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- Karched, M, et al.
(författare)
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A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 68:1, s. 46-51
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- 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.
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| 2. |
- Paul-Satyaseela, M, et al.
(författare)
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Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein.
- 2006
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Ingår i: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 55:7, s. 931-942
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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