Effects of Steroid Hormones on Neisseria Gonorrhoeae PAUL G
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1980, p. 281-288 Vol. 18, No. 2 0066-4804/80/08-0281/08$02.00/0 Effects of Steroid Hormones on Neisseria gonorrhoeae PAUL G. LYSKO AND STEPHEN A. MORSE* Department ofMicrobiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201 Various steroids were tested for their effects upon gonococcal 02 consumption and glucose catabolism. The ability to inhibit gonococcal 02 uptake appeared to be related to the molecular configuration of the steroid. The presence oflipophilic groups enhanced inhibition, whereas the addition of hydrophilic groups markedly diminished inhibition. Steroid inhibition decreased with an increasing number of polar groups. Glucose catabolism was inhibited by steroid hormones, and the degree of inhibition was influenced by pH and medium composition. Changes in growth medium and pH also resulted in differential steroid inhibition of 02 uptake. Under certain conditions, lactate partially relieved this inhibition. Gon- ococci that were grown in one environment and shifted to a new environment were inhibited by steroids to the same extent as if they had been originally grown in the new environment. The differential effects of medium and pH upon steroid inhibition may be due to structural rearrangements involving membrane phase transitions or to altered receptor affinity. The percentage of positive cultures obtained grown in either a complex medium (CM; 19) lacking from females with gonorrhea varies with the day soluble starch or in a defined medium (DM) supple- of the menstrual cycle (8, 10, 11). Maximum mented with hypoxanthine and uracil (17). Both CM recovery occurs at menstruation, whereas mini- and DM were buffered with 30 mM N-2-hydroxyethyl- mum piperazine-N'-2-ethanesulfonic acid (HEPES) and ad- recovery coincides with the peak plasma justed to the desired pH with NaOH or HCL. Liquid progesterone levels seen during week 3 of the media were inoculated with cells obtained by suspend- cycle. Plasma levels of f3-estradiol are biphasic, ing the growth on corresponding solid agar medium with a peak at midcycle and a secondary peak after incubation overnight at 37°C with increased CO2 during week 3 (24, 25). (4% CO2). All liquid cultures were incubated at 37°C Previous reports from our laboratory have with shaking. Turbidity was measured at 540 nm with indicated that Neisseria gonorrhoeae is unusual a Klett-Summerson colorimeter. among gram-negative bacteria in that its growth Chemicals and radioisotopes. Steroid hormones is inhibited by progesterone (3, 18). Progesterone were obtained from Sigma Chemical Co., St. Louis, Mo. [1-'4C]glucose (specific activity, 9.6 mCi/mmol) bound to both protein and lipid components of was obtained from New England Nuclear Corp., Bos- the cytoplasmic membrane, where it inhibited ton, Mass. All other reagents were of analytical grade. the activities of respiratory enzymes (14, 18). 02 uptake. 02 uptake was measured with a Yellow This study investigates the inhibitory activi- Springs Instrument Co. model 53 oxygen monitor at- ties of various steroids and their derivatives on tached to a circulating water bath adjusted to 37°C. gonococcal respiration to ascertain the molecu- Exponential-phase cultures of gonococci were diluted lar characteristics necessary for inhibition. In with fresh medium, sparged, and added to the elec- addition, environmental parameters which trode chambers at a cell density of ca. 2.0 x 108 in a might influence the outcome of steroid-cell in- volume of 4 ml. The number of cells was determined teractions were examined. by visual counts with a Petroff-Hausser counting chamber. Diplococci were counted as two cells. When CM was used for diluent, the growth factor supple- MATERIALS AND METHODS ment (19) was omitted since its presence caused 02 Organisms. N. gonorrhoeae CS-7 was used uptake. When DM was used for diluent, solution III throughout this study. Isolation, characteristics, and was omitted. Each diluent contained 5 mM glucose. maintenance of this strain have been described previ- When the rate of 02 uptake was linear, steroids ously (16, 19). Strains 1384 and 2024 are auxotrophic dissolved in absolute ethanol were added to the cham- for arginine, hypoxanthine, and uracil (AHU strains) bers to a final concentration of 40 jig per 5 x 107 cells. and were isolated from disseminated infections (17). Inhibition was recorded as the percentage of the orig- Strain F62, colony types 1 and 4, was obtained from inal rate of 02 uptake 10 min after steroid addition. the Neisseria Reference Laboratory, Public Health With ,8-estradiol diacetate, inhibition was biphasic and Service Hospital, Seattle, Wash. was recorded from the linear portion of each curve Medium and growth conditions. Gonococci were regardless of the time. The final concentration of 281 282 LYSKO AND MORSE ANTimICROB. AGENTS CHEMOTHER. ethanol was <0.4% and did not inhibit growth or 02 TABLE 1. Effect of various steroids on 02 uptake of uptake. N. gonorrhoeae CS- 7a The relative concentrations of 02 in each medium Steroid ~~~%Original at each pH were determined by comparison of air- Steroid 02 uptakeb saturated media to air-saturated 0.02 M sodium phos- Estrane derivatives phate buffer (pH 7.4). The absolute concentration of stradiol 81 02 in the phosphate buffer was determined by the a-Estradiol 85 con- method of Robinson and Cooper (23). Absolute ,6-Estradiol diacetate .. 56-94C centrations of 02 in the growth media could not be Bi-Estradiol dipropionate ................ 96 directly determined by this method. Relative 02 con- ,B-EEthynyl estradiol ...................... 71 centrations in each medium were within 2% of each Ethynyl estradiol-3-methyl ether ........ 47 other. Norethynodrel 72 Respiratory quotients (Q02) were determined for Norethindrone acetate .................. 14 cells under each set of conditions and are expressed as Pregnane derivatives Progesterone .......................... 25 microliters of 02 consumed per minute per 10i cells. lla-Hydroxyprogesterone 81 Radiorespirometry. Cultures were harvested dur- 17a-Hydroxyprogesterone 95 ing the exponential phase of growth (120 Klett units), lla-Acetoxyprogesterone ........ ....... 77 centrifuged, and resuspended in the appropriate me- 5/8-Pregnan-3,20-dione .................. 54 dium to a cell density of 5 x 10' cells per ml. The Pregnenolone 100 techniques for measuring C02 release from radiola- Pregnenolone acetate ................... 98 beled glucose have been previously described (19). Medroxyprogesterone acetate ........... 100 Samples of respiratory '4CO2 were collected every 10 Cortisone ............................. 100 min for 80 min. At 30 min, steroid hormones dissolved Hydrocortisone ........................ 100 Corticosterone ......................... 81 in ethanol were added to the cell suspensions absolute Deoxycorticosterone .................... 88 to a concentration of 60 ,tg per 5 x 10' cells. An Androstane derivatives identical amount of ethanol was added to a cell sus- Testosterone .......................... 64 pension not receiving hormones. Testosterone acetate .......... ......... 15 Testosterone propionate ........ ........ 19 Testosterone-17#-hemisuccinate ......... 86 RESULTS 19-Nortestosterone 96 Androstandione 84 on 02 Effect of various steroids uptake. Androstendiol ......................... 79 A spectrum of inhibitory activity was observed Dehydroisoandrosterone 84 when various steroids were tested for their abil- Miscellaneous Cholesterol 95 ity to inhibit 02 uptake of exponential-phase gonococci (Table 1). Among the steroid hor- Diethylstilbestrol ............ .......... 4 mones, the estrane derivatives exhibited the a Gonococci were grown in a complex liquid medium (pH inhibition. Notable exceptions were ethy- 7.2) with glucose at 37°C with shaking. Cells were diluted with least fresh medium to approximately 5 x 107 cells per ml. Steroids nyl estradiol and its 3-methyl ether, norethynod- were added to a final concentration of 40ug per 5 x 107 cells. rel, and norethindrone acetate, all of which are 'Determined from the linear rate 10 min after steroid used as estrogenic components of birth control addition. pills. Norethindrone acetate possesses both an 'Inhibition was biphasic. acetyl chain and an a-ethynyl group (-C=CH) at C-17 (Fig. 1). Norethynodrel and ethynyl ocorticosteroids tested were not very inhibitory. estradiol and its 3-methyl ether, which possess Cortisone and hydrocortisone had no effect on the a-ethynyl group but lack the acetyl group, 02 uptake. Corticosterone and deoxycorticos- were far less inhibitory toward gonococci than terone were only slightly inhibitory, which is was norethindrone acetate.,B-Estradiol diacetate interesting since deoxycorticosterone only dif- was unusual in that its inhibition was biphasic; fers from progesterone by the presence of a the dipropionate derivative did not exhibit bi- hydroxyl group at C-21. phasic inhibition. Inhibition by testosterone was enhanced by Progesterone was the most inhibitory preg- the addition of acetate or propionate groups; the nane derivative tested. The addition ofhydroxyl hemisuccinate form was not very inhibitory. The or acetoxy groups at C-11 or C-17 reduced the C-19 methyl group was apparently important for inhibition. The addition of a methyl group at C- inhibition, since 19-nortestosterone did not in- 6 and an acetoxy group at C-17 (medroxyproges- hibit 02 uptake. Androstandione, androstendiol, terone acetate) completely abolished inhibition. and dehydroisoandrosterone,