Effect of iron regulation on expression and hemin-binding function of outer-sheath proteins from Treponema denticola

Abstract

The effect of the iron-chelating compounds EDDA and BPD on polypeptide regulation in the putative oral pathogen Treponema denticola was studied. SDS-PAGE analysis of the T. denticola strains grown in the presence of EDDA or BPD, i.e. iron-limiting environmental conditions, revealed the expression of 44 and 43 kDa polypeptides in the outer sheath, a 73 kDa polypeptide in the cell membrane, and a 16 kDa polypeptide in the soluble cell fraction. The hemin-binding activity of purified outer sheaths from T. denticola TD-4 grown in the presence of 6.4 mM EDDA was significantly greater than that observed in control (absence of EDDA) outer sheaths. Both activities were inhibited by proteinase K. SDS-PAGE, LDS-PAGE and TMBZ staining revealed the 44 and 43 kDa outer-sheath polypeptides to be expressed by T. denticola strains GM-1. MS-25, ATCC 33520 and ATCC 33404 (TD-4), strains which possessed strong hemin-binding activity. The 44 kDa hemin-binding polypeptide was purified by 1% CHAPS solubilization, HPLC, and SDS-preparative electrophoresis. N'-terminal sequence analysis indicated the purified 44 kDa polypeptide to belong to a new, undescribed group of polypeptides possessing hemin-binding activity.