[Objective] To investigate the molecular mechanism by which Listeria monocytogenes regulates the expression of mitochondrial calcium uptake 2 (MICU2) through the virulence factor listeriolysin O (LLO) and their interaction, thereby affecting mitochondrial calcium homeostasis and bacterial intracellular proliferation. [Methods] Western blotting was employed to analyze MICU2 expression in HeLa cells infected with L. monocytogenes EGD-e or Δhly. Gene silencing and eukaryotic overexpression approaches were used to examine how MICU2 regulated the intracellular proliferation of L. monocytogenes. AlphaFold3 was used to predict the interaction sites between LLO and MICU2, and co-immunoprecipitation (Co-IP) was performed to verify their interaction. Mitochondrial calcium fluorescence probe (Rhod-2 AM) was used to analyze the regulatory role of MICU2 in calcium homeostasis. [Results] EGD-e infection upregulated MICU2 expression at 4 h and 6 h post-infection (P<0.001), whereas Δhly showed no effect (P>0.05). The silencing of MICU2 enhanced bacterial proliferation (P<0.01) and elevated mitochondrial calcium levels (P<0.05), whereas overexpression of MICU2 reduced bacterial proliferation (P<0.01) and decreased mitochondrial calcium levels (P<0.05). AlphaFold3 predicted that alanine (Ala) at position 462 of LLO interacted with glutamate (Glu) at position 119 of MICU2 via a hydrogen bond, and Co-IP confirmed their interaction. [Conclusion] L. monocytogenes upregulates MICU2 expression via LLO, and MICU2 inhibits bacterial intracellular proliferation by reducing mitochondrial calcium levels. The interaction between LLO and MICU2 is a key molecular basis for this process. These findings provide insights into the pathogenic mechanism of L. monocytogenes.

[Objective] We isolated endophytes from the roots of Isatis indigotica Fort. and investigated the effects of endophytes on the growth, physiology, and medicinal quality of I. indigotica, aiming to provide a reference for microbial regulation of the cultivation and production of this plant. [Methods] The relevant culture media were selected to analyze the plant growth-promoting activities of the endophytes. Three endophyte strains (Bacillus sp. BC00, Bacillus sp. BV11, and Pseudomonas sp. PA28) were selected and applied singly to I. indigotica four times. The physiological indexes of the leaves were measured at different time points after the last treatment, and the growth and physiological indexes and the content of active ingredients of the roots and the leaves were measured at the harvesting stage. [Results] The photosynthetic pigment content and nutrient-metabolizing enzyme activities of I. indigotica leaves in each treatment group were higher than those in the control group at each time point. The three strains of endophytic bacteria significantly promoted the growth and biomass accumulation of I. indigotica. Strain BC00 had the best promoting effect on the dry weight of the above-ground part, which was increased by 74.16% compared with that of the control. Strain BV11 had the most significant promoting effect on the dry weight of the below-ground part, which was increased by 216.02% compared that of the control. The treatments with endophytic bacteria significantly affected the content of endogenous hormones and soluble substances. The three strains of endophytic bacteria had different effects on the active ingredients. Compared with the control, strain BC00 increased the content of indigo and epigoitrin by 7.83% and 5.64%, respectively, while strain BV11 slightly decreased the content of indigo and epigoitrin. The total active biomass of each index ingredient in the strain treatments was significantly higher than that of the control. [Conclusion] The endophytic bacterial strains BC00, BV11, and PA28 had significant promoting effects on the growth, physiological indexes, and active ingredients of I. indigotica, and strain BC00 demonstrated the best comprehensive promoting effect. The findings provide a basis for the later development of microbial fertilizers conducive to the growth of I. indigotica.

[Objective] To investigate the effects of calcium ion-free (Ca²⁺-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca²⁺ in culture media to create Ca²⁺-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca²⁺-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca²⁺-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca²⁺-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca²⁺-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca²⁺-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

[Objective] To deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang. [Methods] Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined. [Results] Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588. [Conclusion] The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.

[Objective] To achieve efficient expression of the alkaline laccase PIE5 in Coprinopsis cinerea with molasses as a substitute for glucose as the carbon source. [Methods] We enhanced the laccase production in C. cinerea by either exogenous addition of the invertase GspInv or endogenous co-expression of GspInv to hydrolyze sucrose in molasses. The fermentation conditions were optimized based on the laccase activity. [Results] With 40 g/L molasses as the carbon source, strain CcPIE5-14 achieved the laccase activity of (11.9±1.2) U/mL, while sucrose remained unutilized in the fermentation liquid. Upon addition of exogenous GspInv, sucrose in the fermentation liquid was hydrolyzed into fructose and glucose, and strain CcPIE5-14 exhibited the peak laccase activity of (14.8±0.7) U/mL in the medium with 30 g/L molasses. Co-expression of GspInv in CcPIE5-14 generated the engineered strain CcPIE5-14-GspInv-12, which demonstrated the maximum laccase activity of (28.1±2.4) U/mL in the mKjalke medium. When using molasses as the carbon source for laccase production, strain CcPIE5-14-GspInv-12 achieved the peak laccase activity of (20.1±2.7) U/mL, which represented a 2.24-fold increase over that of the parental strain CcPIE5-14. Following fermentation condition optimization, strain CcPIE5-14-GspInv-12 attained the maximum laccase activity of (44.6±2.6) U/mL, which marked a 2.22-fold enhancement over the pre-optimization level. [Conclusion] The engineered strain CcPIE5-14-GspInv-12, co-expressing laccase and invertase, demonstrates efficient production of the alkaline laccase PIE5 in C. cinerea with cost-effective molasses as the carbon source.

Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are two precursors of the redox coenzyme nicotinamide adenine dinucleotide (NAD). Due to their ability to effectively increase NAD levels without toxic side effects, they have garnered significant attention. [Objective] This study employed an integrated approach combining metabolomics and 16S rRNA gene sequencing to investigate and compare the effects of NMN or NR supplementation on growth performance, intestinal health, gut microbiota, and metabolites in mice. [Methods] Male C57 mice were randomly allocated into a control group and two experimental groups. The experimental groups received drinking water supplemented with NMN or NR via gavage. Non-targeted metabolomics analysis and 16S rRNA gene sequencing were conducted to systematically investigate the alterations in endogenous metabolites and gut microbiota composition. Furthermore, the expression levels of associated factors were quantified by PCR. [Results] Compared with the control group, supplementation with NMN or NR increased the richness and diversity and improved the composition of gut microbiota in mice, with NMN showing greater efficacy. Additionally, both NMN and NR supplementation significantly upregulated the expression levels of metabolites associated with anti-inflammatory and antioxidant activities. PCR results indicated that both NMN and NR suppressed the expression of pro-inflammatory cytokines. Furthermore, NMN supplementation significantly increased the number of goblet cells in the colon, thereby enhancing the intestinal barrier function. [Conclusion] Dietary supplementation with NMN or NR improves the gut microbiota composition in mice, elevates the levels of beneficial metabolites, and inhibits the expression of pro-inflammatory cytokines.

Sauropus androgynus has high medicinal and edible values. However, its growth is threatened by various viral diseases, which severely affect both the yield and quality of S. androgynus. Since research is limited regarding the viral diseases affecting S. androgynus in China. [Objective] To isotation, identifying the viral pathogens of S. androgynus in China. [Methods] The small RNA sequencing (sRNA-seq) data of S. androgynus leaves from our previous study were analyzed. RT-PCR was employed to detect the datura yellow vein virus (DYVV) in leaf samples of 10 different varieties of S. androgynus. With the total RNA of positive S4 leaves as a template, reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), and Sanger sequencing were employed to determine the full-length genome sequence of the DYVV isolate from S. androgynus (named DYVV-sa). [Results] The analysis of the sRNA-seq data revealed the presence of DYVV in S. androgynus. RT-PCR detection of different varieties showed that only S4 and S12 tested positive for DYVV, and the full-length sequence of DYVV-sa was cloned based on S4. The genome of DYVV-sa was 13 185 nt in length and contained six open reading frames (ORFs). The DYVV-sa showed the identity as high as 95.8%-98.1% with the DYVV sequences isolated from Thunbergia alata. Moreover, the phylogenetic tree also demonstrated that DYVV-sa shared the closest genetic relationship with DYVV, clearly indicating that DYVV-sa was an isolate of DYVV. In addition, the majority of DYVV-sa virus-derived small interfering RNA (vsiRNA) were 21 nt and 22 nt, and those of 21 nt were more abundant. The first nucleotide at the 5′ termini of vsiRNAs derived from DYVV-sa preferred U and C. The proportion of vsiRNAs derived from the negative strand was higher than that from the positive strand. The distribution of vsiRNAs along the viral genome was generally even, with some hot spots formed in local regions. [Conclusion] This study found that DYVV can infect S. androgynus and successfully obtains the full-length genomic sequence of the DYVV-sa isolate. These findings expand the known natural host range of DYVV, provide crucial theoretical foundations for research on its genetic diversity and phylogenetic relationship, and offer clues for the prevention and control of viral diseases attacking S. androgynus.

[Objective] To investigate whether Fusobacterium nucleatum promotes the development of lung cancer and find out the underlying mechanisms. [Methods] The cell experiment proved that F. nucleatum can promote the proliferation and metastasis of lung cancer cells; Animal experiments showed that F. nucleatum can promote the development of lung cancer; 16S rRNA gene sequencing of alveolar lavage fluid and transcriptome sequencing of lung tissue were performed to analyze the effects of F. nucleatum on the lung flora of mice with lung cancer and the activation of relevant pathways in lung tissue. [Results] The cell experiment showed that F. nucleatum significantly promoted the proliferation and metastasis of A549 cells. The animal experiments confirmed that F. nucleatum infection in the lungs increased the number of nodules and enhanced the fluorescence intensity in the lung tissue. The 16S rRNA gene sequencing revealed the enrichment of Muribaculum and Simplicispira in the lung of infected mice. The transcriptome sequencing results showed that the mouse model of lung cancer activated the interleukin-17 signaling pathway, cytokine-cytokine receptor interactions, Staphylococcus aureus infection pathway, tumor necrosis factor signaling pathway, and other signaling pathways related to inflammation or immunity after being infected with F. nucleatum. [Conclusion] F. nucleatum infection in mice with lung cancer disrupts the microbiota and activates the immunity- and inflammation-related signaling pathways in the lung, thereby promoting the metastasis of lung cancer in mice.

Cotton Verticillium wilt, caused by the soil-borne fungal pathogen Verticillium dahliae, is a devastating disease that severely impacts global cotton production. [Objective] To investigate the biocontrol potential and mechanism of endophytic Pseudomonas sp. NWSUAF303 against cotton Verticillium wilt and provide novel microbial resources for managing soil-borne diseases in cotton. [Methods] The strain was identified by phylogenetic analysis based on 16S rRNA gene sequences and phenotypic characterization. Its antifungal spectrum was evaluated via dual-culture and volatile organic compounds (VOCs) inhibition assays. VOCs were detected by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Pot experiments were carried out to assess the disease control efficacy of the strain. RT-qPCR and enzymatic activity assays were employed to elucidate the resistance mechanism of the strain against cotton Verticillium wilt. [Results] Strain NWSUAF303 was identified as Pseudomonas alvandae, exhibiting plant growth-promoting properties including nitrogen fixation, phosphate solubilization, and indole-3-acetic acid (IAA) production. Its non-volatile metabolites inhibited six phytopathogenic fungi, whereas VOCs demonstrated broader antifungal spectrum against seven pathogens, showing the inhibition rates >95% against Sclerotinia sclerotiorum and of 89.27% against V. dahliae 592. The VOCs of this strain downregulated the expression of virulence genes (VdPR1, Vdpf, and VdGAL4) in V. dahliae (P<0.05). Three key antifungal VOCs were identified, including 2,3-butanedione, 2-nonanol, and 6-methyl-2-heptanol, with the inhibitory effect of 2,3-butanedione on V. dahliae being first reported. Pot experiments revealed the control efficacy of 54.40% against Verticillium wilt, which was comparable to that of carbendazim. Strain NWSUAF303 activated the salicylic acid/jasmonic acid (SA/JA) signaling pathway, upregulating the expression of defense-related genes GhPAL, Gh4CL, and GhCHI (P<0.01), while enhancing the activities of peroxidase (POD), polyphenol oxidase (PPO), and superoxide dismutase (SOD). [Conclusion] P. alvandae NWSUAF303 combats Verticillium wilt through dual mechanisms: producing novel antifungal VOCs and activating systemic resistance via SA/JA signaling and defense enzyme coordination. With broad-spectrum antifungal activity and plant growth-promoting properties, this strain represents a promising biocontrol agent for sustainable management of cotton Verticillium wilt.

[Objective] To study the relieving effects and potential mechanisms of Lactiplantibacillus plantarum DY6 and the soybean meal fermented with this strain on constipation in mice and to develop non-toxic feed additives with probiotic potential. [Methods] Constipation was induced in male BALB/c mice by gavege of loperamide hydrochloride. The modeled mice underwent long-term feeding with the soybean meal fermented with DY6 or gavage with DY6 suspension. Linaclotide served as the positive control. We then systematically assessed the effects of fermented soybean meal on intestinal functions, immune factors, metabolic profiles, and gut microbiota in constipated mice. [Results] Compared with the model group, long-term intake of the soybean meal fermented with DY6 increased fecal moisture content and intestinal motility (P<0.05), ameliorated goblet cell reduction and lymphocyte aggregation in the colon tissue, lowered the serum levels of tumor necrosis factor-α, interleukin-6, and vasoactive intestinal peptide (P<0.05), and elevated the fecal levels of acetic acid and butyric acid (P<0.01). The fermented soybean meal modulated the gut microbiota structure. Specifically, the significantly enriched probiotics Odoribacter and Blautia were positively correlated with indicators of relieving constipation, while the significantly reduced pathogenic bacteria Helicobacter and Colidextribacter were negatively correlated with indicators of relieving constipation. [Conclusion] The soybean meal fermented with DY6 alleviates constipation by modulating the gut microbiota structure and metabolic profiles, restoring intestinal electrolyte homeostasis, and suppressing inflammation-associated signaling pathways. This intervention establishes novel applications and a theoretical framework for developing functional biological feed additives capable of both preventing and managing constipation in animals.