Department of Experimental Anesthesiology, Clinic for Anesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany.
Mol Oral Microbiol. 2013 Jun;28(3):192-203. doi: 10.1111/omi.12015. Epub 2012 Dec 26.
We have previously shown that benzamidine-type compounds can inhibit the activity of arginine-specific cysteine proteinases (gingipains HRgpA and RgpB); well-known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important periodontopathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, because pentamidine, which is a 20-fold less efficient inhibitor of gingipain than 2,6-bis-(4-amidinobenzyl)-cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine-derived compounds other than Arg-gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5-1-2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine-Sepharose column to identify proteins interacting with benzamidine. In addition to HRgpA and RgpB the analysis revealed heat-shock protein GroEL as another ligand for benzamidine. To better understand the effect of benzamidine-derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine-derived compounds caused significant increases in GroEL, at both the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also by pentamidine, a relatively weak gingipain inhibitor. This effect may depend not only on gingipain inhibition but also on interaction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment.
我们之前已经表明，苯甲脒类化合物可以抑制精氨酸特异性半胱氨酸蛋白酶（牙龈蛋白酶 HRgpA 和 RgpB）的活性；这些是牙龈卟啉单胞菌的重要毒力因子。它们还可以抑制这种重要的牙周致病菌的体外生长。显然，生长停滞与它们抑制这些蛋白酶的能力无关，因为戊二脒对牙龈蛋白酶的抑制作用比 2,6-双（4-脒基苄基）环己酮（ACH）低 20 倍，但它更有效地阻止了牙龈卟啉单胞菌的生长。为了确定除 Arg-牙龈蛋白酶以外的苯甲脒衍生化合物的靶标，并解释其抑菌作用，我们用苯甲脒-琼脂糖柱进行亲和层析，分析了牙龈卟啉单胞菌 ATCC 33277 和牙龈卟啉单胞菌 M5-1-2（临床分离株）细胞提取物，以鉴定与苯甲脒相互作用的蛋白质。除了 HRgpA 和 RgpB 之外，分析还显示热休克蛋白 GroEL 是苯甲脒的另一个配体。为了更好地了解苯甲脒衍生化合物对牙龈卟啉单胞菌的影响，我们将细菌暴露于苯甲脒、戊二脒、ACH 和热中，然后确定牙龈蛋白酶和 GroEL 的表达情况。暴露于热和苯甲脒衍生化合物会导致 GroEL 在 mRNA 和蛋白质水平上的显著增加。有趣的是，尽管牙龈蛋白酶被证明是感染受精卵模型中的主要毒力因子，但死亡率不仅被 ACH 显著降低，而且被戊二脒（一种相对较弱的牙龈蛋白酶抑制剂）显著降低。这种效果可能不仅取决于牙龈蛋白酶的抑制作用，还取决于苯甲脒衍生物与 GroEL 的相互作用。因此，这些化合物可能在支持性牙周炎的治疗中得到应用。
