Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form.
BACKGROUND
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 mum), 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 mum) 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.
Karched
Maribasappa
M
Oral Microbiology, Department of Odontology, Umeå University, SE-90187 Umeå, Sweden. maribasappa.karched@odont.umu.se
Ihalin
Riikka
R
Eneslätt
Kjell
K
Zhong
Deyu
D
Oscarsson
Jan
J
Wai
Sun N
SN
Chen
Casey
C
Asikainen
Sirkka E
SE
D016976:Aggregatibacter actinomycetemcomitans / Q000472:pathogenicity* / Q000502:physiology* / Q000648:ultrastructure; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000648:ultrastructure; D006801:Humans; D008074:Lipoproteins / Q000276:immunology / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins