Karched Maribasappa, Ihalin Riikka, Eneslätt Kjell, Zhong Deyu, Oscarsson Jan, Wai Sun N, Chen Casey, Asikainen Sirkka E
Oral Microbiology, Department of Odontology, Umeå University, SE-90187 Umeå, Sweden.
BMC Microbiol. 2008 Jan 28;8:18. doi: 10.1186/1471-2180-8-18.
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 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.
Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.
伴放线聚集杆菌是一种与侵袭性牙周炎相关的口腔细菌。有研究表明，细菌外膜蛋白的细胞外释放主要通过外膜囊泡进行。本研究调查了伴放线聚集杆菌菌株中肽聚糖相关脂蛋白（AaPAL）的存在及保守性、AaPAL的免疫刺激作用，以及活细胞是否以独立于囊泡的游离可溶形式释放这种结构性外膜脂蛋白。
通过聚合酶链反应-限制性片段长度多态性和免疫印迹法在临床伴放线聚集杆菌菌株中证实了pal基因座及其基因产物。在不同生长条件下培养未发现AaPAL表达有明显需求。野生型菌株（D7S）及其自发实验室变体（D7SS）中pal基因的失活导致了多效性细胞效应。在细胞培养插入模型（滤膜孔径0.02μm）中，当D7S和D7SS菌株在插入物中的血清或肉汤中孵育时，从滤液中检测到了AaPAL。电子显微镜显示，伴放线聚集杆菌囊泡（0.05 - 0.2μm）大于滤膜孔径，滤液中无囊泡。滤液对细胞质标记物环磷酸腺苷（cAMP）受体蛋白的免疫印迹呈阴性。体外模型表明AaPAL可刺激人全血产生细胞因子。
游离可溶的AaPAL可通过独立于囊泡的过程在细胞外释放。
