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1.
Mol Cell Biochem ; 165(2): 111-20, 1996 Dec 20.
Article in English | MEDLINE | ID: mdl-8979259

ABSTRACT

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro. Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [32P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and 32P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 microM of a synthetic peptide corresponding to the sequence around Ser1029 of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser1029 of the STaR/GC remains a candidate phosphorylation site by PKC.


Subject(s)
Colon/enzymology , Escherichia coli/metabolism , Guanylate Cyclase/metabolism , Protein Kinase C/metabolism , Receptors, Peptide/metabolism , Bacterial Toxins/metabolism , Binding Sites , Cell Line , Enterotoxins/metabolism , Enzyme Activation , Escherichia coli Proteins , Humans , Peptides/chemical synthesis , Peptides/metabolism , Phorbol 12,13-Dibutyrate/pharmacology , Phosphorylation , Precipitin Tests , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled
2.
Infect Immun ; 64(6): 1950-5, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8675292

ABSTRACT

Haemophilus ducreyi expresses an 18,000-molecular-weight outer membrane protein that contains a conserved surface-exposed epitope recognized by monoclonal antibody 3B9. Monoclonal antibody 3B9 cross-reacts with proteins of similar molecular weight found in many Haemophilus sp. strains, including P6, a candidate vaccine for Haemophilus influenzae. The gene encoding the 18,000-molecular-weight outer membrane protein was identified by screening a lambdagt11 genomic library with 3B9. The coding sequence of the gene was localized to a 471-bp open reading frame, designated pal (peptidoglycan-associated lipoprotein). Translation of pal predicted a mature polypeptide with a molecular weight of 15,000 that had extensive homology with P6 and Escherichia coli PAL. The predicted signal peptide had features characteristic of a prokaryotic lipoprotein, and processing of PAL was sensitive to globomycin in H. ducreyi. The sequences encoding mature H. ducreyi PAL were subcloned into the vector pRSET B and expressed as a polyhistidine-containing fusion protein that bound 3B9. In Western blot (immunoblot) analysis, serum samples obtained from healthy subjects and patients with chancroid or other genital ulcer diseases contained antibodies to purified PAL. Antibodies that bound to PAL were removed by absorption with a lysate of Haemophilus sp. antigens, suggesting that patients with chancroid do not develop an H. ducreyi-specific antibody response to PAL.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Haemophilus ducreyi/chemistry , Lipoproteins/chemistry , Peptidoglycan/chemistry , Proteoglycans , Amino Acid Sequence , Antibodies, Bacterial/blood , Base Sequence , Chancroid/immunology , Escherichia coli Proteins , Haemophilus ducreyi/immunology , Humans , Lipoproteins/immunology , Molecular Sequence Data , Molecular Weight , Peptidoglycan/immunology , Sequence Homology, Amino Acid
3.
Infect Immun ; 62(11): 4909-14, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7927771

ABSTRACT

Moraxella (Branhamella) catarrhalis, a mucosal pathogen closely related to Neisseria species, is a prominent cause of otitis media in young children and lower respiratory tract infections in adults. In this study, we investigated whether M. catarrhalis can compete for iron bound to human transferrin or human lactoferrin in a manner similar to that utilized by Neisseria meningitidis and Neisseria gonorrhoeae. Our studies demonstrated that M. catarrhalis obtains iron from these serum carrier proteins and also maintains growth with ferric nitrate in vitro. Furthermore, we report that when M. catarrhalis is grown under iron-limited conditions, the bacteria express new outer membrane proteins that are not detected in membranes of organisms cultured in an iron-rich environment. We have shown that these are iron-repressible proteins since they are not induced by other environmental stresses and the expression of these proteins is repressed when a source of iron is provided for iron-limited bacteria. The iron-repressible proteins are expressed in the absence of any detectable siderophore production. These iron-repressible proteins may be important for the acquisition and utilization of iron in vivo, which could allow M. catarrhalis to colonize and survive on human mucosal surfaces.


Subject(s)
Iron/metabolism , Lactoferrin/metabolism , Moraxella catarrhalis/growth & development , Transferrin/metabolism , Antibodies, Bacterial/immunology , Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Biological Transport , Humans , In Vitro Techniques , Molecular Weight , Moraxella catarrhalis/metabolism , Siderophores/metabolism
4.
Infect Immun ; 61(4): 1346-51, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8454337

ABSTRACT

Haemophilus ducreyi contains a major outer membrane protein (MOMP) whose apparent molecular weight is 39,000 to 42,000 for all strains tested. Two monoclonal antibodies (MAbs), designated 9D12 and 2C7, bound to the MOMP for all strains of H. ducreyi tested. As reported previously, MAb 9D12 was H. ducreyi specific (E. J. Hansen and T. A. Loftus, Infect. Immun. 44:196-198, 1984). MAb 2C7 bound to all members of the family Pasteurellaceae tested, suggesting that the MAbs bound to distinct epitopes on the MOMP. The MOMP was purified by extraction of whole cells with Zwittergent and ion-exchange chromatography. A peak eluted from a cation-exchange column contained three bands. All three species bound both MAbs, and the fraction yielded a single N-terminal amino acid sequence, suggesting that the bands represented different conformations of the MOMP. The MOMP was heat modifiable, contained two cysteine residues, and was cationic at pH 8.0, features not usually associated with classical porin proteins. The N-terminal amino acid sequence and total amino acid content of the MOMP were homologous to the OmpA proteins of members of the family Enterobacteriaceae and the OmpA-like protein of Actinobacillus actinomycetemcomitans. An OmpA-specific polyclonal serum bound to the MOMP, and MAb 2C7 bound to Haemophilus influenzae protein 5, an OmpA-like protein, indicating that the MOMP was antigenically related to OmpA. These data indicated that the most abundant protein in the outer membrane of H. ducreyi was not a classical porin and belonged to the OmpA family of proteins.


Subject(s)
Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Haemophilus ducreyi/chemistry , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/immunology , Haemophilus ducreyi/immunology , Molecular Sequence Data , Peptide Mapping , Sequence Alignment
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