Adhesins, Escherichia coli ; genetics ; Bacterial Typing Techniques ; Escherichia coli Proteins ; genetics ; Fimbriae Proteins ; genetics ; Genes, Bacterial ; Genotype ; Uropathogenic Escherichia coli ; classification ; genetics

Escherichia coli (E. coli) has ability to express thin aggregative fimbriae, known as curli, on the cell surface. Previously, a few example of curli expression in serogroup O157:H7 of enterohemorrhagic E. coli (EHEC) were reported, compared to other E. coli groups. However, significance of curliation in the EHEC pathobiology has not been described well in the literature. A highly curliated O157:H7 strain was used in this study in order to elucidate role of curliation in EHEC adherence to cultured HEp-2 cells. The expression of curli in the EHEC isolate was consistent with strong positive indication of Congo-red (CR) binding and formation of clumps in the bottom of the tube containing Luria-Bertani (LB) broth when cultured overnight at 37 degress C. A few CR-binding negative (CR-) colonies occurred spontaneously within the population of CR+ isolate. The CR+ EHEC showed massive aggregative adhesion pattern, whereas the spontaneous CR- strain showed typical localized adherence on HEp-2 cells. Electron microscopy confirmed highly curliated bacteria in the CR+ EHEC sample. Interestingly, the curliation disappeared in a msbB1 and msbB2 double mutant derived from the CR+ EHEC. These results suggest that the compromised outer membrane integrity caused by msbB mutations may abrogate curli production in the CR+ EHEC harbouring penta-acylated lipid A structure in their outer membrane.
Bacterial Adhesion/*physiology ; Bacterial Outer Membrane Proteins/*physiology ; Cell Aggregation ; Cells, Cultured ; Epithelial Cells/*microbiology ; Escherichia coli O157/pathogenicity/*physiology/ultrastructure ; Fimbriae, Bacterial/*metabolism/ultrastructure ; Humans ; Larynx/cytology/microbiology ; Microscopy, Electron

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.
Adhesins, Escherichia coli ; metabolism ; Amino Acid Sequence ; Animals ; Enterotoxigenic Escherichia coli ; metabolism ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Fimbriae Proteins ; metabolism ; Fimbriae, Bacterial ; chemistry ; Histones ; genetics ; metabolism ; Host-Pathogen Interactions ; Intestinal Mucosa ; metabolism ; Molecular Sequence Data ; Receptors, Cell Surface ; genetics ; metabolism ; Swine

To investigate the inflammatory cytokine production of human epithelial cell lines stimulated with uropathogenic E. coli strains showing 3 different adherence patterns, differential expression of inflammatory cytokine (IL-1a, IL-lB, IL-8, TNFa, and TGFB) mRNA were detected by RT-PCR. IL-1a, IL-1B, IL-8, and TGFB mRNAs constitutively expressed in epithelial cell lines, but not TNFa. The expression of IL-1a and IL-1B mRNA was increased in J-82 cells stimulated with E. coli strains showing DA, LA, or AggA pattern. The expression of IL-8 mRNA was increased, whereas TGFj3 mRNA was decreased in J-82 cells stimulated with E. coli strain showing AggA.pattern. Treatment with crude bacterial adhesins (CBA) isolated from E. coli strains showing DA or LA pattern increased IL-la, IL-lB, IL-S, and TGFj3 mRNA expressions in J-82 cells and HeLa cells. IL-la, IL-lB, and TGFB mRNA expressions were decreased in epitheUal cells stimulated with CBA from E. coli strain showing AggA pattern, whereas IL-8 mRNA expression was significantly increased. The expressions of cytokine mRNAs showed little differences between epithelial ceRs used, but great differences between CBA from DA or LA and AggA strain. LPS stimulation was little changed cytokine mRNA expressions in epithelial cells. This study suggests that cytokine gene expression of epithelial cells by the bacterial stimulation mainly depends on the bacterial adhesins recognized by the respective receptors of epithelial cells.
Adhesins, Bacterial ; Epithelial Cells* ; Gene Expression ; HeLa Cells ; Humans ; Interleukin-8 ; RNA, Messenger ; Uropathogenic Escherichia coli*

Bacterial Outer Membrane Proteins ; chemistry ; genetics ; physiology ; Bacteriophages ; chemistry ; Humans ; Receptors, Virus ; chemistry ; Vibrio cholerae ; virology

<b>OBJECTIVEb>To evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs).

<b>METHODSb>CJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD.

<b>RESULTSb>CJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD.

<b>CONCLUSIONb>Improved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.

Bacterial Outer Membrane Proteins ; analysis ; metabolism ; Campylobacter jejuni ; growth & development ; metabolism ; Culture Media

<b>OBJECTIVEb>To investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

<b>METHODSb>Mutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

<b>RESULTSb>When grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

<b>CONCLUSIONb>YpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

PURPOSE: Porphyromonas gingivalis and Tannerella forsythia have been implicated as the major etiologic agents of periodontal disease. These two bacteria are frequently isolated together from the periodontal lesion, and it has been suggested that their interaction may increase each one's virulence potential. The purpose of this study was to identify proteins on the surface of these organisms that are involved in interbacterial binding. METHODS: Biotin labeling of surface proteins of P. gingivalis and T. forsythia and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed to identify surface proteins involved in the coaggregating activity between P. gingivalis and T. forsythia. RESULTS: It was found that three major T. forsythia proteins sized 161, 100, and 62 kDa were involved in binding to P. gingivalis, and P. gingivalis proteins sized 35, 32, and 26 kDa were involved in binding to T. forsythia cells. CONCLUSIONS: LC-MS/MS analysis identified one T. forsythia surface protein (TonB-linked outer membrane protein) involved in interbacterial binding to P. gingivalis. However, the nature of other T. forsythia and P. gingivalis surface proteins identified by biotin labeling could not be determined. Further analysis of these proteins will help elucidate the molecular mechanisms that mediate coaggregation between P. gingivalis and T. forsythia.
Bacteria ; Bacterial Outer Membrane Proteins ; Biotin ; Biotinylation ; Forsythia* ; Mass Spectrometry ; Membrane Proteins ; Membranes ; Periodontal Diseases ; Periodontitis ; Porphyromonas gingivalis* ; Porphyromonas* ; Virulence

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD(50) of 4.0 x 10(5) CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD(50) of 1.2 x 10(12) CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.
Adhesins, Escherichia coli/*genetics/*metabolism ; Animals ; Chickens ; Escherichia coli/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Plasmids/*genetics/metabolism ; Poultry Diseases/*microbiology ; Sepsis/*microbiology

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh(s)) and a 33 kDa beta-domain (Tsh(beta)). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh(s) (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh(beta) (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37degrees C or 42degrees C. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26degrees C, 37degrees C, or 42degrees C, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37degrees C in mucin agar, and it was not detected when the bacteria were grown at 26degrees C in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.
Adhesins, Escherichia coli/*metabolism ; Brazil ; Escherichia coli/*metabolism ; *Gene Expression Regulation, Bacterial ; Hemagglutination ; Mucins/metabolism ; Protein Transport ; Recombinant Proteins/isolation & purification/metabolism