Antibody response of rabbits and cystic fibrosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa

doi:10.1006/mpat.1994.1004

Microbial Pathogenesis

Volume 16, Issue 1, January 1994, Pages 43-51

https://doi.org/10.1006/mpat.1994.1004 Get rights and content

Abstract

The aim of this work was to study the immunogenicity of an outer membrane (OM) protein (AIgE; 54 kDa) which is produced solely by mucoid, i.e. alginate-producing strains of P. aeruginosa. The source of AIgE used for our study was the mucoid strain CF3/M1 originally isolated from sputum of a cystic fibrosis (CF) patient. The purified non-denatured protein served as antigen to raise polyclonal monospecific anti-AIgE antibodies in rabbits and to assay sera from 41 cystic fibrosis (CF) patients for anti-AIgE antibodies. According to clinical protocols the sputa of 22 CF patients were positive for P. aeruginosa , 18 were negative and one case was unknown. Our ELISA studies showed that high titers of anti-AIgE antibodies (IgG) corresponded well with the infection status of the CF patients. None of 23 control sera derived from healthy volunteers contained significant levels of anti-AIgE antibodies. Thus the ELISA should be considered as a sensitive diagnostic tool for the early detection of mucoid P. aeruginosa infections in CF patients. Furthermore, we suggest to include AIgE in a multicomponent experimental vaccine for potential protection of non-colonized CF patients from colonization with mucoid P. aeruginosa.

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Cited by (25)

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2019, Journal of Molecular Biology
Citation Excerpt :
Some bacteria belonging to the Pseudomonas and Azotobacter genera also produce alginate. Bacterial alginates differ from those produced by brown algae in that they have little or no G-blocks and can be O-acetylated at some of the C-2 and C-3 carbons of the mannuronic acid residues [6–8]. Alginate lyases catalyze the degradation of alginate, targeting the glycosidic 1, 4 O-linkage via acid-base catalysis of β-elimination [9].
Alginate lyases, which are important in both basic and applied sciences, fall into ten polysaccharide lyase (PL) families. PL36 is a newly established family that includes 39 bacterial sequences and one eukaryotic sequence. Till now, the structures or catalytic mechanisms of PL36 alginate lyases have yet to be revealed. Here, we characterized a novel PL36 alginate lyase, Aly36B, from Chitinophaga sp. MD30. Aly36B is a polymannuronate specific endolytic alginate lyase. To probe the catalytic mechanism of Aly36B, the structures of wild-type Aly36B and its mutants (K143A/Y185A in complex with alginate tetrasaccharide and K143A/M171A with trisaccharide) were solved. The overall structure of Aly36B belongs to the β-jelly roll scaffold, adopting a typical β-sandwich fold. Aly36B contains a Ca²⁺, which is far away from the active center and plays an important role in stabilizing the structure of Aly36B. Based on structural and mutational analyses, the catalytic mechanism of Aly36B for alginate degradation was explained. During catalysis, Arg¹⁶⁹, Tyr¹⁸⁵, and Tyr¹⁸⁷ are responsible for neutralizing the negative charge of the substrate, and Lys¹⁴³ acts as both the catalytic base and the catalytic acid, which represents a new kind of catalytic mechanism of alginate lyases. Sequence alignment shows that these four residues involved in catalysis are highly conserved in all PL36 sequences, suggesting that PL36 alginate lyases may adopt a similar catalytic mechanism. Taken together, this study reveals the molecular structure and catalytic mechanism of a PL36 alginate lyase, broadening our knowledge on alginate lyases and facilitating future biotechnological applications of PL36 alginate lyases.
A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and AlgT/U activity results in the loss of alginate production
2012, Gene
Citation Excerpt :
Alginate overproducing strains have outer membrane protein profiles with a prominent 54-kDa protein that is not observed in nonmucoid strains (Goldberg and Ohman, 1987; Grabert et al., 1990). The identity of the 54-kDa protein has been shown to be the product of the algE gene (Mathee et al., 1999), which is transcribed as part of an 18-kb operon of biosynthetic genes and appears to encode the porin component of a secretory complex involved in polymer export to the bacterial surface (Chu et al., 1991; Hay et al., 2010; Rehm et al., 1994a, 1994b). The mobility of the AlgE protein in SDS-PAGE is variable (Grabert et al., 1990).
Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg⁺) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg⁺ due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.
Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation
2009, Journal of Biotechnology
The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361–387 and 393–416. Accordingly, the extended cytosolic loop could be located at positions 71–361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid–base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.
Alg44, a unique protein required for alginate biosynthesis in Pseudomonas aeruginosa
2006, FEBS Letters
Here the putative alginate biosynthesis gene alg44 of Pseudomonas aeruginosa was functionally assigned. Non-polar isogenic alg44 deletion mutants of P. aeruginosa were generated and did neither produce alginate nor released free uronic acids. No evidence for alginate enrichment in the periplasm was obtained. Alginate production was restored by introducing only the gene alg44. PhoA fusion protein analyses suggested that Alg44 is a soluble protein localized in the periplasm. Hexahistidine-tagged Alg44 was detected by immunoblotting. The corresponding 42.6 kDa protein was purified and identified by MALDI/TOF-MS analysis. Alg44 might be directly involved in alginate polymerization presumably by exerting a regulatory function.
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Regular ArticleAntibody response of rabbits and cystic fibrosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa

Abstract

Regular Article
Antibody response of rabbits and cystic fibrosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa