Pyroptosis a lytic form of programmed cell death, is a crucial host defense mechanism against bacterial pathogens. While caspase-mediated pathways are central to pyroptosis, the involvement of apoptotic regulators such as Bak, Bax, and MCL-1 in bacterial infection-induced pyroptosis remains unclear. Here, we investigated how these BCL-2 family proteins modulate pyroptosis induced by Vibrio vulnificus and Salmonella enterica serovar Typhimurium in murine cells. In mouse embryonic fibroblasts (MEFs), both pathogens strongly induced Gbp2 expression and activated caspase‑11, whereas activation of caspase‑1 occurred only in macrophages, indicating engagement of both non-canonical and canonical pyroptosis pathways. Importantly, Bak^-/- and Bax^-/- MEFs exhibited significantly reduced Gbp2 upregulation and caspase-11 activation-an effect most pronounced in Bak-deficient cells leading to attenuated pyroptotic cell death. These data suggest that pro-apoptotic proteins, Bak and Bax, act as positive regulators that amplify the Gbp2-caspase-11 axis. Conversely, overexpression of the anti-apoptotic protein MCL‑1 had no significant impact on Gbp2 expression, caspase activation, membrane integrity, or LDH release, indicating that pyroptosis proceeds independently of MCL‑1 regulation. Collectively, our findings uncover a novel role for Bak and Bax in promoting Gbp2-driven pyroptosis during Gram-negative bacterial infections, while MCL‑1 does not impede this process. This work expands our understanding of the crosstalk between apoptotic and pyroptotic pathways in innate immune responses.

Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.

The pho regulon plays a critical role in maintaining phosphate homeostasis in bacteria, with the PhoU protein functioning as a regulator that bridges the PhoB/PhoR two-component system and the PstSCAB₂ phosphate transporter. While PhoU is known to suppress PhoR autophosphorylation under high phosphate conditions via interaction with its PAS domain, its broader regulatory functions remain elusive. Here, we investigated the role of the PhoU Ala147 residue in Salmonella enterica serovar Typhimurium using a phoU^A147E substitution mutant. Bacterial two-hybrid and immunoprecipitation assays confirmed that Ala147 is essential for PhoU-PhoR PAS domain interaction, and its substitution leads to derepression of pho regulon genes, even in high phosphate conditions. This disruption impaired Salmonella survival inside macrophages and mouse virulence, demonstrating the importance of PhoU-PhoR interaction in Salmonella pathogenesis. However, unlike the phoU deletion mutant, the phoU^A147E mutant does not exhibit growth defects or polyphosphate accumulation, indicating that the PhoU-PhoR interaction is not involved in these phenotypes. Our findings reveal PhoU as a multifaceted regulator, coordinating phosphate uptake and pho regulon expression through distinct molecular interactions, and provide new insights into its role in bacterial physiology and virulence.

Actinobacteria, a phylum of Gram-positive bacteria, are renowned for their remarkable ability to produce antibacterial natural products. The National Institute of Biological Resources (NIBR) of Korea maintains a collection of Korean native actinobacteria. In this study, we explored the phylogenetic and biosynthetic diversity of the NIBR actinobacteria collection to assess its potential as a source of new antibacterial natural products. A 16S rDNA-based phylogenetic analysis revealed a high level of genetic diversity within the collection, with a predominance of Streptomyces, along with rare actinobacterial genera such as Kitasatospora and Micromonospora. Additionally, genetic network analysis of biosynthetic gene clusters (BGCs) from 15 sequenced NIBR actinobacterial strains demonstrated extensive BGC diversity, with many clusters identified as cryptic. Screening of culture extracts for antibacterial activity, followed by dereplication of active extracts, suggested the presence of potentially novel antibacterial natural products. Activity-guided isolation and whole-genome sequencing of the active strain KU57 led to the isolation of one new and three known svetamycin congeners along with their BGC. Overall, our findings highlight the NIBR actinobacteria collection as a valuable source for the discovery of new antibacterial natural products.

Nosocomial infections caused by Pseudomonas aeruginosa (P. aeruginosa) have become increasingly common, particularly among immunocompromised individuals, who experience high mortality rates and prolonged treatment durations due to the limited availability of effective therapies. In this study, we screened for anti-ExoS compounds targeting P. aeruginosa and identified pycnogenol (PYC) as a potent inhibitor of the type III secretion system (T3SS), a major virulence mechanism responsible for the translocation of effectors such as ExoS. Using ELISA, western blotting, and real-time PCR analyses in both P. aeruginosa and infected H292 cells, we found that PYC significantly reduced T3SS activity. Mechanistically, PYC suppressed the transcription of T3SS-related genes by downregulating exsA expression in P. aeruginosa. Furthermore, pretreatment with PYC attenuated the cytotoxic effects and reduced the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18), in P. aeruginosa-infected H292 cells. These effects were associated with the inhibition of NF-κB signaling and inflammasome activation. Taken together, our findings suggest that PYC may serve as a promising therapeutic candidate against P. aeruginosa infections by targeting T3SS-mediated virulence and modulating host inflammatory responses.

Yeast prion [PSI+], an amyloid form of the translation termination factor Sup35p/eRF3, causes translational stop codon readthrough by sequestering functional Sup35p. This unique phenotype may be analyzed via [PSI+]−suppressible nonsense alleles, and has greatly contributed to the advancement in yeast prion research. For comparing canonical reporters, like chromosomal ade1−14 or ade2−1, and plasmid-borne ura3−14, the de novo generation and characteristics of [PSI+] was investigated across common yeast laboratory strains (BY4741, 74D−694, and 779−6A). The results showed significant variability in [PSI+] induction frequency among strains. [PSI+] was successfully induced in BY4741 and frequently in 74D−694 (via Ade⁺ selection), but not in 779−6A. Notably, [PSI+] clones, even from identical genetic backgrounds, displayed vastly different nonsense suppression phenotypes depending on the reporter allele used; resulting in diverse growth patterns and suppression levels. Quantitative analyses revealed that prion seed counts fluctuated significantly based on the detection allele and observed phenotype. Furthermore, Sup35p aggregate visualization revealed distinct structural patterns between BY4741 and 74D−694, indicating strain-specific differences. Transferring [PIN+] prion variants from different strains into a common [psi−][pin−] background yielded similar [PSI+] inducibility and seed numbers, suggesting that the observed phenotypic and quantitative diversities of [PSI+] prions stem primarily from the interplay between the specific reporter detection system and the host strain's genetic background rather than solely from inherent differences in the initial [PIN+] prion or fundamental changes in the [PSI+] protein itself. This study underscores the crucial need to consider both the detection methodology and host genetic context for accurate prion variant characterization.

Human papillomaviruses (HPVs) cause abnormal cellular proliferation, leading to malignant or benign lesions, such as cervical cancer and warts. The genome of HPV16, the most prevalent high-risk oncogenic genotype within the Alphapapillomavirus genus, encodes two oncoproteins. One of these proteins, E7, interacts with multiple host proteins and modulates their functions through distinct pathways. The CR2 domain of HPV16 E7 was recently reported to interact with the μ2 subunit of clathrin-adaptor protein 2 (AP2-μ2), an adaptor complex involved in cargo internalization during clathrin-mediated endocytosis. In this study, to provide molecular insights into their intermolecular interactions, we determined the crystal structures of AP2-μ2 in complex with the HPV16 E7-derived peptides. Subsequent biochemical analyses revealed that this interaction is primarily maintained by the Y-x-x-Φ motif and further supported by acidic cluster residues of HPV16 E7. Finally, sequence alignment of the E7 CR2 domains from various HPV genotypes showed that the AP2-μ2-binding motif is largely conserved in Alpha-, Beta-, and Mupapillomaviruses, but not in Nu- and Gammapapillomaviruses.

CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.

The 2015 Zika virus (ZIKV) outbreak in Brazil and its global spread underscored the urgent need for effective and broadly protective vaccines. While C57BL/6 and BALB/c mice are widely used in preclinical vaccine research, direct comparisons of their ability to elicit ZIKV-specific neutralizing antibodies (nAbs) remain limited. This study aimed to systematically evaluate and compare the immunogenic potential of these two common mouse strains across diverse vaccine platforms, focusing on their capacity to generate functional neutralizing antibody responses. We assessed nAb and IgG responses following four vaccination strategies: (1) DNA vaccine encoding prMEΔTM followed by E protein domain III boost, (2) recombinant EΔTM protein expressed using baculovirus system, (3) formalin-inactivated ZIKV, and (4) live ZIKV. Although both strains generated detectable ZIKV- and E protein-specific IgG, the magnitude and quality of responses varied by vaccine platform and strain. Notably, C57BL/6 mice consistently mounted significantly higher nAb titers than BALB/c mice across all immunization groups, including subunit- and whole-virus-based vaccines. In contrast, BALB/c mice showed lower or undetectable nAb responses, despite comparable or higher total IgG levels in some cases. These findings show that host genetic background is a critical determinant of vaccine-induced neutralization and underscore the importance of selecting appropriate animal models in ZIKV vaccine development. C57BL/6 mice, due to their robust nAb responses, represent a reliable model for evaluating vaccine immunogenicity. Conversely, the limited nAb responses in BALB/c mice position them as a potential low-responder model, offering a stringent system to test the potency and breadth of protective immunity under suboptimal conditions.

Strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2, identified as coagulase-negative, Gram-stain-positive, halotolerant, non-motile coccoid bacteria, were isolated from traditional Korean soybean foods. Strains Mo2-6^T and S9 were both catalase- and oxidase-negative, whereas KG4-3^T and 50Mo3-2 were catalase-positive but oxidase-negative. The optimal growth conditions for Mo2-6^T and S9 were 30°C, 2% NaCl, and pH 7.0, while KG4-3^T and 50Mo3-2 grew best at 35°C, 2% NaCl, and pH 7.0. All strains contained menaquinone-7 as the predominant isoprenoid quinone, with anteiso-C_15:0 and iso-C_15:0 as the major cellular fatty acids (> 10%). Additionally, anteiso-C_13:0 was a major fatty acid in strain KG4-3^T. The DNA G + C contents of strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2 were 33.4%, 33.3%, 32.5%, and 32.7%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, formed distinct lineages within the genus Staphylococcus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, belonged to the same species. Meanwhile, dDDH and ANI values between strains Mo2-6^T and KG4-3^T, as well as comparisons with other Staphylococcus type strains, were below the species delineation thresholds, indicating they represent novel species. Based on phenotypic, chemotaxonomic, and molecular data, we propose strain Mo2-6^T as the type strain of Staphylococcus parequorum sp. nov. (=KACC 23685^T =JCM 37038^T) and strain KG4-3^T as the type strain of Staphylococcus halotolerans sp. nov. (=KACC 23684^T =JCM 37037^T).

Akkermansia muciniphila (AKK, A. muciniphila) fortifies the intestinal barrier, inhibits the colonization of pathogenic bacteria, and protects the host’s health. Nevertheless, the existing literature offers inadequate evidence to ascertain whether A. muciniphila can effectively treat Candida albicans (C. albicans) infections in vitro, and the underlying mechanisms remain ambiguous. This study, animal models were established through gavage with clinical isolates of C. albicans to induce gastrointestinal tract colonization and subsequent translocation infection. The models were subsequently administered A. muciniphila. We examined the analysis of 16S rRNA gene sequencing, metabolomics of colonic contents, and transcriptomics of colonic tissue. The intestinal barrier, inflammatory responses, and immune cell infiltration are analyzed. This study revealed that A. muciniphila markedly mitigated C. albicans translocation infection and modified the intestinal microbial community structure and metabolic attributes in model mice. After administering A. muciniphila to the translocation infection group, there was a notable increase in the prevalence of bacteria that produce short-chain fatty acids, including Eubacterium_F. Moreover, there was a significant increase in the levels of specific pathogens, including Faecalibaculum, Turicibacter, and Turicimonas. The study demonstrated that A. muciniphila treatment can improve the composition of intestinal microbiota and metabolites, augment the tight junctions of colonic tissue and diminish systemic inflammatory response. This presents an innovative therapeutic approach for the potential treatment of intestinal C. albicans infection using A. muciniphila.

Condensin plays a central role in mitotic chromosome organization and segregation by mediating long-range chromatin interactions. However, the extent to which cellular metabolic status influences condensin function remains unclear. To gain insights into the relationship of metal ion homeostasis and the function of condensin, we conducted genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) using Schizosaccharomyces pombe under iron- or zinc-deficient conditions. Under iron- or zinc-deficient conditions, ChIP-seq results revealed a selective reduction in condensin binding at high-affinity target loci, particularly genes regulated by Ace2 and Ams2, while cohesin binding remained largely unaffected. Hi-C analysis showed that iron depletion weakened chromatin interactions at these condensin targets and centromeres, without disrupting global genome architecture. DNA fluorescence in situ hybridization (FISH) confirmed that iron deficiency impaired long-range associations between centromeres and Ace2 target loci at the single-cell level. Notably, iron deficiency led to chromosome segregation defects during mitosis, suggesting that diminished condensin occupancy compromised genome stability. These changes occurred without significant alterations in condensin protein levels or global transcription, indicating a direct effect of metal ion availability on condensin activity. Collectively, our findings revealed a previously unrecognized regulatory axis in which cellular metal ion homeostasis modulated condensin-dependent chromatin organization and mitotic chromosome segregation, offering new insights into the integration of metabolic state with genome maintenance.

CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.

Two Gram-stain-negative, obligately aerobic, non-motile, short rod-shaped bacteria, designated IMCC43871^T and IMCC45206^T, were isolated from coastal surface seawater collected from the Yellow Sea and the South Sea of Korea, respectively. The two strains shared 99.2% 16S rRNA gene sequence similarity with each other and exhibited ≤ 98.4% similarity to three described Rubrivirga species. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between IMCC43871^T and IMCC45206^T were 88.5% and 36.3%, respectively, confirming that they represent two distinct species. Their ANI (≤ 77.7%) and dDDH (≤ 21.4%) values relative to the type strains of the genus Rubrivirga further supported the recognition of strains IMCC43871^T and IMCC45206^T as two novel species within the genus. The complete genomes of IMCC43871^T (4.17 Mb, 71.8% G + C content) and IMCC45206^T (4.17 Mb, 72.8% G + C content) fall within the known genomic range of the genus. Cellular fatty acid, quinone, and polar lipid profiles were consistent with the chemotaxonomic features of the genus Rubrivirga, supporting their affiliation with the genus. Based on phylogenetic, genomic, and phenotypic evidence, strains IMCC43871^T and IMCC45206^T are proposed as two novel species, Rubrivirga aquatilis sp. nov. and Rubrivirga halophila sp. nov., respectively. The type strains are IMCC43871^T (= KCTC 102072^T = NBRC 116463^T) and IMCC45206^T (= KCTC 92925^T = NBRC 116172^T = CCTCC AB 2023136^T).

Extracellular vesicles derived from probiotics have received considerable attention for their pivotal role in bacterial‒host communication. These nanosized, bilayer-encapsulated vesicles carry diverse bioactive molecules, such as proteins, lipids, nucleic acids, and metabolites. Currently, ample evidence has emerged that probiotic extracellular vesicles may modulate several processes of host physiological hemostasis and offer therapeutic benefits. This review examines the biogenesis, composition, and immunomodulatory functions of probiotic-derived extracellular vesicles in probiotic–host interactions, highlighting the therapeutic potential of probiotic extracellular vesicles in the diagnosis and treatment of conditions such as cancer and inflammatory bowel disease. We further summarize the techniques for the separation and purification of extracellular vesicles, providing a methodological foundation for future research and applications. Although the field of probiotic extracellular vesicle research is still in its infancy, the prospects for their application in the biomedical field are broad, potentially emerging as a novel therapeutic approach.