Nutritional requirements of Treponema denticola and Treponema vincentii
Treponema denticola and Treponema vincentii were grown in a medium supplemented with 0.4% (wt/vol) alpha globulin in place of whole serum. Other serum fractions did not support growth. The growth factors in alpha globulin were destroyed by trypsin and by lipase. Lipid extraction of alpha globulin showed that both a protein and a lipid fraction were required for growth. Sodium salts of either oleic acid (cis-18:1) or elaidic acid (trans-18:1), added to 0.4% delipified alpha globulin supplemented media at a final concentration of 0.04 mg/ml, replaced the alpha globulin lipids required for optimal growth of these two oral treponemes. Tween 80 (polysorbitan monooleate) also supported growth in a medium containing protein. Short chain fatty acids plus 25 µg/ml thiamine pyrophosphate, added to either a basal medium or a medium containing 0.4% albumin, supported limited growth. The principal cellular fatty acids of T. denticola grown in an oleate medium were myristic, pentadecanoic, and palmitic acids. Treponema denticola appears capable of limited synthesis of cellular fatty acids from oleate.
Fifty percent of the total protein content of commercial alpha globulin was found to be albumin. The protein required for T. denticola growth was separated from the other alpha globulin proteins by Affi-Gel Blue (Bio-Rad Laboratories) affinity chromatography which selectively adsorbed albumin. Serum albumin, added to a medium containing oleate, substituted for the alpha globulin protein required by these two treponemes. Trypsin destroyed the growth promoting activity of albumin. A weight ratio of albumin to sodium oleate of 50:1 (0.4% delipified albumin - 0.08 mg/ml oleate) supported optimal growth of T. denticola and T. vincentii. Starch, added to media containing oleate, could not replace albumin for optimal growth. Serum albumin solutions tightly bound added thiamine pyrophosphate (TPP). Optimal growth was achieved only when the TPP concentrations in albumin-oleate media were sufficient to provide excess TPP, unbound to albumin. Whole cells of T. denticola were shown to have proteolytic activity toward casein and alpha globulin proteins. Alpha globulin proteins were also found avidly attached to T. denticola cells that had been suspended in alpha globulin.