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Vesicle-independent extracellular release and bioactivity of peptidoglycan-associated lipoprotein from Aggregatibacter actinomycetemcomitans
Umeå University, Faculty of Medicine, Odontology.
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.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 101
Keyword [en]
Peptidoglycan-associated lipoprotein, outer membrane proteins, immunoproteomics, periodontitis, cytokines, Aggregatibacter actinomycetemcomitans.
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-1419ISBN: 978-91-7264-441-0 (print)OAI: oai:DiVA.org:umu-1419DiVA: diva2:140976
Public defence
2007-11-23, Hall C, Tandläkahögskolan, 9 Tr, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2007-11-06 Created: 2007-11-06 Last updated: 2009-05-26Bibliographically approved
List of papers
1. A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
Open this publication in new window or tab >>A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
2007 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, 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.

Identifiers
urn:nbn:se:umu:diva-12977 (URN)10.1016/j.mimet.2006.06.004 (DOI)16904783 (PubMedID)
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2017-12-14Bibliographically approved
2. Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein.
Open this publication in new window or tab >>Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein.
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2006 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, 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.

Identifiers
urn:nbn:se:umu:diva-12987 (URN)10.1099/jmm.0.46470-0 (DOI)16772422 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2017-12-14Bibliographically approved
3. Characterization of immunoaffinity purified peptidoglycan-associated lipoprotein of Actinobacillus actinomycetemcomitans.
Open this publication in new window or tab >>Characterization of immunoaffinity purified peptidoglycan-associated lipoprotein of Actinobacillus actinomycetemcomitans.
2006 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 831, no 1-2, 116-125 p.Article in journal (Refereed) Published
Abstract [en]

Peptidoglycan-associated lipoprotein (PAL) is a highly conserved structural outer membrane protein among Gram-negative bacteria. In some species, it is proinflammatory and released extracellularly. We purified a newly identified PAL (AaPAL) of a periodontal pathogen Actinobacillus actinomycetemcomitans by using AaPAL antipeptide antibodies coupled to immunoaffinity chromatography column. No protein impurities originating in A. actinomycetemcomitans were found in the final product. Sera from patients infected by A. actinomycetemcomitans recognized the purified AaPAL. The present purification method seems to be suitable for isolation of AaPAL and probably PALs of other bacterial species, and applicable in studies investigating proinflammatory mechanisms of A. actinomycetemcomitans.

Identifiers
urn:nbn:se:umu:diva-12998 (URN)10.1016/j.jchromb.2005.11.052 (DOI)16378764 (PubMedID)
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2017-12-14Bibliographically approved
4. Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form
Open this publication in new window or tab >>Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form
Show others...
2008 (Swedish)In: BMC Microbiology, ISSN 1471-2180, E-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.

National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-8838 (URN)10.1186/1471-2180-8-18 (DOI)18226201 (PubMedID)
Available from: 2008-02-18 Created: 2008-02-18 Last updated: 2017-12-14Bibliographically approved

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