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Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form
Umeå University, Faculty of Medicine, Department of Odontology.
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2008 (Swedish)In: BMC Microbiology, ISSN 1471-2180, Vol. 28, no 8:18Article in journal (Refereed) Published
Abstract [sv]

Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 um), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 um) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSIONS: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

Place, publisher, year, edition, pages
2008. Vol. 28, no 8:18
National Category
URN: urn:nbn:se:umu:diva-8838DOI: 10.1186/1471-2180-8-18PubMedID: 18226201OAI: diva2:148509
Available from: 2008-02-18 Created: 2008-02-18 Last updated: 2011-08-29Bibliographically 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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Eneslätt, KjellOscarsson, JanWai, Sun NyuntChen, CAsikainen, Sirkka
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Department of OdontologyOral MicrobiologyDepartment of Molecular Biology (Faculty of Medicine)Umeå Centre for Microbial Research (UCMR)
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