The thioredoxin family plays crucial roles in bacterial oxidative stress defenses and virulence regulation, while the function of its member YbbN in Vibrio parahaemolyticus remains unclear. [Objective] To elucidate the regulatory role of YbbN in the biological characteristics and pathogenicity of Vibrio parahaemolyticus, providing potential targets for developing novel anti-infection strategies. [Methods] The ybbN knockout strain (ΔybbN) and complementary strain (CΔybbN) of Vibrio parahaemolyticus SH112 were constructed by homologous recombination. The strains were compared regarding the growth characteristics, motility, biofilm formation, bacterial competition, cell adhesion, cytotoxicity, and pathogenicity in mice. [Results] Although the knockout of ybbN showed no significant effects on bacterial growth, motility, cell adhesion, or colonization, it markedly attenuated key pathogenic traits. Specifically, it decreased the biofilm formation (by 19%-30%), killing efficiency against competitive bacteria (*: P<0.05; ****: P<0.000 1), and cytotoxicity in HeLa cells (by 27%), while increasing the survival rate of mice by 87.5%. [Conclusion] This study demonstrates for the first time that YbbN specifically regulates critical aspects involved in biofilm formation, bacterial competition, and cytotoxicity in host cells, significantly influencing the biological characteristics and pathogenicity of Vibrio parahaemolyticus. These findings not only expand the understanding about the functional diversity of the thioredoxin family proteins but also provide new molecular targets and a theoretical basis for preventing Vibrio parahaemolyticus infections.

[Objective] To identify and develop a phage-derived lyase that can be heterologously expressed with high activity and stability and determine its optimal working conditions. [Methods] We employed the turbidity reduction assay to evaluate the bacteriolytic activity and identify the optimal parameters. [Results] Genome annotation and protein prediction of the Vibrio alginolyticus phage phiV208 showed that ORF30 encoded a lyase, named lysV208. This enzyme demonstrated soluble expression in Escherichia coli BL21(DE3), reaching a purified concentration of 204 μg/mL after 16 h induction with 0.25 mmol/L IPTG. Its bacteriolytic activity (turbidity reduction rate) increased from 24.2% to 68.0% in the presence of 0.5 mmol/L EDTA. Enzymatic characterization revealed that lysV208 exhibited the maximum bacteriolytic activity (75.6%) at 45 ℃ while maintaining high activity (52.8%-71.9%) within the temperature range of 25-37 ℃, which is typical for bacterial disease outbreaks in aquatic and terrestrial animals. The enzyme showed the maximum activity at pH 7.0 and retained substantial bacteriolytic activity (44.0%-63.2%) under alkalescence conditions (pH 7.0-9.0), demonstrating adaptability to marine and freshwater aquaculture environments. Divalent metal ions including Zn²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ at 0.1-1.0 mmol/L moderately enhanced the bacteriolytic activity of lysV208, whereas those at 10.0 mmol/L reduced the activity (P<0.01). In addition, lysV208 displayed broad-spectrum lytic effects, showing the bacteriolytic activity of 59.7% against V. alginolyticus V039, 68.9% against Vibrio vulnificus H1, 65.8% against Vibrio parahaemolyticus GH32, and 38.0% and 65.6% against Vibrio harveyi TY13 and G1, respectively. [Conclusion] The recombinant lyase lysV208 demonstrates robust and stable in vitro bacteriolytic activity and a broader spectrum than its source phage. These findings highlight its potential for the control of bacterial infections and the development of phage-lyase synergistic agents.

[Objective] Microorganisms play a critical role in the treatment of food waste. Elucidating their community structure and functional characteristics is essential for optimizing treatment processes and improving operational efficiency. [Methods] We employed high-throughput 16S rRNA gene amplicon sequencing to investigate the microbial community structures and potential functions in three systems: the anaerobic digestion system, the wastewater treatment system, and the air purification system (including biofilters and deodorization towers), in a food waste treatment facility. [Results] The microbial communities differed significantly in diversity and composition across systems. The anaerobic digestion system exhibited lower microbial diversity and richness, primarily influenced by environmental factors such as temperature, pH, and biochemical oxygen demand (BOD). Functional analysis indicated that organic matter degradation was the dominant microbial function across the facility. Specifically, the anaerobic system was enriched with genera such as Defluviitoga, Methanothermobacter, and Lentimicrobium, which were involved in hydrolysis, fermentation, and methanogenesis. The wastewater treatment system was dominated by Candidatus Anammoximicrobium, Nitrolancea, and Truepera, which drove organic matter degradation and nitrogen transformation processes, including anaerobic ammonium oxidation (anammox), nitrification, and denitrification. The air purification system was enriched with Chryseobacterium and Paracoccus, which were associated with biofilm formation and the degradation of volatile organic compounds and sulfur-containing compounds. [Conclusion] The microbial community characteristics in each treatment system was closely associated with system-specific operational conditions. The enrichment of functionally distinct microbial taxa reflected their ecological roles. In the anaerobic digestion system, methanogens and syntrophic bacteria cooperatively facilitated organic matter degradation and methane production. In the wastewater treatment system, nitrogen-cycling bacteria ensured efficient nitrogen removal. In the air purification system, the enrichment of heterotrophic degraders and sulfur compound-metabolizing genera (e.g., Sphingobium and Flavobacterium) enhanced deodorization performance. These findings provide a theoretical basis for microbial community regulation and process optimization in food waste treatment facilities.

[Objective] To construct a PK15 cell line stably expressing the prolyl oligopeptidase (POP) by using the PiggyBac transposon system and systematically investigate its regulatory role in the proliferation of foot-and-mouth disease virus (FMDV). [Methods] A recombinant PiggyBac vector carrying the POP gene was constructed and transfected into PK15 cells, and the expression of the target protein was detected by Western blotting. The monoclonal cell line stably expressing POP was isolated via the limiting dilution method. The stability of mRNA and protein levels of POP was verified by RT-qPCR and Western blotting, respectively. The viability of the selected cell line was assessed by the Cell Counting Kit-8 (CCK-8) assay. FMDV replication kinetics in the stable cell line were comprehensively analyzed by Western blotting, RT-qPCR, and the 50% tissue culture infective dose (TCID₅₀) assay. [Results] A PK15 cell line stably expressing POP was established. No significant differences in cell viability were observed between the stable cell line and the control cell line. The protein level of POP remained stable in the established cell line after 30 passages. The results of the viral infection assay demonstrated that the FMDV proliferation level in the PK15 cell line stably expressing POP was significantly higher than that in the control group, with this stimulatory effect being maintained across multiple passages. [Conclusion] We successfully constructed a PK15 cell line stably expressing POP. The results reveal that POP overexpression enhances FMDV replication in a passage-independent manner. These findings provide a valuable experimental model for elucidating the molecular mechanism underlying the role of POP in FMDV replication.

Lipases are a group of biocatalysts that efficiently catalyze the hydrolysis, alcoholysis, acidolysis, ester exchange, and synthesis of esters at the oil-water interface. These enzymes play crucial roles in various fields, including pharmaceuticals and chemical engineering. Microbial lipases are more readily available and hold greater value for research and application than animal and plant lipases. [Objective] To isolate lipase-producing microorganisms from natural environments, optimize their culture conditions, and characterize the structure of the produced lipase. [Methods] We used bromocresol purple as an indicator to screen lipase-producing strains from oil-contaminated soil and identified the strains by morphological observation and 18S rRNA gene sequencing. We optimized the culture conditions of the strains by single-factor experiments and response surface methodology. We performed proteomic sequencing on the active proteins to identify the potential lipase. We amplified and sequenced the gene of the potential lipase by PCR and analyzed its multi-level structure. [Results] A lipase-producing fungal strain FA3 was isolated from oil-contaminated soil and identified as Aspergillus sp. The strain was cultured with olive oil as the sole carbon source, and the p-nitrophenol colorimetric method revealed that the intracellular enzyme activity of strain FA3 was 263.75 U/g. The optimized culture conditions were as follows: emulsified olive oil at 4 mL/L, peptone at 18 g/L, K₂HPO₄ at 1 g/L, NaCl at 10 g/L, MgSO₄ at 0.5 mmol/L, and pH 5.3. After culture at 30 ℃ for 77.5 h, the specific activity reached 2 120.27 U/g, which was about 8.04 times higher than that before optimization. The proteomic analysis and structural prediction revealed the conserved Gly-Asp-Ser motif, confirming the lipase as a typical GDSL lipase. The enzyme contained strictly conserved Ser, Gly, Asn, and His residues and it was thus classified as a member of the N-sulphoglucosamine sulphohydrolase (SGNH) hydrolase family. [Conclusion] After optimization of the culture conditions, strain FA3 demonstrated significantly enhanced lipase production, showing great potential for application. We clarify the functions and structural characteristics of the lipase, providing a basis for the engineering of related proteins.

[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 compare the bacterial growth, oxidative resistance, and bacterial infection in cells and host among Listeria monocytogenes EGD-e, lmo0175 (LPXTG motif-anchored protein)-deleted and complementary strains, so as to investigate the roles of Lmo0175 in anti-oxidative resistance and bacterial infection. [Methods] The lmo0175-deleted and complementary strains were constructed to compare the difference in bacterial growth, oxidative resistance, adhesion, invasion, intracellular proliferation, survival of infected mice, and bacterial loads in organs of Listeria monocytogenes. [Results] The deletion of lmo0175 remarkably decreased oxidative resistance, cell proliferation, colonization in the liver and spleen, and pathogenicity in mice. However, it had no significant impact on bacterial growth, adhesion or invasion. [Conclusion] The LPXTG motif-anchored protein Lmo0175 contributes to the anti-oxidative resistance, proliferation, and colonization in specific organs of Listeria monocytogenes.

[Objective] To investigate the pathogenicity of Lichtheimiaramosa LYSF001 and ITS resistance to common fungicides, providing experimental evidence for the diagnosis and treatment of fungal infections in animals. [Methods] The strain was identified by morphological characterization and molecular biology techniques. PCR was employed to detect virulence genes in strain LYSF001, and an animal infection model was established to evaluate the pathogenicity of the strain. Histopathological examination, Grocott’s methenamine silver (GMS) staining, and ITS sequences amplification were employed to analyze pathological changes in the livers and kidneys of infected animals. Additionally, antimicrobial susceptibility tests were conducted to assess the sensitivity of strain LYSF001 to caspofungin, amphotericin B, and itraconazole. [Results] Strain LYSF001 was identified as L. ramosa. PCR analysis revealed that strain LYSF001 carried five virulence genes (CalA, PKP2, LaeA, Alp2, and AspF1). Animal experiments demonstrated that the strain led to the mortality of 40% and caused visceral hyperemia in mice, with the most significant pathological changes (inflammatory cell infiltration and tissue necrosis) observed in the livers and kidneys. Severe infections led to animal mortality. GMS staining revealed the presence of dark-colored hyphae in the heart and liver, and ITS sequences amplification further confirmed fungal infection. The antimicrobial susceptibility test results indicated that strain LYSF001 was resistant to caspofungin but sensitive to amphotericin B and itraconazole. [Conclusion] L. ramosa LYSF001 exhibits strong pathogenicity, capable of causing severe infections and even death in animals. Additionally, the strain showed resistance to caspofungin but sensitivity to amphotericin B and itraconazole. The findings provide important experimental evidence and technical support for the diagnosis and treatment of fungal infections in animals.

[Objective] To elucidate the virulence-associated biological characteristics of a transposon insertion mutant with impaired biofilm formation of Avibacterium paragallinarum. [Methods] The wild-type strain JZIC-005 and a biofilm-deficient mutant Tn-1504 (from a pre-established transposon mutant library) were selected for determination of insertion sites biological characterization. [Results] PCR results demonstrated site-specific transposon integration into TonB of A. paragallinarum. While mutant Tn-1504 exhibited no growth defects compared with the wild-type strain (P>0.05), it demonstrated reductions of 46.57% in adhesion to DF-1 cells and 77.61% in cellular invasion. Cytotoxicity assays revealed a 34.97% reduction of Tn-1504 in inducing host cell damage. All of the above phenotypic differences were statistically significant (P<0.01). [Conclusion] We demonstrate that inactivation of TonB specifically impairs the adhesion, invasion, and virulence characteristics of A. paragallinarum, providing a key target for analyzing the pathogenic mechanism and developing anti-virulence strategies for this pathogen.

[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.