Treponema pallidum subsp. pallidum and related organisms are helically- shaped bacteria which cause venereal syphilis, endemic syphilis, yaws and pinta in humans. Although the incidence of sexually transmitted diseases would be expected to decrease with changing sexual behavior as a result of the AIDS epidemic, the number of reported primary and secondary syphilis cases in the United States increased 30% in 1987, due primarily to apparent epidemics in New York, Florida, and California. Further understanding of T. pallidum is needed to aid in the prevention, diagnosis, and treatment of syphilis. In the proposed research, we will continue studies on the physiology, structure, and antimicrobial susceptibility of T. pallidum. The principal focus will be efforts to continuously culture T pallidum in vitro, using as a starting point a tissue culture system which supports 20- to 100-fold multiplication of the organism over a 12 to 15 day period of incubation. Mammalian cells, in particular Sf1Ep cottontail rabbit epithelial cells, are required for the in vitro multiplication of T. pallidum in this system, but recent studies indicate that rapid growth or high metabolic rates of these cells inhibit treponemal multiplication. Furthermore, removal of amino acids and vitamins from the culture medium enhances the multiplication of T. pallidum, apparently by inhibiting the growth and metabolism of the tissue culture cells. In the coming grant period, conditions inhibitory to mammalian cell metabolism, including irradiation, addition of cycloheximide, and use of nutrient-deficient media, will be tested for their ability to enhance the multiplication of T. pallidum If conditions are identified which significantly increase and prolong treponemal growth, they will be combined with subculture or medium replacement techniques in an attempt to produce continuous growth. Through the deletion of medium components, dialysis of serum, and removal of tissue extract, it should be possible to define the nutritional requirements of T pallidum. In addition, the biosynthetic capacity of T. pallidum will be assessed by measuring the incorporation of radiolabelled D-glucose, fatty acids, and inorganic salts into proteins, nucleic acids, and lipids. To assess the antimicrobial susceptibility pattern of T. pallidum, the in vitro culture system will be utilized to determine the minimal inhibitory and bactericidal concentrations of a variety antimicrobial agents against the Nichols strain and other pathogenic strains. In our continuing studies of the major structural elements of T. pallidum, we will attempt to purify and characterize the outer membrane, cytoplasmic membrane, and cytoplasmic fibrils of pallidum using isoelectric focusing, hydrophobic interaction chromatography, sucrose gradient sedimentation, and other methods. Immunoelectron microscopy of ultrathin sections will be used to localize both internal and external proteins in the overall structural studies will be integrated with recent immunologic and recombinant DNA studies to improve our understanding of the host-parasite interactions of this unusual organism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI020006-06
Application #
3129470
Study Section
Bacteriology and Mycology Subcommittee 1 (BM)
Project Start
1982-08-01
Project End
1993-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
You, Y; Elmore, S; Colton, L L et al. (1996) Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum. J Bacteriol 178:3177-87
Walker, E M; Howell, J K; You, Y et al. (1995) Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols). J Bacteriol 177:1797-804
Norris, S J (1993) Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group. Microbiol Rev 57:750-79
Walker, E M; Arnett, J K; Heath, J D et al. (1991) Treponema pallidum subsp. pallidum has a single, circular chromosome with a size of approximately 900 kilobase pairs. Infect Immun 59:2476-9
Houston, L S; Cook, R G; Norris, S J (1990) Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli. J Bacteriol 172:2862-70
Norris, S J; Charon, N W; Cook, R G et al. (1988) Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis. J Bacteriol 170:4072-82
Norris, S J; Edmondson, D G (1988) In vitro culture system to determine MICs and MBCs of antimicrobial agents against Treponema pallidum subsp. pallidum (Nichols strain). Antimicrob Agents Chemother 32:68-74
Norris, S J; Edmondson, D G (1986) Serum requirement for the multiplication of Treponema pallidum in a tissue-culture system: association of growth-promoting activity with the protein fraction. Sex Transm Dis 13:207-13
Norris, S J; Edmondson, D G (1986) Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system. Infect Immun 53:534-9