RESUMO
An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUC19. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Ácidos Graxos/imunologia , Haemophilus influenzae/imunologia , Acilação , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Eletroforese em Gel de Poliacrilamida , Haemophilus influenzae/genética , Imunização , Dados de Sequência Molecular , Ácido Palmítico , Ácidos Palmíticos , Sinais Direcionadores de Proteínas/genética , Coelhos , Ratos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.
Assuntos
Proteínas da Membrana Bacteriana Externa , Haemophilus influenzae/análise , Lipoproteínas/isolamento & purificação , Peptidoglicano/isolamento & purificação , Proteoglicanas , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Detergentes , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Escherichia coli/análise , Proteínas de Escherichia coli , Ácidos Graxos/análise , Temperatura Alta , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência do Ácido Nucleico , SolubilidadeRESUMO
The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5beta-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [35S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [14C]glutamate and [14C]formate was observed, leaving the origin of the five-carbon unit still in doubt.
Assuntos
Salmonella typhimurium/metabolismo , Enxofre/metabolismo , Tiamina/biossíntese , Cisteína/metabolismo , Modelos QuímicosRESUMO
The mechanism of biosynthesis of 4-methyl-5-beta-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various 14C-labeled precursors was determined. We found that;e1Me-14C]methionine, [2-14C]methionine, [U-14C]alanine, and [2-14C]glycine were not incorporated whereas [2-14C]tyrosine was incorporated. Degradation of the 4-methyl-5-beta-hydroxyethyl thiazole obtained after [2-14C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-beta-hydroxyethyl thiazole biosynthesis.