Effects of reproductive hormones/gonococcal pathogenesis

Investigators

Abstract

Summary: Infections due to Neisseria gonorrhoeae represent a significant cause of morbidity and mortality in women and children throughout the world. The pathogenesis of gonococcal infections is not well understood, particularly with respect to the effect of patient hormonal status on the infectious disease process. Currently, there are no licensed vaccines for prevention of gonorrhea. This project is investigating the effect of reproductive hormones, estradiol and progesterone, and oral contraceptives, on the expression of gonococcal outer membrane antigens, and whether or not these hormones affect attachment and/or invasion to human cervical and endometrial tissue culture cells. The goal is to identify potential new antigens that may be particularly relevant in gonococcal infections in women and to determine if hormonal status may be a factor to be considered in the evaluation of models of infection. The specific aims of this project are: 1. to assess the effect of estradiol and progesterone on expression of gonococcal outer membrane antigens; and 2. to determine whether estradiol and progesterone alter gonococcal adherence and/or invasion to human cells. Progress to date on specific aims: 1. We have determined the effects of reproductive hormones on the cultured cell line Hec1B: estradiol was not inhibitory to the growth of Hec1B cells in culture over a range of concentration from 1x 10-6 to 1 x 10 -12 M/L, and the cell line grew optimally at concentrations of 1x 10 -8 and -9 M/L. These concentrations are similar to the physiologic levels measured in serum taken from women during the early proliferative stage of the menstrual cycle. Progesterone and combined progesterone and estradiol are slightly inhibitory to cell proliferation. Phenol red has a weak estrogenic-like activity and we have identified this common component of cell culture media as a possible confounder in gonococcal adherence assays. 2. Outer membrane proteins from HEC1B cells grown with and with out estradiol were analyzed and at least two proteins at 80kDa and 180 kDa demonstrated increased expression when exposed to estradiol. These proteins will be excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. 3. Gonococcal adherence assays were conducted and an increase in gonococcal adherence in the presence of 1 x 10-9 M/L estradiol was observed. Our laboratory has recently developed an assay that will more accurately quantitate the number of bacteria bound to cultured cells. 4. Outer membrane proteins isolated from gonococci bound to Hec1B cells in the presence or absence of estradiol revealed an altered protein profile by SDS-PAGE. Several proteins were down-regulated and one protein was up-regulated. These proteins were excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. Sequenced proteins were used to search the FA1090 Neisseria genome at the University of Oklahoma and the NCBI-Blast database. The down regulated genes were identified as groEL, dnaK, L23, and a gene encoding a putative lipoprotein. The up regulated protein was identified as neisserial catalase. The gene for each identified protein has been cloned from MS11mkC genomic DNA. Promoterless b-galactosidase (lacZ) and GFP reporters were constructed which will be used to evaluate differences in gene transcription of identified genes in the presence and absence of hormones. Our laboratory is in the process of identifying other proteins that differed in their expression when in the presence of estradiol. High precentage SDS-PAGE is being used to isolate lower molecular weight proteins.