DEMONSTRATION OF ADHERENCE PROPERTIES OF PORPHYROMONAS-GINGIVALIS OUTER-MEMBRANE VESICLES USING A NEW MICROASSAY

Vesicles made by Porphyromonas gingivalis possess several biological activities, including the ability to adhere to oral surfaces and to bacteria. In this study, a new and simple method was developed to measure the adherence capability of outer membrane vesicles from P. gingivalis. Vesicles were conjugated to fluorescent microspheres (0.7 mu m) and added to well of a Teflon-coated microscope slide previously covered with a variety of soluble ligands. After incubation and washes, the number of fluorescent microspheres per microscopic field were counted. Vesicle-coated microspheres attached best to gelatin (>200 per field), whereas other compounds (such as fibronectin, fibrinogen, collagen and laminin) provided moderate attachment, and no attachment was observed to bovine serum albumin. Adherence to any of the tested ligands was not observed when fluorescent microspheres were conjugated to bovine serum albumin or lipopolysaccharides from P. gingivalis. The adherence of vesicle-coated microspheres to ligands was not significantly affected when the pH of the reaction mixture was between 4 and 10. None of the tested carbohydrates lowered the attachment capability of vesicle-coated microspheres to substrates. When vesicle-coated microspheres were treated with trypsin and chymotrypsin or heated, this resulted in a significant loss of attachment, suggesting a possible involvement of proteinaceous molecules in the process. The present study confirms that vesicles of P. gingivalis are capable of attachment to various molecules and indicate their potential role in colonization.