The basic goals of this proposal are to achieve continuous in vitro culture of Treponema pallidum and to characterize the proteins associated with the structural components of the organism. Of highest priority will be the attempted continuous passage of T. pallidum in vitro utilizing the tissue culture system developed by Fieldsteel et al. The approach to extending the limited multiplication obtained in this system will be: 1) to identify the nutritional and environmental requirements for T. pallidum growth, and use this information to optimize culture conditions; and 2) to maintain optimal conditions through subcultivation, continuous flow, or chemostatic techniques. Because of our recent success in achieving significant multiplication in secondary cultures, subcultivation as a means of prolonging in the in vitro growth of T. pallidum will be investigated. Stabilization of culture conditions by continuous flow medium replacement or chemostatic techniques will eliminate the depletion of nutrients and accumulation of toxic products as possible causes of the cessation of growth. High density tissue culture techniques utilizing hollow fiber perfusion cultures, sepharose microcarriers or collagen matrices will be used in combination with continuous flow medium replacement to determine whether these 'tissue-like' conditions will enhance treponemal growth. In defining the nutritional requirements of T. pallidum, the growth-promoting factors present in serum and tissue extracts will be purified and characterized. Because the requirement for serum is associated with serum proteins, it will be determined whether albumin complexed with lipids and other compounds is capable of replacing the serum requirement, as it is for other parasitic spirochetes. The medium currently used for cultivation studies could be deficient in required compounds; therefore, the effects of supplementation with fatty acids and vitamins required by related bacteria will be assessed. Monitoring of numerous cultural parameters during in vitro cultivation of T. pallidum will be used as a means for identifying changes leading to the cessation of treponemal growth. Identification of the proteins associated with the outer membrane, cytoplasmic membrane, flagella, and cytoplasm of T. pallidum will aid in understanding their functional properties and their potential roles as protective immunogens. Therefore, the structural distribution of the major polypeptides of T. pallidum will be determined by structural fractionation and immunoelectron microscopy techniques.
