Fungal extracellular vesicles (EVs) have emerged as critical mediators of fungal physiology, virulence, and host–pathogen interactions. Since their first description in Cryptococcus neoformans, EVs have been identified in several fungal species and shown to transport a broad repertoire of bioactive cargo. Increasing evidence indicates that fungal EVs participate in multiple biological processes, including cell wall remodeling, stress adaptation, biofilm formation, antifungal resistance, and modulation of host immune responses. Recent advances in cryo-electron microscopy, multi-omics approaches, and functional genetics have substantially expanded our understanding of the molecular mechanisms governing EV biogenesis, cargo selection, and extracellular trafficking. These studies have further revealed that EV cargo loading is a highly regulated process linked to intracellular proteostasis, glycosylation, lipid homeostasis, and environmental adaptation. In parallel, the intrinsic immunogenicity and structural stability of fungal EVs have highlighted their translational potential as diagnostic biomarkers, vaccine platforms, therapeutic targets, and nanoscale delivery systems. Given the increasing global burden of invasive fungal infections, this review focuses on EVs derived from clinically relevant human fungal pathogens. We summarize recent advances in EV biogenesis, cargo regulation, their roles in pathogenesis, highlight emerging translational applications, and discuss key unresolved questions and future research directions in the field.

Small regulatory RNAs (sRNAs) are short noncoding RNAs that can fine-control the expression of target genes in trans at the post-transcriptional level in prokaryotes. Since there is a big challenge in constructing gene-knockout libraries, synthetic sRNAs have attracted considerable interest in synthetic biology and metabolic engineering, as they enable targeted gene knockdown without requiring chromosomal modifications. However, the development of high-efficiency synthetic sRNAs remains a demanding task that requires careful consideration of multiple design factors. Here, we provide a detailed protocol for the design and construction of synthetic sRNAs, detailing key design principles and critical optimization factors, including scaffold selection, target mRNA binding affinity, target mRNA secondary structure, and Hfq expression levels. This strategy can be broadly applied across E. coli and other bacterial hosts to modulate gene expression, thereby supporting versatile applications in synthetic biology and metabolic engineering.

This study compared the rhizosphere microbial communities of two closely related Monotropastrum species (M. humile, Mh; and M. humile var. glaberrima, Mhg) and identified key soil factors associated with their assembly. Bacterial and fungal communities were profiled by Illumina high-throughput sequencing, and soil physicochemical properties were assessed across multiple sites in Zhejiang Province, China. The bacterial communities of both species were dominated by Proteobacteria and Acidobacteriota at the phylum level, while the dominant fungal groups belonged to Ascomycota and Basidiomycota. The two plants shared several dominant bacterial genera, including Serratia, Burkholderia-Caballeronia-Paraburkholderia, and Bradyrhizobium, as well as common dominant fungal genera such as Saitozyma and Podila. Despite these similarities, species-specific enrichment patterns were observed. The rhizosphere of Mhg contained higher abundances of Acidothermus and Lactarius, whereas Mh preferentially enriched Cedecea, Klebsiella, and Russula. Bacterial communities were shaped by pH, soil organic matter (SOM), available potassium (AK), and available phosphorus (AP), whereas fungal communities were primarily influenced by pH, alkali-hydrolyzable nitrogen (AN), and SOM (p < 0.05). These results suggest that both host identity and soil properties contribute to rhizosphere microbial assembly, with clear host-associated differentiation in microbial communities. Notably, the identified host-associated microbial taxa, particularly key mycorrhizal fungi, may serve as potential microbial inoculants, providing new opportunities for the conservation and cultivation of mycoheterotrophic plants.

Dual specificity phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases that regulate diverse cellular processes through dephosphorylation of phosphorylated substrates. DUSPs are commonly found in eukaryotes, bacteria, archaea, and viruses. However, structural and biochemical characterization of bacterial DUSP remains limited, as only one bacterial DUSP has been identified thus far. In this study, we investigated a novel putative bacterial DUSP from Candidatus Chlorohelix allophototropha, referred to as CCaDUSP. The crystal structure of CCaDUSP showed the presence of a well-conserved catalytic motif with a characteristic phosphate-binding loop. Biochemical analyses further confirmed that CCaDUSP exhibits phosphatase activity and contains dual general acid/base residues, both of which contribute to its enzymatic activity. These findings not only represent the first characterization of a novel bacterial DUSP with dual general acid/base residues but also provide a foundation for understanding the diversity of DUSP proteins in bacteria.

The global rise of multidrug-resistant bacteria poses a critical threat to public health, and bacteriophage-derived endolysins have emerged as promising alternatives to conventional antibiotics. The engineered endolysin LNT113, derived from the Escherichia coli phage PBEC131 endolysin EC340, exhibits potent lytic activity against Gram-negative bacteria. This study investigated the transcriptomic responses of E. coli to sublethal LNT113 stress and identified genetic determinants required for bacterial adaptation to endolysin-induced stress. Transcriptomic analysis identified 552 differentially expressed genes (DEGs) following sublethal LNT113 exposure. Thirteen DEGs associated with stress response and envelope maintenance were individually deleted to generate thirteen mutant strains and to functionally evaluate their roles in bacterial stress tolerance. Among these, the ΔfabB and Δ(prmB–yfcL) mutants exhibited significantly reduced survival under sublethal LNT113 exposure, indicating increased susceptibility to the endolysin. Regarding the prmB–yfcL operon, individual genes were deleted to determine the gene critical for bacterial tolerance. Deletion of aroC and mepA rendered E. coli more susceptible to LNT113. Furthermore, 1-N-phenylnaphthylamine uptake assays demonstrated increased membrane permeability in the ΔfabB, ΔaroC, and ΔmepA mutants. Complementation with pWSK129::fabB, pWSK129::aroC, and pWSK129::mepA restored membrane integrity in the respective mutant strains. These findings suggest that fabB-mediated unsaturated fatty acid biosynthesis and mepA-dependent peptidoglycan remodeling are critical for maintaining envelope integrity under endolysin stress, whereas aroC may indirectly support bacterial tolerance to LNT113 via metabolic adaptation. This study provides insights into bacterial responses to LNT113 and offers a foundation for optimizing endolysin-based therapeutic strategies.

Reliable quantification of neutralizing antibodies (nAb) against human adenovirus type 55 (HAdV-55) is critical for the evaluation of emerging vaccine candidates. While the plaque reduction neutralization test (PRNT) is currently the reference standard, its utility for large-scale studies is limited by low throughput, labor-intensive plaque counting, and prolonged assay times. In this study, we established and analytically validated a microneutralization assay based on cytopathic effect (MN-CPE) as a scalable alternative for HAdV-55-specific nAb quantification. Comparative performance analysis revealed that both assays maintain high dilution linearity, with coefficients of determination (R²) of 0.988 for MN-CPE and 0.9926 for PRNT. Relative accuracy assessments using high-, middle-, and low-titer reference sera demonstrated acceptable responses across the dynamic range. Notably, the MN-CPE assay allowed for the definition of a negative-control acceptance range, providing a distinct statistical advantage over PRNT, where negative-control values were consistently zero. Furthermore, both assays successfully detected HAdV-55-specific nAbs in immunized cynomolgus macaques, with no cross-reactivity observed against other HAdV types such as HAdV-4. These findings indicate that the MN-CPE assay is analytically comparable to PRNT and serves as a practical, relatively high-capacity alternative for HAdV-55 neutralization testing in clinical and preclinical vaccine research.