HycB, PutP, TrpB), rsmF (EvgA) and rsmG (OppC). Known rRNA modification sites for rluD (Ψ) and rsmG (m7G) were detected from analysis of nanopore electrical signal; however, only a weak signal was apparent for m6A (rlmF, rlmJ) and m5C (rsmF) modifications. The inhibition of rRNA modifications resulted in mRNA modification changes, including for genes ompC, cspC, dbhA, dbhB and secY. Our work provides an approach for unravelling the epitranscriptome of E. coli to gain insight into its functional role.

Targeted, genome-scale gene perturbation screens using clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call CRISPR transposition and overexpression (CRISPRtOE). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CASTs) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.IMPORTANCESystematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.

Pathological microenvironment of diabetic wounds greatly increases bacterial infection, hyperinflammation, vasculopathy and neuropathy, impeding wound healing. To address these issues, a Cu and Mg codoped ZIF-8 (CuMg@ZIF) nanoplatform with pH-responsive degradation and multi-enzyme activities (peroxidase (POD)-like activity, superoxide dismutase (SOD)- and catalase (CAT)- activity) was constructed. Density functional theory (DFT) calculations reveal that Cu and Mg doping induce stretching of metal-N bonds and electron rearrangement in ZIF framework, which enhance degradation and subsequent ion releasing in acidic microenvironment, and increase Cu[+]/Cu[2+] ratio to improve multi-enzyme activities. CuMg@ZIF exhibits excellent antibacterial efficiencies (100% against S. aureus and 99.98% against E. coli) at a low concentration (50 μg/ml) via rapid metal ion releasing and •OH generation. After sterilization, the SOD- and CAT-like activities, along with the moderate release of ions from the CuMg@ZIF scavenge ROS and facilitate macrophage recovery and M2 phenotype polarization to inhibit inflammatory reaction and secrete anti-inflammatory cytokines, which further enhance the biofunctions of endothelial cells, neural cells, and fibroblasts. This study provides a promising pH sensitive therapy to eliminate infection, promote immunoregulation, neuroangiogenesis and ECM remodeling, thereby accelerating the diabetic wound repair process.

Immunosenescence, the age-related decline in immune function, varies widely among individuals. Although both genetic and environmental factors contribute to variation in immunosenescence, the extent to which age-specific genetic effects shape age-related immune decline remains poorly understood. Here, we tested whether age-related variation in bacterial clearance is shaped by age-dependent genetic effects by measuring clearance of Escherichia coli infection in 175 genotypes at 1 week of age and 183 genotypes at 5 weeks of age. Clearance ability declined with age, but the magnitude of decline differed markedly among genotypes. Age-specific genetic effects explained substantially more phenotypic variation than age-independent genetic differences, and the genetic correlation in clearance ability across ages was modest, indicating that genetic effects on immune performance were only partially shared across ages. Consistent with these findings, genome-wide analyses identified largely distinct sets of candidate genes associated with clearance in young and old flies, including genes exhibiting genotype-by-age effects. Network and gene ontology analyses revealed enrichment for plasma membrane-associated genes and pathways involved in cell adhesion and cell migration. Functional follow-up of 14 candidate genes using hemocyte-specific RNA interference produced limited effects overall but confirmed that nebulosa contributes to bacterial clearance. Together, our results demonstrate that immunosenescence is shaped by age-dependent genetic effects and identify conserved pathways and candidate genes for future mechanistic studies of age-related immune decline.

Escherichia coli exhibits remarkable genetic diversity that enables it to adapt to the intestinal environment. Here we establish an in vivo CRISPR interference platform that leverages bacterial gene fitness as a high-resolution functional reporter of E. coli adaptations within mice harbouring a defined minimal microbial community (OligoMM12). The screen revealed that diet profoundly shapes the metabolic landscape of E. coli and the essential gene profile identified cross-feeding interactions. Comparison between a laboratory strain (MG1655), a uropathogenic strain (CFT073) and an adherent-invasive E. coli (AIEC LF82) identified distinct genetic requirements for intestinal colonization, highlighting divergent motility, stress response and respiration strategies. In a host inflammatory environment, we found that AIEC LF82 preferably colonized the small intestine with a mobile genetic element, Gally prophage, playing an important role in modulating fitness. These findings provide a high-resolution genetic atlas of E. coli's functional adaptation and demonstrate the utility of functional genomics to probe the gut environment itself.