Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Cattle ; Animals ; Mice ; Bacterial Proteins/metabolism* ; Bacillus cereus/metabolism* ; Hemolysin Proteins/metabolism* ; Virulence Factors/metabolism* ; Enterotoxins/metabolism*

Oral squamous cell carcinoma (OSCC) is the most prevalent and most commonly studied oral cancer. However, there is a void regarding the role that the oral microbiome may play in OSCC. Although the relationship between microbial community composition and OSCC has been thoroughly investigated, microbial profiles of the human microbiome in cancer are understudied. Here we performed a small pilot study of community-wide metatranscriptome analysis to profile mRNA expression in the entire oral microbiome in OSCC to reveal molecular functions associated with this disease. Fusobacteria showed a statistically significantly higher number of transcripts at tumour sites and tumour-adjacent sites of cancer patients compared to the healthy controls analysed. Regardless of the community composition, specific metabolic signatures were consistently found in disease. Activities such as iron ion transport, tryptophanase activity, peptidase activities and superoxide dismutase were over-represented in tumour and tumour-adjacent samples when compared to the healthy controls. The expression of putative virulence factors in the oral communities associated with OSCC showed that activities related to capsule biosynthesis, flagellum synthesis and assembly, chemotaxis, iron transport, haemolysins and adhesins were upregulated at tumour sites. Moreover, activities associated with protection against reactive nitrogen intermediates, chemotaxis, flagellar and capsule biosynthesis were also upregulated in non-tumour sites of cancer patients. Although they are preliminary, our results further suggest that Fusobacteria may be the leading phylogenetic group responsible for the increase in expression of virulence factors in the oral microbiome of OSCC patients.
Carcinoma, Squamous Cell ; microbiology ; Humans ; Metagenome ; Microbiota ; Mouth Neoplasms ; microbiology ; Phylogeny ; Pilot Projects ; RNA, Messenger ; metabolism ; Transcriptome ; Virulence ; Virulence Factors ; metabolism

To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.
Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; isolation & purification ; physiology ; Host Specificity ; Virulence Factors ; genetics ; metabolism ; Yersinia enterocolitica ; genetics ; metabolism ; virology

The presence of hyl gene in 364 PFGE clones of Enterococcus faecium was detected by colony hybridization under conditions of high stringency. The isogenic hyl-deficient mutant (* hyl) was constructed with suicide pTX4577 and screened by allelic replacement. Moreover, an in vitro study was made on the effect of hyl gene detection on the growth ability of hylgene detection on the mutant, and an in vivo study was made on the decrease of virulence in the mouse peritonitis model. The results showed, in the clinical isolates, the positive percentage of hyl gene was 32.8%, which was significantly higher than that (5.3%) in the non-clinical isolates. The * hyl was selected by kanamycin and identified by PCR, pulsed field gel electrophoresis (PFGE) and Southern blot. The experimental evidence indicated that the growth ability of * hyl was remarkably reduced in comparison with that of the wild-type strain. The percentage survival of mice in TX2466 groups was 0, while that of * hyl groups was 50% at the same inoculum in mouse peritonitis. The differences were significant. These data suggest that hyl gene in specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Being important in the pathogenesis, hyl gene is probably a major virulence factor of Enterococcus faecium.
Animals ; Enterococcus faecium ; genetics ; pathogenicity ; Genes, Bacterial ; Gram-Positive Bacterial Infections ; microbiology ; Hyaluronoglucosaminidase ; genetics ; Male ; Mice ; Mice, Inbred ICR ; Mutation ; Peritonitis ; microbiology ; Virulence ; Virulence Factors ; genetics ; metabolism

Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.
Base Sequence ; Chronic Periodontitis ; Clone Cells ; Dental Plaque ; Drug Resistance ; Energy Metabolism ; Heme ; Humans ; Iron ; Mass Screening ; Periodontitis ; Porphyromonas gingivalis ; Porphyromonas ; Virulence ; Virulence Factors

Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47phox in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling.
Cell Degranulation ; Cell Line ; *Exocytosis ; Humans ; Mast Cells/*drug effects/*metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; Reactive Oxygen Species/*metabolism ; Trichomonas vaginalis/*metabolism ; Virulence Factors/*metabolism

OBJECTIVETo study the distribution of Yersinia enterocolitica and its virulence factors in Nantong, Jiangsu.

METHODSYersinia strains were isolated from livestock and poultry. Conventional PCR was used to detect the virulence factors of all strains and strain 0:8 was analyzed by pulsed-field gel electrophoresis(PFGE).

RESULTSThe combined isolation rate of Yersinia enterocolitica from livestock and poultry was 31.06% and the gene distribution characters were: 39.57% of them were ail-, ystA- , ystB-, yadA- , virF-; 60.43% were ail- , ystA- , ystB + , yadA- , virF- respectively. The two reference strains from America and Denmark showed similar electrophoresis patterns but were significantly different with O:8 strains isolated from China while the serotypes of Yersinia enterocolitica O:3 and O:9 which were the main epidemic strains in China, were not found in this area.

CONCLUSIONThe pathogenic Yersinia enterocolitis O:3 and O:9 were not found in Nantong,Jiangsu province.

Animals ; Animals, Domestic ; microbiology ; China ; Electrophoresis ; Poultry ; microbiology ; Virulence Factors ; genetics ; metabolism ; Yersinia enterocolitica ; genetics ; isolation & purification ; pathogenicity

OBJECTIVETo investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.

METHODSMurine macrophage J774A cells were co-cultured with TcpC producing wild type E. coli strain CFT073 (TcpC(wt)) or tcpc gene-deleted CFT073 mutant (TcpC(mut)) in Transwell system, respectively. Apoptosis of J774A cells co-cultured with TcpC(wt) or TcpC(mut) was analyzed by Annexin/PI double staining. The levels of reactive oxygen species (ROS) in J774A cells were determined by DCFH-DA staining after treatment with TcpC(wt) or TcpC(mut) at 6 h, 12 h,24 h or 36 h. After the ROS was scavenged by N-acetylcysteine (NAC), the changes of J774A cell apoptosis were also examined. The expression of caspase-3 in J774A cells co-cultured with TcpC(wt) or TcpC(mut) in the presence or absence of 0.1 mmol NAC was detected by Western blot.

RESULTSJ774A cells co-cultured with TcpC(wt) for 24 h or 36 h showed significantly increased apoptosis (27.39% ± 4.05% and 28.45% ± 4.55%,respectively) when compared to control group (7.96% ± 1.63% and 10.55% ± 1.44%,P<0.01) or TcpC(mut) group (11.45% ± 2.77% and 19.26%± 2.89%,P<0.01). Levels of ROS in J774A cells treated with TcpC(wt) for 24 h (108.8 ± 9.73) or 36 h (100.3 ± 10.11) were significantly higher than those in control group (56.8 ± 4.11 and 52.8 ± 4.42,P<0.01) or TcpC(mut) (69.7 ± 5.66 and 62.6 ± 4.56, P < 0.01). The pro-apoptotic effects of TcpC(wt) on J774A cells were reversed by 0.1 or 1 mMol NAC treatment. Expression of caspase-3 in J774A cells co-cultured with TcpC(wt) (0.43 ± 0.04) decreased significantly when compared to control group (0.75 ± 0.08,P<0.05) or TcpC(mut) group (0.80 ± 0.12,P<0.05). However,total caspase-3 expression was restored in J774A cells co-cultured with TcpC(wt) in the presence of 0.1 mmol NAC (0.80 ± 0.09).

CONCLUSIONTcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.

Acetylcysteine ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Reactive Oxygen Species ; metabolism ; Virulence Factors ; pharmacology

In order to clone lvgA gene (Legionella virulence gene) of Legionella pneumophila and detect its expression in prokaryotic cell, we amplified the lvgA gene from the total cell DNA of Legionella pneumophila with PCR,and then inserted it into the coloning vector pUC18. The recombinant plasmid pUlvgA was obtained. After the recombinant plasmid pUlvgA was identified with restriction enzyme analysis, polymerase chain reaction and nucleotide sequencing analysis, the lvgA gene was subcloned into the prokaryotic expression vector pGEX-4T-1. The prokaryotic expression recombinant plasmid pGlvgA was constructed. After IPTG induction, the E. coli JM109 containing the recombinant plasmid pGlvgA expressed fusion protein. The expression of lvgA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicated that the lvgA gene of 627 bp long was amplified, the recombinant plasmids pUlvgA and pGlvgA were constructed successfully, and the GST-LvgA fusion protein of approximately 36 KDa in size was expressed in prokaryotic cell efficiently as expected.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Cytoplasm ; metabolism ; Escherichia coli ; metabolism ; Legionella pneumophila ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Virulence Factors ; biosynthesis ; genetics

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.
Animals ; Rats ; Bacteremia/metabolism* ; Blood-Brain Barrier/microbiology* ; Caveolin 1/metabolism* ; Gingipain Cysteine Endopeptidases/metabolism* ; Permeability ; Porphyromonas gingivalis/pathogenicity* ; Transcytosis ; Virulence Factors/metabolism*