Brevibacillus laterosporus (B. laterosporus) is a significant probiotic bacterium known for producing multiple secondary metabolites with notable antibacterial activity. In this study, bioinformatic tools such as FastANI and Roary were employed to analyze genomic similarity and construct the B. laterosporus pan-genome. The software platforms antiSMASH and BAGEL4 were used to predict biosynthetic gene clusters (BGCs) and identify bacteriocins. Core candidate bacteriocins were subsequently subjected to heterologous expression in E. coli, followed by nickel column purification and concentration. Their antibacterial activity was systematically evaluated using agar diffusion assays, minimum inhibitory concentration (MIC) determinations, time-kill curves, scanning electron microscopy (SEM), and live/dead bacterial staining in Listeria monocytogenes (L. monocytogenes). Pan-genome analysis revealed an open genome for B. laterosporus, comprising 2,840 core genes. KEGG enrichment analyses indicated that these core genes were significantly associated with pathways responsible for the biosynthesis of antimicrobial compounds, including secondary metabolite, pan-quinone, and terpenoid quinone biosynthesis. AntiSMASH predicted 1,654 BGCs, predominantly non-ribosomal peptide synthase (NRPS), polyketide synthase (PKS), and heterocyclic amine-associated antimicrobial protein synthesis clusters. Moreover, BAGEL4 predicted three putative core candidate bacteriocin variants (BreL1, BreL2, and BreL3), all of which exhibited pronounced antibacterial activity against L. monocytogenes. Among these, BreL1 demonstrated the most robust activity, causing morphological changes consistent with membrane-associated damage. Collectively, this study elucidates the open pan-genome architecture and the metabolically rich secondary metabolome of B. laterosporus, identifies three putative bacteriocin variants with active antibacterial efficacy against L. monocytogenes, which provides a theoretical foundation for developing natural antimicrobial agents.

Salmonella Typhimurium (S. Typhimurium) is a major foodborne Gram-negative pathogen, presenting significant health risks to both humans and animals. LuxR family regulatory factors are critically involved in bacterial adaption to environmental changes and virulence regulation. Nevertheless, the biological function of YjjQ, a member of this family, remains uncharacterized in S. Typhimurium. To clarify the regulatory function of YjjQ in S. Typhimurium, λ-Red homologous recombination was employed to construct the yjjQ gene deletion strain (SL1344-ΔyjjQ) and its complementation strain (SL1344-ΔyjjQ/comp). Then, the growth characteristics, biofilm formation, motility, cell adhesion and invasion, intracellular survival and proliferation, as well as virulence, were systematically analyzed, respectively. Furthermore, the expression profiles of those genes associated with biofilm, flagella, and virulence were evaluated, and the interaction between YjjQ and predicted target promoter was experimentally validated. Compared to SL1344 and SL1344-ΔyjjQ/comp, SL1344-ΔyjjQ exhibited notably enhanced biofilm formation, motility, cell invasion, and intracellular proliferation under hyperosmotic conditions, in addition to heightened virulence in mice. RT-qPCR analysis revealed these genes related to flagellar (flhD, fliA), biofilm (csgD, fliC), and SPI-1 (invF) were significantly upregulated. The EMSA confirmed that YjjQ protein directly binds the flhDC promoter region, suppressing transcription of the flhDC operon, thereby negatively regulating flagellar, biofilm, and virulence. These findings provide new insights into the molecular mechanisms underlying the coordinately modulated strategy of biofilm formation, flagellar biosynthesis and virulence in S. Typhimurium.

Mycoplasma bovis is a major etiological agent of bovine respiratory disease, causing substantial economic losses to the cattle industry. To address this challenge, we performed a pan-genome analysis of 80 globally sourced M. bovis genomes and identified 1058 core genes. Reverse vaccinology and immunoinformatics approaches were subsequently integrated to screen three potential vaccine candidates: namely lipoate protein ligase 2 (LplA), elongation factor 4 (LepA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Codon-optimized gene sequences were cloned into the pET-28a vector and successfully expressed and purified using E. coli expression system. Thirty female BALB/c mice were randomized into five groups (n = 6 per group). For primary immunization, each mouse received 100 μg of recombinant protein emulsified in Freund's complete adjuvant via subcutaneous injection, followed by booster immunizations with 50 μg of protein formulated with Freund's incomplete adjuvant at weeks 2 and 4. Fourteen days after primary immunization, all vaccinated groups exhibited significantly elevated antigen-specific antibody levels, which remained high through week 6. Notably, rLplA and rGAPDH induced higher antibody titers than rLepA. Analysis of the IgG2a/IgG1 subtype ratio indicated that rLplA, rGAPDH, and rLepA predominantly elicited Th2-type immune responses. In addition, all three recombinant proteins significantly increased cytokine production. Moreover, no adverse physiological reactions or histopathological abnormalities were observed throughout the immunization period, confirming the favorable safety profile of the candidates. Collectively, these findings identify promising targets for the development of M. bovis subunit vaccines to address pathogen variability, antibiotic resistance, and the limited efficacy of current vaccines.

This study aimed to identify and characterize a high-efficiency chitinase gene from Brevibacillus laterosporus (B. laterosporus), characterize its enzymatic traits, and assess its degrading activity against Caenorhabditis elegans (C. elegans) and Parascaris equorum (P. equorum) eggs. A nematicidal B. laterosporus isolate was subjected to whole-genome sequencing for chitinase gene screening, cloning, and molecular identification. The target gene was heterologously expressed in Escherichia coli BL21 (DE3), and biochemical properties including temperature, pH, metal ions, and substrate conditions were investigated, its kinetic parameters were determined, and its biological effects on C. elegans and P. equorum eggs were analyzed. The protein BLChi79 (79.3 kDa) belongs to GH18 chitinases with a typical carbohydrate-binding module. Its optimum activity occurred at 60 °C and pH 6.0, with colloidal chitin as the optimal substrate. Mg2+, Fe2+, and Mn2+ boosted its activity, while K+, Cu2+, Zn2+, and Ca2+ suppressed catalysis. Its Km was 6.14 mg·mL-1, Vmax 7.78 μmol·min-1·mg-1, and kcat 10.16 min-1. The purified recombinant enzyme could degradethe C. elegans chitin layer and the P. equorum egg vitelline envelope. In summary, the BLChi79 derived from B. laterosporus targets chitin-enriched eggshells, acting as a green biocatalyst for controlling livestock gastrointestinal nematodes.

Listeria monocytogenes (L. monocytogenes) is a zoonotic foodborne pathogen that triggers life-threatening invasive illnesses. MarR-type transcriptional regulators widely control bacterial stress adaptation, antibiotic tolerance and virulence, playing crucial roles in the pathogenesis of diverse bacterial pathogens. Lmo0840 is a conserved member of this family; however, its physiological functions and regulatory networks in L. monocytogenes remain completely unexplored. To this end, we constructed an lmo0840 deletion mutant and its complemented strain in the L. monocytogenes EGD-e background and systematically characterized the biological functions of Lmo0840 using phenotypic assays, transcriptomic profiling, and electrophoretic mobility shift assays (EMSA). Phenotypic characterization revealed that loss of lmo0840 impaired growth rate under hyperosmotic, oxidative, and iron-limitation stresses, while unexpectedly promoting growth rate at 30 °C and under tetracycline stress. Consistently, Disk diffusion assays further showed that the Δlmo0840 mutant exhibited significantly reduced inhibition zone diameters against tetracycline antibiotics compared to the EGD-e strain. Moreover, the Δlmo0840 mutant exhibited reduced early biofilm formation and attenuated macrophage adhesion, yet showed enhanced cellular invasion and decreased virulence in a murine model. EMSA further demonstrated that Lmo0840 directly binds to the promoter region of lmo0839 and represses its transcription, thereby contributing to bacterial adaptation to tetracycline stress. Collectively, these findings establish Lmo0840 as a pleiotropic regulator involved in environmental stress responses, tetracycline adaptation, and virulence in L. monocytogenes. Notably, this study identifies for the first time an Lmo0840–Lmo0839 regulatory cascade underlying tetracycline adaptation. These insights not only deepen our understanding of the molecular mechanisms governing Lm environmental adaptation and pathogenicity but also provide a potential target for the prevention and control of listeriosis.

The antibacterial activities of numerous medium and long-chain unsaturated fatty acids are well documented. However, the natural short-chain unsaturated fatty acid, 4-pentenoic acid (C5:1 Δ4) has not been systematically evaluated for its antibacterial activity, mechanism of action, or application potential. This study investigated these to provide a basis for novel preservatives. 4-Pentenoic acid showed broad-spectrum activity against 21 strains of pathogenic bacteria, with MICs 1.5-3 mg/mL, MBCs 3-6 mg/mL, and low resistance risk. It disrupted bacterial membrane integrity, inhibited biofilm formation, and regulated the pdu, nar and RND efflux pump genes, thereby affecting bacterial metabolism and stress responses. Checkerboard broth microdilution susceptibility assays confirmed that the combination of 4-pentenoic acid with antibiotics (such as meropenem) exerted additive antibacterial effects. At effective antibacterial concentrations, oral administration of 4-pentenoic acid caused no observable toxicity. It can markedly inhibit the proliferation of pathogenic bacteria in milk, beef, and peanut butter, while dosages close to the minimum inhibitory concentration exerted minimal adverse effects on the sensory properties and pH of these foods. In summary, this study provides theoretical and experimental basis for the development of new food preservatives and antibacterial agents.

Salmonella Typhimurium (S. typhimurium) is a significant zoonotic pathogen responsible for gastroenteritis and severe systemic infections in various hosts. The AraC family transcription factors are key gene expression regulators in prokaryotes, essential for bacterial adaptation to the environment and virulence. Despite their importance, the role of yeaM, a member of this family in S. typhimurium, remains unexplored. To elucidate yeaM regulatory function in virulence and biofilm formation, we engineered mutant and complementary strains of the yeaM gene using homologous recombination. We assessed their capabilities in biofilm formation under different conditions, macrophage adherence and invasion, and virulence in mice. Additionally, we identified potential target genes regulated by yeaM through transcriptome sequencing and confirmed these findings using an electrophoretic mobility shift assay (EMSA) and a dual-luciferase reporter assay. Our results demonstrate that, compared to the parental strain SL1344 and the complemented strain C Delta yeaM, the Delta yeaM strain exhibited significantly enhanced biofilm formation, increased invasion of mouse intestinal epithelial cells, enhanced intracellular proliferation within macrophages, and elevated induction of macrophage apoptosis. Furthermore, the Delta yeaM deletion strain displayed significantly increased virulence in mice and enhanced proliferation in milk. Transcriptome analysis revealed that S. typhimurium pathogenicity island 4 (SPI4) genes (siiA, siiB, siiC, siiD, siiF, and siiE) were significantly upregulated following the deletion of the yeaM gene. EMSA and dual-luciferase reporter assays further showed that the yeaM protein can bind to the promoter of the siiA gene and suppress its expression, thereby modulating the biofilm formation and virulence of S. typhimurium.

Introduction:Nematode-trapping fungi (NTFs) can produce various chitinases to degrade nematode body wall and eggshell chitin during predation. However, the regulatory mechanisms of their expression of chitinases still remain unclear. The primary objective of this study was to elucidate the differential protein profile of A. oligospora, an NTF, in response to chitin. Material and Methods:Colloidal chitin was added to induce the culture of A. oligospora, and the phenotypic differences before and after induction were observed under inverted microscope. The differential proteins before and after mycelium induction were screened by liquid chromatography-tandem mass spectrometry. The differentially expressed chitinase was expressed in Pichia yeast, and the recombinant enzyme was incubated with Caenorhabditis elegans and its egg suspension to explore its biological activity. Results:It was found that there was a significant acceleration in the mycelial growth post chitin interaction in A. oligospora. A total of 1,124 differentially expressed proteins (DEPs) were identified between the control group (AO-c) and the experimental group (AO-e), with 183 upregulated and 941 downregulated. Gene Ontology analysis revealed that the DEPs acted in various metabolic processes with catalysis and binding functions. Kyoto Encyclopedia of Genes and Genomes analysis associated these proteins primarily with signalling pathways related to glucose metabolism. Three chitinases were significantly modulated among DEPs. Moreover, enzymatic activity assays demonstrated that one of them effectively degraded C. elegans and its eggs. Conclusion:These findings suggest that A. oligospora can significantly alter its protein expression profile in response to chitin, thereby facilitating its sugar metabolism and mycelial development. Our study provided new insights into the regulatory mechanisms of nematode predation in A. oligospora.

The nematode-trapping fungus (NTF) Arthrobotrys oligospora (A. oligospora) is a promising biocontrol agent, but the transcriptional regulators governing its predation remain poorly understood. Here, we demonstrated that the APSES transcription factor AoMbp1 is a master regulator of its development and stress adaptation. Deletion of AoMbp1 severely impaired mycelial growth, conidiation, trap formation, and tolerance to oxidative and osmotic stresses. Transcriptome analysis revealed that these defects were associated with the widespread downregulation of genes, including those within the MAPK signaling pathway. Crucially, we showed that AoMbp1 directly binds to the promoter of AoSho1, a key upstream sensor of the MAPK cascade, and activates its expression. This finding establishes a direct AoMbp1-AoSho1 regulatory axis controlling trap morphogenesis and environmental adaptation. Our study provides novel mechanistic insights into the regulation of nematode trapping and identifies a potential target for enhancing the efficacy of A. oligospora as a biocontrol agent.

Exosomes are nanoscale vesicles secreted by cells that play vital regulatory roles in intercellular communication and immune responses. Listeria monocytogenes (L. Monocytogenes, LM) is a notable Gram-positive intracellular parasitic bacterium that infects humans and diverse animal species. However, the specific biological function of exosomes secreted by macrophages during L. Monocytogenes infection (hereafter EXO-LM) remains elusive. Here, we discovered that EXO-LM stimulated the secretion of inflammation-associated cytokines by macrophages, facilitating the intracellular survival of L. monocytogenes within macrophages. Transcriptomic analysis shows that EXO-LM significantly upregulates immune recognition and inflammation-related signaling pathways in macrophages. Furthermore, a ceRNA regulatory network comprising exosomal ncRNAs and macrophage RNAs was constructed through EXO-LM transcriptome sequencing. Utilizing bioinformatics and dual-luciferase reporter assays, we identified two potential binding sites between lncRNA Rpl13a-213 and miR-132-3p. Cell transfection experiments demonstrated that Rpl13a-213 overexpression augmented pro-inflammatory cytokine expression in macrophages, in contrast to the suppression by miR-132-3p overexpression. The decrease in Rpl13a-213 upon EXO-LM stimulation enhances miR-132-3p expression, dampening the inflammatory response in macrophages and aiding L. monocytogenes intracellular survival. This study unveils the immunomodulatory function of exosomal ncRNAs originating from macrophages, which provides fresh perspectives into the mechanisms underlying macrophage inflammatory response regulation by L. monocytogenes-infected cell-derived exosomes.

Introduction. Salmonella Typhimurium (STM) is a food-borne Gram-negative bacterium, which can infect humans and a wide range of livestock and poultry, causing a variety of diseases such as septicaemia, enteritis and abortion.Hypothesis/Gap Statement. We will decipher the impacts of sRNA STnc1280 on STM virulence and provide a theoretical basis to reveal the regulatory role and molecular mechanism of STnc1280.Aim. The main objective of this study was to clarify whether sRNA STnc1280 exerts regulatory roles on STM pathogenicity.Methodology. The STnc1280 gene was amplified and its molecular characteristics were analysed in this study. Then, STnc1280 gene deletion strain (STM-ΔSTnc1280) and the complementary strain (ΔSTnc1280/STnc1280) were constructed by λ-Red homologous recombination method, respectively, to analyse of adhesion and invasive ability and pathogenicity of different strains. Subsequently, the potential target gene regulated by STnc1280 was predicted using target RNA2 software, followed by the verification of the interaction between STnc1280 and target mRNA using the dual plasmid reporter system (DPRS). Furthermore, the mRNA and protein level of target gene was determined using qRT-PCR and Western blot, respectively.Results. The results revealed that the cell adhesion and invasive ability and pathogenicity of STM-ΔSTnc1280 were significantly reduced compared to STM-SL1344 strain, indicating that the deficiency of STnc1280 gene significantly influenced STM pathogenicity. The DPRS results showed that STnc1280 can interact with the mRNA of target gene gldA, thus suppressing the expression of lacZ gene. Furthermore, the level of gldA mRNA was not influenced in STM-ΔSTnc1280, but the expression of GldA protein decreased significantly.Conclusion. Combining the bioinformatic analysis, these findings suggested that STnc1280 may bind to the SD sequence of gldA mRNA, hindering the binding of ribosomes to gldA mRNA, thereby inhibiting the expression of GldA protein to modulate the virulence of STM.

Salmonella Typhimurium (STM) is an important zoonotic Gram-negative pathogen that can cause infection in a variety of livestock and poultry. Meanwhile, as an important foodborne pathogen, the bacterium can survive in various stressful environments and transmits through the fecal-oral route, posing a serious threat to global food safety. To investigate the roles of STM1863, a member of the DUFs protein family, involved in STM environmental adaptation, biofilm formation, and virulence. We analyzed the molecular characteristics of the protein encoded by STM1863 gene and examined intra- and extracellular expression levels of STM1863 gene in mouse macrophages. Furthermore, we constructed STM1863 gene deletion and complementation strains and determined its environmental adaptation under stressful conditions such as acid, alkali, high salt, bile salt, and oxidation. And the capacity of biofilm formation and pathogenicity of those strains were analyzed and compared. In addition, the interaction between the promoter of STM1863 gene and RcsB protein was analyzed using DNA gel electrophoresis migration assay (electrophoretic mobility shift assay [EMSA]). The experiments revealed that acid adaptability and biofilm formation ability of STM1863 gene deletion strain were significantly weakened compared with the parental and complementary strains. Moreover, the adhesion and invasion ability of STM1863 deletion strain to mouse macrophages was significantly decreased, while the median lethal dose (LD50) increased by 2.148-fold compared with the parental strain. In addition, EMSA confirmed that RcsB protein could bind to the promoter sequence of STM1863 gene, suggesting that the expression of STM1863 gene might be modulated by RcsB. The present study demonstrated for the first time that STM1863, a member of the DUFs protein family, is involved in the modulation of environmental adaptation, biofilm formation, and virulence.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it’s target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

As important post-transcriptional regulators of gene expression, sRNAs play important modulatory roles in the environmental adaptation and virulence of bacteria. To investigate the regulatory role of sRNA STnc3020 in the virulence of Salmonella typhimurium (S. typhimurium). This study analyzed the impacts of STnc3020 deletion on adherence, invasion, intracellular survival, macrophage apoptosis, and pathogenicity of S. typhimurium in mice. Furthermore, potential regulatory target genes of STnc3020 were identified and its regulatory mechanism was validated. The results showed that at the cellular level, the deletion of STnc3020 significantly reduced the adhesion ability of S. typhimurium to intestinal epithelial cells (P < 0.01), as well as its proliferation and apoptosis-inducing abilities within macrophages (P < 0.01). Meanwhile, animal experiment results indicated that the deletion of STnc3020 significantly reduced the colonization rate of S. typhimurium in the liver and cecum of mice (P < 0.01), and increased the median lethal dose (4.28 × 105) in mice. Regulatory mechanism research results showed that STnc3020 can interact with the target gene prgJ of the Type III secretion system (T3SS), and the protein level of PrgJ significantly decreased after the deletion of STnc3020 (P < 0.01). These findings offer new insights into sRNA-mediated virulence control and may aid in developing new vaccines and drugs for S. typhimurium.

Abstract Introduction Listeria monocytogenes (LM) is an important food-borne pathogen, and the risk of its ingestion is a serious public health issue. The better its environmental adaptation mechanisms and pathogenicity are understood, the better the risk it poses can be countered. The regulatory role of the small non-coding RNA (sRNA) rli106 in the environmental adaptation and pathogenicity of LM is still unclear and this study investigated that role through its biological function. Material and Methods An LM-Δrli106 gene deletion strain and an LM-Δrli106/rli106 gene complementation strain were constructed using the homologous recombination technique. Then, the adaptation of these strains to temperature, alkalinity, acidity, salinity, ethanol and oxidative stressors, their biofilm-forming ability and their pathogenicity in mice were investigated to show the regulatory roles of sRNA rli106 in LM. The target gene of rli106 was also predicted, and the interaction between it and rli106 was verified by a two-plasmid co-expressing system based on E.coli and Western blot analysis. Results The adaptation of LM-Δrli106 to environmental stressors of pH 9, 5% NaCl and 8% NaCl, 3.8% ethanol and 5 mM H2O2 was significantly reduced when compared to the parental (LM EGD-e) and complementation strains. Also, the biofilm formation, cell adhesion, invasion, intracellular proliferation and pathogenicity of LM-Δrli106 in mice were significantly reduced. The results of two-plasmid co-expression and Western blot showed that rli106 can interact with the mRNA of the predicted DegU target gene. Conclusion The sRNA rli106 may positively regulate the expression of the DegU gene in LM. This study sheds light on its regulatory roles in environmental adaptation and pathogenicity, providing new insights into the molecular mechanism of sRNA mediation in LM .

Small regulatory RNAs (sRNAs) play important roles as post-transcriptional regulators throughout the life activities of bacteria. To explore the roles of the regulatory sRNA rli41 identified in Listeria monocytogenes (LM), we analyzed the molecular characteristics and expression profiles of sRNA rli41 during cell infection. Then the deletion strain LM-Δrli41 and complementation strain LM-Δrli41-rli41 were constructed and used to investigate the effects of deficiency of rli41 gene on the adhesion, invasion and pathogenicity of LM. Furthermore, sRNA rli41-regulated potential target genes were predicted in silico, followed by the verification of sRNA-mRNA interaction using a dual plasmid reporter system. The results revealed that sRNA rli41 was highly conserved in LM and its expression level was 15.78-fold up-regulated during cell infection as compared to extracellular environment. Compared with the parental strain and complementation strain, cell adhesion and invasion of the deletion strain were significantly reduced. Moreover, LD50 of LM increased and the pathogenicity weakened significantly due to the deficiency of rli41 genes. Additionally, sRNA rli41 could complementarily base pairing with lmo2178 mRNA 5'-UTR, which could facilitate the expression of peptidoglycan-binding protein Lmo2178, which in turn promotes the cell adhesion, invasion and pathogenicity of LM. Taken together, these findings demonstrated for the first time that the regulatory sRNA rli41 is implicated in cell adhesion, invasion and pathogenicity by modulating the expression of virulence factor Lmo2178 in LM.

Fascioliasis, a global zoonotic parasitic disease, is mainly caused by Fasciola hepatica (F. hepatica) parasitizing in the livers of hosts, mainly humans and herbivores. Glutathione S-transferase (GST) is one of the important excretory- secretory products (ESPs) from F. hepatica, however, the regulatory roles of its Omega subtype in the immunomodulatory effects remain unknown. Here, we expressed F. hepatica recombinant GSTO1 protein (rGSTO1) in Pichia pastoris and analyzed its antioxidant properties. Then, the interaction between F. hepatica rGSTO1 and RAW264.7 macrophages and its effects on inflammatory responses and cell apoptosis were further explored. The results revealed that GSTO1 of F. hepatica owned the potent ability to resist oxidative stress. F. hepatica rGSTO1 could interact with RAW264.7 macrophages and inhibit its cell viability, furthermore, it may suppress the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, but promote the expression of anti-inflammatory cytokine IL-10. In addition, F. hepatica rGSTO1 may down-regulate the ratio of Bcl-2/Bax, and increase the expression of pro-apoptotic protein caspase-3, thereby eliciting the apoptosis of macrophages. Notably, F. hepatica rGSTO1 inhibited the activation of nuclear factor-κB (NF-κB) and mitogen‑activated protein kinases (MAPKs p38, ERK and JNK) pathways in LPS-activated RAW264.7 cells, exerting potent modulatory effects on macrophages. These findings suggested that F. hepatica GSTO1 can modulate the host immune response, which provided new insights into the immune evasion mechanism of F. hepatica infection in host.

Background: Listeria monocytogenes (LM) is a facultative intracellular pathogen that causes food-borne infections in humans and animals. To invade and multiply within host cells, LM utilizes various strategies to precisely modulate its gene expression and to adapt to the in vivo environment. Objectives: To investigate the regulatory roles of Rli82 sRNA in the motility and pathogenicity of LM EGD-e. Methods: The Rli82 gene knock-out mutant strain, LM-ΔRli82, and the complementation strain, LM-ΔRli82/Rli82, were constructed using homologous recombination technology, and their motility and virulence, respectively, were determined. Moreover, the potential target mRNA regulated by Rli82 was predicted using TargetRNA2 software, and then the interaction between the target mRNA and Rli82 was verified by the two-plasmid reporter system. Results: The results showed that the motility of LM-ΔRli82 was significantly increased at 25°C, facilitated by the production of more flagella than LM EGD-e and LM-ΔRli82/Rli82. Furthermore, LD50 in LM-ΔRli82-infected mice was significantly increased as compared to LM EGD-e and LM-ΔRli82/Rli82, suggesting that the virulence of LM was weakened when the Rli82 gene was deleted. In addition, the mRNA level of flaA was not significantly elevated, but flaA protein was significantly higher in LM-ΔRli82 than in LM EGD-e and LM-ΔRli82/Rli82, suggesting that Rli82 might modulate the translation of flaA mRNA at the post-transcriptional level. Conclusions: Taken together, our findings for the first time revealed that Rli82 sRNA might be involved in the modulation of the expression of flaA protein, thereby influencing the mobility and pathogenicity of LM.

克隆目的基因LMlmo2486,分析目的基因及其编码蛋白的分子特征,并验证其编码蛋白的反应原性,为进一步研究LM lmo2486的生物学特性奠定前期的基础.通过PCR扩增单核细胞增生李斯特菌(LM)新型转录调节因子lmo2486基因,对该基因进行测序,预测分析其编码蛋白质的二、三级结构、理化特性、蛋白结构域等分子特征,并进行遗传进化分析.PCR克隆lmo2486基因的抗原集中区lmo2486K,构建重组载体pET-32a(+)-lmo2486K转化至大肠埃希氏菌感受态细胞BL21(DE3)并使用IPTG低温诱导表达,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blot)检测重组蛋白.结果显示,LM lmo2486基因全长1212 bp,共编码403个氨基酸;lmo2486基因编码的蛋白具有亲水性和跨膜结构,无信号肽,预测发现lmo2486含有PspC和DUF4097结构域.序列同源对比发现,LM lmo2486测序所得的核苷酸序列与LM不同分离株属于同一分支,亲缘关系较近,表明lmo2486基因在LM中高度保守.SDS-PAGE检测表明,重组蛋白Lmo2486K的分子质量约为64.6 ku,Western blot分析发现,Lmo2486K蛋白具有一定的反应原性.