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Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein.
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
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2006 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, Vol. 55, no 7, 931-942 p.Article in journal (Refereed) Published
Abstract [en]

In a search for novel bioactive cell surface structures of periodontal pathogens, it was found that sera from two patients with Actinobacillus actinomycetemcomitans-associated infections reacted strongly at 17 kDa on immunoblots of A. actinomycetemcomitans outer-membrane protein (OMP) preparations. The 17 kDa antigen was also recognized by anti-CsgA (Escherichia coli curli major subunit) antibody. The 17 kDa A. actinomycetemcomitans protein was identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by two-dimensional immunoblotting and subsequent sequence analysis by mass spectrometry and bioinformatics tools. AaPAL was an OMP and a lipoprotein, and it had an OmpA-like domain. In a group of middle-aged subjects (n = 26), serum reactivity to AaPAL was associated with the presence of periodontitis but not with the oral detection of A. actinomycetemcomitans. Both human sera and rabbit antisera against three different types of antigens, the gel-purified AaPAL, A. actinomycetemcomitans whole-cell antigens, and CsgA, recognized putative PALs of oral haemophili in addition to AaPAL. The results demonstrated that the novel AaPAL is a conserved bacterial lipoprotein. It is expressed in vivo and is strongly immunoreactive. The antigenic cross-reactivity found between AaPAL and oral haemophili may enhance local and systemic immuno-inflammatory reactions in periodontitis.

Place, publisher, year, edition, pages
2006. Vol. 55, no 7, 931-942 p.
URN: urn:nbn:se:umu:diva-12987DOI: 10.1099/jmm.0.46470-0PubMedID: 16772422OAI: diva2:152658
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2011-04-14Bibliographically approved
In thesis
1. Vesicle-independent extracellular release and bioactivity of peptidoglycan-associated lipoprotein from Aggregatibacter actinomycetemcomitans
Open this publication in new window or tab >>Vesicle-independent extracellular release and bioactivity of peptidoglycan-associated lipoprotein from Aggregatibacter actinomycetemcomitans
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden.

We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation.

When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate.

To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array.

A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism.

The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

Place, publisher, year, edition, pages
Umeå: Odontologi, 2007. 63 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 101
Peptidoglycan-associated lipoprotein, outer membrane proteins, immunoproteomics, periodontitis, cytokines, Aggregatibacter actinomycetemcomitans.
National Category
urn:nbn:se:umu:diva-1419 (URN)978-91-7264-441-0 (ISBN)
Public defence
2007-11-23, Hall C, Tandläkahögskolan, 9 Tr, Umeå, 13:00 (English)
Available from: 2007-11-06 Created: 2007-11-06 Last updated: 2009-05-26Bibliographically approved

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Borén, ThomasArnqvist, AnnaAsikainen, Sirkka
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Oral MicrobiologyDepartment of Medical Biochemistry and Biophysics
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