BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.
ADP Ribose Transferases/genetics ; Bacterial Proteins/*genetics ; Bacterial Toxins/*genetics ; Clostridium difficile/isolation & purification/*metabolism ; DNA, Bacterial/genetics/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Prospective Studies ; Real-Time Polymerase Chain Reaction ; Triose-Phosphate Isomerase/genetics

Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.
ADP Ribose Transferases ; genetics ; Bacterial Toxins ; genetics ; DNA, Bacterial ; analysis ; Exotoxins ; genetics ; Fluorescent Dyes ; Fluorometry ; methods ; Polymerase Chain Reaction ; methods ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; Sensitivity and Specificity ; Taq Polymerase ; Virulence Factors ; genetics

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.
ADP Ribose Transferases ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cell Proliferation ; drug effects ; Exotoxins ; genetics ; pharmacology ; Granzymes ; genetics ; pharmacology ; HeLa Cells ; Humans ; Recombinant Fusion Proteins ; pharmacology ; Virulence Factors ; genetics ; pharmacology

OBJECTIVETo study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).

METHODSOne hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.

RESULTSAmong the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.

CONCLUSIONSThe TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Secretion Systems ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Retrospective Studies ; Virulence

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.
ADP Ribose Transferases ; metabolism ; secretion ; Bacterial Proteins ; genetics ; metabolism ; secretion ; Bacterial Toxins ; metabolism ; Gene Expression Regulation, Bacterial ; Mutation ; Peptide Hydrolases ; genetics ; metabolism ; Pore Forming Cytotoxic Proteins ; genetics ; secretion ; Protease Inhibitors ; pharmacology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity

AIMTo investigate wether the corresponding protein of mono-ADP-ribosyltransferase 3 (ART3) mRNA is expressed in human testes and, if so, whether the expression is cell type-specific.

METHODSART3 mRNA was determined in human testes and sperm by reverse transcription-polymerase chain reaction (RT-PCR). The glycosyl-phosphatidylinositol linkage of ART3 was shown by treating ART3-transfected HEK-293-T cells with phospholipase C. Fluorescent activated cell sorter (FACS)-analyses were used to detect ART3 on mature spermatozoa and immunohistological studies to detect the protein in testes.

RESULTSART3 protein was shown to be present in testes. It was found on spermatocytes only. It was absent from spermatogonia, spermatids and spermatozoa. The absence of ART3 from spermatozoa was confirmed by FACS-analysis. ART3 protein was detected neither within a seminoma nor on Leydig cells.

CONCLUSIONHere we show for the first time that ART3 protein is expressed in testes in particular on spermatocytes, indicating that ART3 exerts a specific function only required at a particular stage of spermatogenesis.

ADP Ribose Transferases ; genetics ; Cell Line ; Flow Cytometry ; GPI-Linked Proteins ; Humans ; Male ; Membrane Proteins ; genetics ; Organ Specificity ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatocytes ; enzymology ; Spermatozoa ; enzymology ; Testis ; enzymology ; Transfection

OBJECTIVEPseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1.

METHODSBy allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis.

RESULTSPull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS.

CONCLUSIONOrf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Base Sequence ; Blotting, Western ; DNA Primers ; Electrophoresis, Polyacrylamide Gel ; Kinetics ; Molecular Chaperones ; genetics ; metabolism ; Protein Binding ; Pseudomonas aeruginosa ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo explore the relationship between genetic polymorphisms in apurinic/apyrimidinic endonuclease (APE1) and ADP ribosyltransferase (ADPRT) and individuals' susceptibility to chronic benzene poison ing (BP).

METHODSA case-control study was conducted. One hundred and fifty-two B P patients and 152 workers occupationally exposed to benzene without poisoning manifestations were investigated. The mismatched bases combined to create restriction site with restrained fragment length polymorphism technique (CRS-RFLP) was used for detecting the single nucleotide polymorphisms (SNPs) at Asp148Glu of APE1 gene and Val762Ala of ADPRT gene.

RESULTSThere was no significant difference in the distribution of genotypes of APE1Asp148Glu and ADPRTVal762Ala between the patients and the control groups. Compared with individuals having genotype of APE1Asp148Glu T/T without habit of alcohol consumption, there was a 4.13 times increased risk of BP for the alcohol user with genotype of APE1Asp148Glu T/T (OR = 4.13, 95% CI: 1.07 - 15.85, P = 0.03). The analysis of Logistic regression showed that smoking may play some role in modifying the risk of cironic benzene poisoning (OR = 0.33, 95% CI: 0.14 - 0.75, P = 0.01).

CONCLUSIONThe genetic polymorphisms in APE1Asp148Glu, ADPRTVal762Ala are not related to the risk of BP. Potential interaction is found between alcohol consumption and polymorphism of APE1Asp148Glu. Further study is needed to elucidate this interaction.

ADP Ribose Transferases ; Alcohol Drinking ; genetics ; Benzene ; poisoning ; Case-Control Studies ; Chronic Disease ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Genetic Predisposition to Disease ; Genotype ; Humans ; Occupational Exposure ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.
ADP Ribose Transferases ; biosynthesis ; genetics ; Bacterial Toxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Immunotoxins ; genetics ; isolation & purification ; Interleukin-10 ; biosynthesis ; genetics ; Pseudomonas aeruginosa ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Virulence Factors ; biosynthesis ; genetics