Objective: To analyze the characteristics of super-antigen (SAg) of group A Streptococcus pyogenes (GAS), isolated from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017. Methods: Throat swab specimens from patients with scarlet fever or pharyngeal infections were collected and tested for GAS. Eleven currently known SAg genes including SpeA, speC, speG, speH, speI, speJ, speK, speL, speM, smeZ and ssa were tested by real-time PCR while M protein genes (emm genes) were amplified and sequenced by PCR. Results: A total of 377 GAS were isolated from 6 801 throat swab specimens, with the positive rate as 5.5%. There were obvious changes noticed among speC, speG, speH and speK in three years. A total of 45 SAg genes profiles were observed, according to the SAgs inclusion. There were significant differences appeared in the frequencies among two of the highest SAg genes profiles between emm1 and emm12 strains (χ(2)=38.196, P<0.001; χ(2)=72.310, P<0.001). There also appeared significant differences in the frequencies of speA, speH, speI and speJ between emm1 and emm12 strains (χ(2)=146.154, P<0.001; χ(2)=52.31, P<0.001; χ(2)=58.43, P<0.001; χ(2)=144.70, P<0.001). Conclusions: Obvious changes were noticed among SAg genes including speC, speG, speH and speK from patients with scarlet fever or pharyngeal infections in Beijing between 2015-2017. SAg genes including speA, speH, speI and speJ appeared to be associated with the emm 1 and emm 12 strains. More kinds of SAg genes profiles were isolated form GAS but with no significant differences seen in the main SAg genes profiles, during the epidemic period.
Antigens, Bacterial/genetics* ; Bacterial Outer Membrane Proteins ; Bacterial Proteins ; Beijing/epidemiology* ; China/epidemiology* ; Exotoxins ; Female ; Humans ; Membrane Proteins ; Pharyngitis/microbiology* ; Pharynx/microbiology* ; Pregnancy ; Pregnancy Complications, Infectious/microbiology* ; Real-Time Polymerase Chain Reaction ; Scarlet Fever/microbiology* ; Streptococcal Infections ; Streptococcus pyogenes/isolation & purification* ; Superantigens/genetics*

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.
Actinobacillus pleuropneumoniae/genetics/*pathogenicity/*physiology ; Animals ; *Apoptosis ; Bacterial Proteins/genetics/metabolism ; Blotting, Southern ; Exotoxins/*genetics ; Hemolysin Proteins/genetics/metabolism ; *Hemolysis ; Macrophages, Alveolar/metabolism/*microbiology ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Swine ; Virulence

OBJECTIVETo investigate drug-resistance and carriage of virulence factors of Pseudomonas aeruginosa (Pa) isolated from children.

METHODThirty-eight strains of Pa were collected and isolated in pediatric clinic during 2006-2009, and tests were undertaken to identify bacteria and susceptibility test was performed using VITEK-2 COMPACT GNI and AST-GN13 cards. The virulence factors were confirmed by using polymerase chain reaction (PCR) and sequencing.

RESULTAll the 38 strains of Pa were resistant to ampicillin, ampicillin/sulbactam, cefazolin, nitrofurantoin, trimethoprim/sulfamethoxazole, resistance rates were 100%. Except for ceftriaxone (60.53%), the resistance rates to other antibiotics were all below 16%. PCR test showed that all the 38 strains of Pa carried exotoxin A(toxA) and nitric oxide reductase A (norA), however, detective ratio of the other virulence factors, exoenzyme Y (exoY) was 84.21% (32/38), exoenzyme S (exoS) 57.89% (22/38), pyocyanin (pyp) 42.11% (16/38), exoenzyme U (exoU) 34.21% (13/38), and 38 strains of Pa did not carry exoenzyme T (exoT) and elastase B (lasB) without exception. By analyzing tests, we discovered that 3 pan-drug resistant strains of Pa were all combination of exo U+/pyp+, there were 4 strains of Pa which were moderately-resistant to imipenem, including exoU+/pyp+/exoY+ (2 isolates), exo U+/pyp+ (1 isolate), and exoY+/exoS+ (1 isolates). It indicated that the drug-resistance rate of exoU+/pyp+ is much higher, compared with exoS+ and exoY+. Molecular epidemiological detection revealed that 2 of 3 extensive-resistance strains of Pa were the same clone, but another one had 96.3% of homology with them.

CONCLUSIONThe above mentioned 34.21% of Pa isolated from children carried virulence factors toxA, norA, exoS, exoY, pyp and exoU. The strains with exoU/pyp had rather high resistance. The strains with pyp had strong toxicity, they easily cause generalized infection, the patients with them had very high mortality.

ADP Ribose Transferases ; genetics ; Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; Carrier State ; epidemiology ; microbiology ; Child ; Drug Resistance, Multiple, Bacterial ; genetics ; Exotoxins ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Pseudomonas Infections ; epidemiology ; genetics ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Virulence Factors ; genetics

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.
Animals ; Bacterial Toxins/genetics/metabolism ; Cat Diseases/epidemiology/*microbiology ; Cats ; Chromosomes, Bacterial/genetics/metabolism ; Dog Diseases/epidemiology/*microbiology ; Dogs ; Electrophoresis, Gel, Pulsed-Field/veterinary ; Enterotoxins/genetics/metabolism ; Exfoliatins/genetics/metabolism ; Exotoxins/*genetics/metabolism ; Hospitals, Animal ; Humans ; Medical Staff, Hospital ; Molecular Sequence Data ; Pets/microbiology ; Polymerase Chain Reaction/veterinary ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus/genetics/isolation & purification ; Staphylococcus intermedius/*genetics/*isolation & purification

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.
AMP Deaminase ; genetics ; Animals ; Anthrax ; pathology ; Antigens, Bacterial ; toxicity ; Bacterial Toxins ; toxicity ; Base Sequence ; Blotting, Western ; Cell Death ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Cells, Cultured ; Exotoxins ; toxicity ; Macrophages ; cytology ; drug effects ; enzymology ; Mice ; Molecular Sequence Data ; Polymerase Chain Reaction

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.
ADP Ribose Transferases/*pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Bacterial Toxins/*pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell/drug therapy/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Chromatography, Liquid ; Cyclin B/metabolism ; Cyclin-Dependent Kinase 2/metabolism ; Drug Resistance, Neoplasm/*drug effects ; E2F1 Transcription Factor/metabolism ; Electrophoresis ; Exotoxins/*pharmacology ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Mouth Neoplasms/drug therapy/*metabolism ; Tandem Mass Spectrometry ; Tumor Suppressor Protein p53/metabolism ; Virulence Factors/*pharmacology

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.
ADP Ribose Transferases ; genetics ; Animals ; Bacterial Toxins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Exotoxins ; genetics ; Female ; Gene Expression ; Immunization ; Influenza A virus ; immunology ; physiology ; Lung ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Matrix Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Virulence Factors ; genetics

OBJECTIVETo investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.

METHODSRecombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.

RESULTSThe tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.

CONCLUSIONImmunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.

ADP Ribose Transferases ; genetics ; Animals ; Apoptosis ; Bacterial Toxins ; genetics ; Bone Neoplasms ; metabolism ; pathology ; Caspase 6 ; genetics ; metabolism ; Cell Line, Tumor ; Exotoxins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Random Allocation ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; Virulence Factors ; genetics

OBJECTIVETo investigate the pathogen causing soft-tissue pyogenic infection in neonate.

METHODSThe isolates of Staphylococcus aureus were obtained from liquor puris and blood by routine method. The Automated Microbiology Analyzer was used for identification and antimicrobial susceptibility test of the isolates. Panton-Valentine leukocidin (PVL) genes were determined by multiplex PCR in the isolates of Staphylococcus aureus. Multilocus sequence typing (MLST) was used to determine the sequence types (STs) of the isolates. The genotypes of SCCmec were also determined by another multiplex PCR in the isolates of methicillin-resistant Staphylococcus aureus (MRSA).

RESULTSIn 3 cases of neonate with soft-tissue pyogenic infection, 2 strains of Staphylococcus aureus isolated from liquor puris in 2 cases. 2 strains of Staphylococcus aureus were isolated from liquor puris and blood from another case. All 4 isolates were methicillin-resistant Staphylococcus aureus (MRSA) strains carrying PVL genes. Their SCCmec types were SCCmec IIIA. The STs of 4 isolates were ST88. The antimicrobial-resistance profile of the isolates were the same except erythromycin.

CONCLUSIONSoft-tissue pyogenic infection in the 3 neonates was caused by the same clone of MRSA carrying PVL genes.

Bacterial Toxins ; genetics ; Exotoxins ; genetics ; Humans ; Infant, Newborn ; Leukocidins ; genetics ; Male ; Methicillin-Resistant Staphylococcus aureus ; genetics ; Multilocus Sequence Typing ; Soft Tissue Infections ; microbiology ; Staphylococcal Infections ; microbiology