Biomedical subjects

B Oudega

Publications and source records attributed to B Oudega.

At least 55 records · Page 3Linked to original sources

PubMed1994

Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: effect of LPS core mutations.

Tn10 transposon mutagenesis of Escherichia coli producing K99 fimbriae was carried out to identify host factors involved in regulation of biosynthesis of fimbriae. Two chromosomal mutants were obtained that showed a strongly reduced cell surface expression of K99 fimbriae upon colony blotting and ELISA. Analysis by inversed PCR and nucleotide sequencing showed that one mutant (EP14) contained the Tn10 transposon in rfaQ, affecting the expression of the rfaQGP gene cluster, whereas the other mutant (EP35) was affected in a, to date, unknown region of the genome. Immunoblotting analysis confirmed a Rd1 type of LPS of mutant strain EP14. These findings for the first time indicated an effect of LPS core biosynthesis on the biogenesis of fimbriae at the cell surface. Preliminary experiments indicated that K99 major subunits, in contrast to K88 subunits, strongly bind LPS molecules.

Antibodies, Monoclonal↗

PubMed1994

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY.

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha-subunit of the eukaryotic SRP receptor ('docking protein') in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta-lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY-depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre-beta-lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.

Bacterial Outer Membrane Proteins↗

PubMed1994

Morphological appearances of K88ab fimbriae and optical diffraction analysis of K88 paracrystalline structures.

K88ab fimbriae are filamentous protein structures at the surface of certain enterotoxigenic Escherichia coli strains. Electron microscopy analysis of K88ab fimbriae showed that these structures have different morphological appearances dependent on the medium in which cells expressing these fimbriae or in which purified fimbriae were suspended. Thin and curled structures, thin and flexible fimbriae, a wider and rigid form of the fimbriae, and, in addition, paracrystalline structures were detected. Optical diffraction analysis of the paracrystalline structures indicated a helical conformation of K88ab fimbriae.

Antigens, Bacterial↗

PubMed1994

Binding of bovine transferrin by Pasteurella multocida serotype B:2,5, a strain which causes haemorrhagic septicaemia in buffalo and cattle.

Pasteurella multocida serotype B:2,5, which causes haemorrhagic septicaemia in buffalo and cattle, was examined for the presence of transferrin-binding proteins. An 82-kDa iron-regulated outer membrane protein was found which specifically binds bovine transferrin. In contrast, P. multocida serotype B:3,4, associated with haemorrhagic septicaemia in feral ruminants, did not express transferrin-binding proteins. These results might indicate a role for transferrin binding in the pathogenesis of haemorrhagic septicaemia in cattle and buffalo.

Animals↗

PubMed1994

Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein.

The pCloDF13-derived bacteriocin release protein (BRP) is synthesized as a prelipoprotein with a signal peptide which remains stable after processing. This signal peptide accumulates in the cytoplasmic membrane and is, together with the mature BRP, required for efficient release of cloacin DF13. We investigated the structural requirements for stability of the BRP signal peptide by constructing hybrid signal peptides consisting of parts of the BRP and Lpp signal peptides. Signal peptide stability was investigated by pulse-labelling and pulse-chase experiments. To study the functioning of the BRP signal peptide, the hybrid constructs were tested for their ability to promote BRP-mediated cloacin DF13-release and their ability to affect the viability of the host cells. The results obtained suggest that the N-terminal part of the BRP signal peptide together with the C-terminal alanine residue are important for stability. When expressed as a separate entity, all mutant signal peptides that contain a part of the BRP signal peptide are capable of affecting cell viability. The results indicated a possible correlation between stability of the BRP signal peptide and cloacin DF13-release.

Alanine↗

PubMed1994

Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone-K88 subunit complex is a heterotrimer.

The interaction of FaeE, a periplasmic chaperone involved in K88 biosynthesis, and the major fimbrial subunit FaeG was investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1. Cells expressing the subcloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion-exchange fast-protein liquid chromatography. SDS-PAGE, native gel electrophoresis, immunoblotting and determination of the N-terminal amino acid sequences and the molar ratio of the N-terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross-linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer.

Antigens, Bacterial↗

PubMed1993

A lipoprotein signal peptide plus a cysteine residue at the amino-terminal end of the periplasmic protein beta-lactamase is sufficient for its lipid modification, processing and membrane localization in Escherichia coli.

By genetic exchange and in vitro mutagenesis a hybrid beta-lactamase was constructed that contained the pCloDF13-encoded bacteriocin release protein signal peptide plus a cysteine residue coupled to the mature portion of beta-lactamase. Immunoblotting, labelling with [3H]palmitate in the presence and absence of globomycin, and pulse-chase experiments revealed that this hybrid construct is modified with lipid and processed into a lipid-modified beta-lactamase. Subcellular localization studies revealed that this hybrid is localized both in the cytoplasmic and outer membranes of Escherichia coli cells. A mutant derivative with an incomplete lipobox (LVG instead of LVAC+1) was not processed and was found in the cytoplasmic membranes.

Amino Acid Sequence↗

PubMed1993

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.

Adenosine Triphosphatases↗

PubMed1992

Characterization and detection of the 40 kDa fimbrial subunit of Bacteroides fragilis BE1.

The amino acid composition and amino-terminal amino acid sequence of the 40 kDa fimbrial subunit of Bacteroides fragilis were determined. No similarity with other known fimbrial subunits or protein sequences was found. The isoelectric point of the fimbriae was determined to be 3.8. The acidic nature of these fimbriae is in agreement with the amino acid composition of the fimbrial subunit. An IgM monoclonal antibody, raised against the denaturated subunit of strain BE1 fimbriae, reacted with native BE1 fimbriae and appeared to be strain specific. Bacteroides fragilis strain BE38 expressed fimbriae which did not react with the polyclonal or monoclonal antibodies directed against strain BE1 fimbriae.

Amino Acid Sequence↗

PubMed1992

The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli.

The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over-expression of the BRP signal peptide was lethal and caused 'lysis'. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane. These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.

Amino Acid Sequence↗

PubMed1992

Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.

Amino Acid Sequence↗

PubMed1991

Localization and function of FanH and FanG, minor components of K99 fimbriae of enterotoxigenic Escherichia coli.

Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively. Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae. Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy. A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins. Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor. Cells defective in FanH produced 1-2% of the K99 fimbriae as compared with wild-type K99 producing cells. These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor. Apparently, FanG and FanH are not required for binding the K99 receptor. These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF/FanG and FanF/FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.

Adhesins, Escherichia coli↗

PubMed1991

Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli.

The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein. Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell-envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.

Amino Acid Sequence↗

PubMed1991

Functioning of the stable signal peptide of the pCloDF13-encoded bacteriocin release protein.

The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.

Amino Acid Sequence↗

PubMed1990

The penultimate tyrosine residue of the K99 fibrillar subunit is essential for stability of the protein and its interaction with the periplasmic carrier protein.

The role of the penultimate and conserved tyrosine residue of the K99 major fibrillar subunit (FanC) in fibrillae biosynthesis and functioning was investigated. By using oligonucleotide-directed in vitro mutagenesis the TAT codon of tyrosine-158 of fanC was changed into a TAG stop codon. The mutant fanC gene encoded a truncated major subunit lacking the two carboxyl-terminal amino acid residues. Furthermore, the tyrosine residue (position 158) was replaced by a serine residue or by a glutamic acid residue. The effect of these mutations on the expression and binding capacity of K99 fibrillae was investigated by using an ELISA, an haemagglutination assay, Escherichia coli minicells and suppressor strains. All mutations completely blocked K99 fibrillae biosynthesis and haemagglutination activity. The mature form of the truncated mutant FanC polypeptide could not be detected in minicells, but its precursor was expressed at a normal level. The results showed that the penultimate tyrosine residue is essential for the expression of mature fibrillar subunits and suggested a function in the interaction with the periplasmic transport protein FanE.

Amino Acid Sequence↗

PubMed1990

K88 fimbriae as carriers of heterologous antigenic determinants.

The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity. To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae. Amino acid residues 163-173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting. Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted. Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163-173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost. Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.

Amino Acid Sequence↗

PubMed1990

Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coli.

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.

Amino Acid Sequence↗

PubMed1990

Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen.

S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the influence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme beta-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-lac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain 536WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain 536WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate had a strong influence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.

Adhesins, Escherichia coli↗