Enzymatic properties of cloacin DF13 and kinetics of ribosome inactivation.

1. The cloacin DF13-induced inactivation of ribosomes in vitro can be described as an enzyme-catalyzed reaction according to the Michaelis-Menten equation. Most probably the cloacin acts as a unique endoribonuclease. 2. At pH 7.8 and 37 degrees C the Km value for the reaction of cloacin DF13 with ribosomes is 13.2 - 10(-6) M. If under these conditions the reaction mixture is supplemented with all components necessary for protein synthesis, the Km changes to 17.7 - 10(-6) M. 3. The in vitro activity of cloacin DF13 has a temperature optimum of 43 degrees C at pH 7.8 and a pH optimum of 8.4 at 37 degtees C. 4. Experiments with cloacin DF13-immunity protein as an inhibitor of the cloacin activity in vitro have indicated that the immunity protein might be considered as a non-competitive and virtually "irreversible" inhibitor.

The use of mutants in the study of structure-function relationships in cloacin DF13.

A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03 . immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 . immunity protein complex and wild-type cloacin complex showed no significant differences. From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity. Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with "immunity" but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82. The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.

Mode of action of the cloacin DF13-immunity protein.

1. Cells of Enterobacter cloacae harbouring the bacteriocinogenic factor Clo DF13 produce an immunity protein which inbhbits the in vitro activity of cloacin DF13. The amino acid composition of purified immunity protein was determined. 2. Experiments about the protection of ribosomes against cloacin DF13 in the presence of the immunity protein show that one molecule of immunity protein neutralized the activity of one molecule cloacin. 3. Direct and specific interaction of cloacin DF13 with the immunity protein has been demonstrated by the analysis of mixtures of both proteins on polyacrylamide gels and by changes in the fluorescence response of cloacin DF13-bound 1-anilinonaphthalene-8-sulfonate in the presence of immunity protein.

Effects of temperature on the activity of cloacin DF13 and cloacin DF13-immunity protein.

During the interaction of cloacin DF13 and sensitive cells the cloacin molecules display different functions which can be distinguished on the basis of their heat-sensitivity. Binding to cell envelope receptors, binding of immunity protein and in vitro inactivation of ribosomes are heat-stable functions in contrast with the entire killing action in vivo. Cloacin DF13-immunity protein appears to be a heat-stable inhibitor of the fibosome inactivation caused by cloacin DF13.

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli.

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli.

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99.

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGc alpha-3Gal beta 4Glc beta Cer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well as thus explain the resistance of adult pigs to infection with E. coli K99.

Characterization of gangliosides of epithelial cells of calf small intestine, with special reference to receptor-active sequences for enteropathogenic Escherichia coli K99.

Glycolipids were prepared from epithelial cells of the small intestine of a newborn calf and assayed for Escherichia coli K99 binding activity on thin-layer chromatograms and in microtiter wells. The bacteria did not bind to any of the non-acid glycolipids, while in the acid fraction several binding-positive glycolipids were detected. The acid glycolipids were isolated and characterized by mass spectrometry, proton NMR spectroscopy and other methods. The following gangliosides were identified, mainly from the epithelial cells from the upper part of the small intestine: NeuAc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuAc-GM3), NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuGc-GM3), GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM2), Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM1), and NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GD1a). A positive binding was demonstrated to NeuGc-GM3, NeuGc-GM2, and NeuGc-GD1a, while NeuAc-GM3 and NeuGc-GM1 were negative. The binding pattern differed somewhat for total acid glycolipids of epithelial cells from three different parts of the small intestine. Based on binding preferences of E. coli K99 to a number of glycolipids of various origins, in comparison with calculated minimum energy conformations, a binding epitope was delineated.

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.

Protection against haemorrhagic septicaemia induced by vaccination of buffalo calves with an improved oil adjuvant vaccine.

An experimental oil adjuvant vaccine was developed against haemorrhagic septicaemia, a disease of cattle and buffalo caused by Pasteurella multocida serotype B and E. Mineral oil, Mercol 52, was used as adjuvant together with Span 85 and Tween 85 as emulsifiers. The vaccine was evaluated by single dose intramuscular immunisation of 1-2 year old buffalo calves. IgG and IgM class antibodies were determined by ELISA. The group of animals immunised with the experimental oil adjuvant vaccine showed a high titre of the IgG class of antibodies measured at 300 days post vaccination. To compare the protective efficacy of the vaccine with the commonly used broth bacterin, another group of buffalo calves was immunised by broth bacterin. This group showed a low level of IgG antibodies. Protection was assessed by challenge with 10(9) viable bacteria of P. multocida type B:2,5 administered subcutaneously, 250 days post vaccination. Animals vaccinated with the experimental oil adjuvant vaccine were fully protected. The other groups of animals, vaccinated with broth bacterin or used as control (non-vaccinated), developed symptoms of haemorrhagic septicaemia. A strong relationship between IgG but not IgM class antibody level and resistance to challenge was observed. The experiment demonstrated that the experimental oil adjuvant vaccine was superior to broth bacterin in providing protection against experimental haemorrhagic septicaemia in young buffalo calves beyond 250 days.

Calf small intestine receptors for K99 fimbriated enterotoxigenic Escherichia coli.

Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae. The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae. Gangliosides capable of binding K99 fimbriated E. coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG. No binding was detected to NeuAc-GM3 and NeuGc-GM1.

Functioning of a hybrid BRP-beta-lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells.

The gene encoding a hybrid BRP-Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of beta-lactamase (Bla) was subcloned in the expression vector pEB112. A similar construct was made using a mutant gene encoding a BRP-Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue. The expression, processing, functioning and subcellular localization of the 'wild-type' and mutant hybrid protein at high-level expression conditions were studied. The 'wild-type' BRP-Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itself; the protein was able to bring about the release of cloacin DF13 and caused apparent cell-lysis after high-level synthesis. The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell-lysis only after strong induction.

Control of temperature-dependent synthesis of K99 fimbriae.

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.

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.

Vacuolating cytotoxic activity of Pasteurella multocida causing haemorrhagic septicaemia in buffalo and cattle.

The toxic activity of Pasteurella multocida strains which cause haemorrhagic septicaemia (HS) in buffalo and cattle was examined in a mouse model. Mice were injected intraperitoneally with 10(2) cells of P. multocida serotype B:2,5. Electron microscopy of peritoneal macrophages obtained 6 h after injection revealed strong induction of cytoplasmic vacuolation, macrophage lysis and death. In vitro experiments with the mouse macrophage cell line RAW 264 incubated with cultures of various HS- and non-HS-associated strains of P. multocida or with culture supernatants revealed macrophage vacuolation when HS-associated strains were used. On pre-incubation of the strains with antiserum obtained from buffalo infected with P. multocida serotype B:2,5 no vacuolation was observed. These results are indicative of the presence of vacuolating cytotoxic activity in HS-associated strains of P. multocida.

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.

Safety and efficacy of an oil-adjuvant vaccine against haemorrhagic septicaemia in buffalo calves: cross-protection between the serotypes B:2,5 and E:2,5.

The safety, efficacy and duration of immunity of an improved oil-adjuvant vaccine against haemorrhagic septicaemia, containing inactivated cells of Pasteurella multocida serotype B:2,5, were tested in young buffalo calves in Pakistan. For safety testing, five buffalo calves were vaccinated intramuscularly with twice the normal dose, and six weeks later with a normal dose. Except for a transient rise in rectal temperature at six hours after the vaccinations, no systemic reactions were observed. The buffaloes remained in good condition and had a normal appetite. No local reactions were observed at the injection site. For efficacy testing two trials were carried out. In the first, buffalo calves were vaccinated intramuscularly either with two doses two-and-a-half months apart, or with a single dose, or left unvaccinated. They were challenged subcutaneously with virulent P multocida after eight, 13 or 15 months. After challenge at eight months the four buffaloes given two doses and the buffalo given one dose were protected, whereas the control animal developed the typical signs of the disease. After the challenges at 13 and 15 months, the vaccinated animals were still protected whereas the control animals died. In the second trial, buffalo calves were vaccinated intramuscularly either with two doses two months apart, or with a single dose at two months or left unvaccinated. The buffaloes were challenged after eight or 14 months. After challenge at eight months the four control animals died, whereas three of the four buffaloes given a single dose were protected. After challenge at 14 months, the three control animals died, whereas four of the five buffaloes given two doses and both the buffaloes given a single dose were protected. To test for cross-protection against the heterologous serotypes E:2,5 and B:3,4, groups of mice were vaccinated once or left unvaccinated. Four weeks later, the vaccinated and control groups were challenged with a dilution series of the different challenge cultures. The vaccine appeared to induce protection against challenge with different strains of serotypes B:2,5 and E:2,5 but not against strains of serotype B:3,4.