Change search
ReferencesLink to record
Permanent link

Direct link
A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
Umeå University, Faculty of Medicine, Odontology.
Umeå University, Faculty of Medicine, Odontology.
Umeå University, Faculty of Medicine, Odontology, Oral Microbiology.
2007 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, Vol. 68, no 1, 46-51 p.Article in journal (Other (popular science, discussion, etc.)) Published
Abstract [en]

Tenacious adherence and autoaggregation of wild-type Actinobacillus actinomycetemcomitans strains jeopardize reliability of determined cell concentrations, e.g. for studies on bacteria-host interactions. We first compared the efficacy of two methods, an indirect and a direct method, for homogenizing cell suspensions of a wild-type, autoaggregating (SA269) strain and of a non-autoaggregating laboratory variant (ATCC 43718) used as a reference. Since the direct method left visible clumps in SA269 suspension, only the indirect method was further tested. In serial dilutions of the homogenized cell suspensions of strains SA269 and ATCC 43718, the OD(600) values (R(2)=0.99, R(2)=0.99, respectively) and protein concentrations (R(2)=0.93, R(2)=0.95, respectively) correlated significantly (all P<0.002) with the dilution factor. There were no differences (P>0.05) in the bacterial viable counts between the two strains or between suspending solutions, i.e., PBS and water, the cell concentrations demonstrating 1x10(9) cells/ml at OD(600)=1. Repeated microscopic cell counts did not differ (P>0.05) from each other. Large aggregates occurred as 1% of cell units counted. Dispersing bacterial mass indirectly to solution leads to homogeneous cell suspensions with repeatable cell concentrations. Viability of A. actinomycetemcomitans was also maintained when cells were suspended in water.

Place, publisher, year, edition, pages
2007. Vol. 68, no 1, 46-51 p.
URN: urn:nbn:se:umu:diva-12977DOI: 10.1016/j.mimet.2006.06.004PubMedID: 16904783OAI: diva2:152648
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2009-09-19Bibliographically 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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Asikainen, Sirkka
By organisation
OdontologyOral Microbiology
In the same journal
Journal of Microbiological Methods

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 33 hits
ReferencesLink to record
Permanent link

Direct link