Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form
1 Oral Microbiology, Department of Odontology, Umeå University, SE-90187 Umeå, Sweden
2 Department of Molecular Biology, Umeå University, SE-90187 Umeå, Sweden
3 Primary Oral Health Care, USC School of Dentistry, University of Southern California, 925 W. 34th Street, Los Angeles CA 90089-0641, USA
4 Department of Biochemistry and Food Chemistry, University of Turku, Turku FIN-20520, Finland
5 Department of Dentistry, Medical School Hospital of Qingdao University, Qingdao, People's Republic of China
BMC Microbiology 2008, 8:18 doi:10.1186/1471-2180-8-18Published: 28 January 2008
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.
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 μm), 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 μm) 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.
Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.