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Isolation of the outer membranes from Treponema pallidum and Treponema vincentii
D R Blanco1, K Reimann, J Skare
1Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
Abstract:
The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified 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 preparations, 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 membranes 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.
Insights
This study isolated outer membranes from Treponema pallidum and Treponema vincentii, revealing unique protein compositions and porin activity in T. pallidum outer membranes, crucial for understanding syphilis pathogenesis.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Treponema pallidum subsp. pallidum causes syphilis, but its outer membrane composition remains poorly understood.
- Treponema vincentii shares some characteristics with T. pallidum, making comparative studies valuable.
Purpose of the Study:
- To isolate and characterize the outer membranes of Treponema pallidum and Treponema vincentii.
- To identify proteins and lipids within these outer membranes.
- To investigate the functional properties, such as porin activity, of the T. pallidum outer membrane.
Main Methods:
- Novel isolation technique for outer membranes using sucrose gradient centrifugation.
- Freeze fracture electron microscopy for visualizing membrane structure.
- Electrophoretic analysis (one- and two-dimensional) with gold staining and immunoblotting for protein identification.
- Reconstitution of solubilized outer membrane into lipid bilayers to assess porin activity.
Main Results:
- Outer membranes of T. pallidum and T. vincentii were successfully purified.
- T. pallidum outer membrane exhibited significantly lower protein particle density than T. vincentii.
- T. pallidum outer membrane contained rare, highly antigenic proteins (65- and 31-kDa) and showed porin activity with two distinct channel sizes (0.35 and 0.68 nm).
- Penicillin-binding proteins were confirmed to be intracellular, excluding inner membrane contamination.
Conclusions:
- The study provides a detailed characterization of T. pallidum and T. vincentii outer membranes.
- Identified potential rare outer membrane proteins in T. pallidum may be targets for future therapeutic strategies.
- The presence of porins in the T. pallidum outer membrane suggests a role in nutrient uptake or molecular transport.