ISOLATION OF THE OUTER MEMBRANES FROM TREPONEMA-PALLIDUM AND TREPONEMA-VINCENTII

The outer membranes from Treponema pallidum subsp. pallidum and Trepon ema vincentii were isolated by a novel method. Purified outer membrane s from T. pallidum and T, vincentii following sucrose gradient centrif ugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze frac ture electron microscopy of purified membrane vesicles from T. pallidu m and T. vincentii revealed an extremely low density of protein partic les; the particle density of T. pallidum was approximately sis times l ess than that of T. vincentii. The great majority of T. vincentii lipo polysaccharide was found in the outer membrane preparation. The T. vin centii outer membrane also contained proteins of 55 and 65 kDa. I-125- penicillin V labeling demonstrated that T. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and w ere not detectable with purified outer membrane material, indicating t he absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundan t 47-kDa lipoprotein known to be associated with the cytoplasmic membr ane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identifi ed by one- and two-dimensional electrophoretic analysis using gold sta ining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously id entified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI ), and 28 kDa were identified. Compared with whole-organism preparatio ns, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membrane s revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.