Vol. 22, No. 3

INFECTION AND IMMUNITY, Dec. 1978, p. 698-708 0019-.9567/78/0022-0698$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Binding of Cholesterol by Neisseria gonorrhoeae RICHARD D. MILLER,' WILLIAM J. WARREN,' ROBERT C. SIZEMORE,' AND STEPHEN A. MORSE2 *

Department ofMicrobiology and Immunology, University of Louisville, School of Medicine, Health Sciences Center, Louisville, Kentucky 40232,' and Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 972012 Received for publication 27 September 1978

The binding of [1,2-3H]cholesterol to Neisseria gonorrhoeae CS-7, Pseudomaeruginosa, and Salmonella typhimurium (smooth and rough strains) was investigated. The kinetics of cholesterol binding to N. gonorrhoeae CS-7 demonstrated that binding occurred slowly with maximum binding by 10 h. Under optimum conditions, a large percentage (65%) of the added cholesterol was associated with the cells. Chemical fractionation revealed that ca. 98% of the labeled cholesterol was associated with the cell membraness. The bound cholesterol was not esterified and was associated primarily with the cytoplasmic membrane. Intact gonococci bound 4 to 30 times more cholesterol than the deep rough mutant S. typhimurium TA1535, the wild-type S. typhimurium DB-21, and P. aeruginosa. In contrast, isolated cell membranes from all organisms rapidly bound cholesterol to the same extent. Therefore, the outer membrane can function as a permeability barrier to cholesterol. Cholesterol binding to both whole cells and isolated cell membranes was influenced by the incubation temperature. The rate of cholesterol binding by whole cells of N. gonorrhoeae decreased markedly at lower temperatures, with almost complete cessation of binding at 0°C. A similar temperature effect on the binding of cholesterol to isolated membranes was not observed. Thus, the effect of temperature on the binding of cholesterol to whole cells was an effect not on the actual binding process but rather on the ability of the cholesterol molecule to penetrate the lipid domain of the gonococcal outer membrane. onas

It was previously reported that the binding of progesterone by Neisseria gonorrhoeae was related to its growth-inhibitory properties (8, 10). Other gram-negative bacteria whose growth was not inhibited by progesterone did not bind this steroid. It was also reported (8) that gonococci bound the inhibitory sterol, cholesterol, although the binding kinetics were somewhat different than the kinetics of progesterone binding. The purpose of the present study was to examine in greater detail the nature of cholesterol binding by N. gonorrhoeae and other gram-negative bacteria to determine how the binding of this sterol differs from that of progesterone, and whether this in vitro binding represents a phenomenon that might be found in vivo. MATERIALS AND METHODS Organisms. N. gonorrhoeae strains CS-7 (colonial type 4) and JW-31 (colonial type 4), Salmonella typhimurium strains DB-21 and TA1535, and Pseudomonas aeruginosa strain PS-7 were used in this study. The description and maintenance of these organisms have been previously described (8, 9, 11). Media and growth conditions. All bacteria were

grown in a liquid medium which contained the following per liter: proteose peptone no. 3 (Difco Laboratories, Detroit, Mich.), 15 g; K2HPO4, 4 g; KH2PO4, 1 g; NaCl, 5 g; and soluble starch, 1 g. The final pH of the medium was 7.2. Glucose (5 g/liter), NaHCO3 (420 mg/liter), and a growth factor supplement, identical in composition to IsoVitaleX enrichment (BBL, Cockeysville, Md.) but lacking glucose, were added after autoclaving. Inocula of N. gonorrhoeae were prepared by adding 2 ml of a thawed stock culture (5) (or alternatively, cells washed from GC agar [Difco] plates) to 25 ml of growth medium. After incubation for 1 h at 37°C in a gyratory water-bath shaker (New Brunswick Scientific Co., New Brunswick, N.J.), the cell suspension was used to inoculate the growth medium (1% vol/vol). For all other organisms, a 1% (vol/vol) inoculum of a suspension of cells from an overnight culture was used to inoculate the growth medium. All liquid cultures were incubated at 37°C in a New Brunswick gyratory shaker. Chemicals and radioisotopes. Cholesterol (A5cholesten-3-ol), cholesterol acetate, cholesterol palmitate, cholesterol oleate, cortisone (A4-pregnen17a,21-diol-3,11,20-trione), testosterone (A4-androsten-17,1-1-3-one), Tween 80, lysozyme, ethylenediaminetetraacetic acid disodiumr salt (EDTA), and bo;98

VOL. 22, 1978 vine serum albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). [1,2-3H]cholesterol (specific activity 60 Ci/mmol) and [8-'4C]adenine (specific activity 43.9 mCi/mmol) were products of New England Nuclear Corp. (Boston, Mass.). All other reagents were of analytical grade and were obtained from standard commercial sources. Analytical procedures. Protein was assayed by the method of Lowry et al. (6) with BSA as the standard. Radioactivity of samples (0.1 ml) was measured in a liquid scintillation spectrometer (Beckman Instrument Corp., Irvine, Calif.) after addition of 15 ml of tritosol scintillation fluid (2). For thin-layer chromatography, 100-,ul samples in chloroform-methanol (2:1) were spotted on heat-activated plasticbacked silica gel sheets without fluorescent indicator (Distillation Products Industries, Rochester, N.Y.) and developed in a benzene solvent. Plates were air dried and cut into 1-cm segments, and radioactivity was measured by liquid scintillation spectrometry. Miscellaneous procedures. Turbidity was measured by Klett-Summerson colorimetry at 540 nm. Dry weights were obtained by drying aliquots of cells to constant weight in tared aluminum dishes at 80'C in a vacuum oven. Buoyant densities of fractions from sucrose gradients were obtained by weighing 10-1d samples in tared micropipettes (Dade Diagnostics, Inc., Miami, Fla.). Cells grown in basal medium containing [8-14C]adenine were chemically fractionated as previously described (11). Approximately 84% of the incorporated adenine was associated with the nucleic acid-containing cell fraction and 13% with the soluble pool material. Oxygen uptake. Late-log-phase cultures were harvested by centrifugation (10,000 x g for 10 min) at 4VC, washed once, and suspended to 470 Klett units (ca. 2 mg [dry weight]/ml) in 25 mM phosphate buffer (pH 7.2) containing 10 mM MgCl2, 85 mM NaCl, and 0.01% (vol/vol) Tween 80. Samples of 3 ml were transferred to an oxygen electrode chamber (Yellow Springs Instrument Co., Yellow Springs, Ohio) maintained at 370C. After a 2-min incubation period with constant stirring to insure consistent oxygenation and temperature equilibrium, the rate of oxygen uptake was followed polarographically. Binding of cholesterol to cell suspensions. Late-log-phase cultures were harvested by centrifugation (10,000 x g for 10 min) at 4VC, washed once, and suspended to 470 Klett units in 25 mM phosphate buffer containing 10 mM MgCl2, 85 mM NaCl, and 0.01% (vol/vol) Tween 80. Samples (1 ml) were transferred to glass centrifuge tubes and warmed to 37°C. An equal volume of labeled cholesterol (0.1 ACi/ml, diluted in the same buffer) was added to each tube and incubation was continued at 37°C. At 30-min intervals, tubes were removed and centrifuged at 12,000 x g for 5 min to sediment the cells. The radioactivity remaining in the supernatant was determined by liquid scintillation spectrometry. Preparation of cell membranes. Cell envelopes were isolated by sonic disruption of whole cells as previously described (8). Outer- and inner (cytoplasmic)-membrane fractions were prepared by the method of Wolf-Watz et al. (21). Outer-membrane

CHOLESTEROL BINDING

699

components were removed from plasmolyzed cells by lysozyme-EDTA treatment and aggregated by lowering the pH to 5.0. The aggregated membranes were collected by centrifugation (12,000 x g for 10 min) and washed four times in cold 5 mM EDTA. The spheroplasts formed as a result of the lysozyme-EDTA treatment were osmotically lysed, and the resulting suspension was centrifuged at low speed (12,000 x g for 15 min) to remove unbroken cells. Cytoplasmic membranes were collected from the resulting supernatant by centrifugation at 100,000 x g for 90 min. Both cytoplasmic- and outer-membrane preparations were further purified by isopycnic centrifugation on 15 to 65% (wt/wt) sucrose density gradients at 250,000 x g for 4 h (SW41 rotor, Beckman model L5-65) as previously described (8). Binding of cholesterol to cell membranes. Cell membrane preparations (ca. 2 mg of protein per ml) were suspended in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4) containing 1 mM EDTA and 0.01% (vol/vol) Tween 80. Samples (0.5 ml) were transferred to glass centrifuge tubes and warmed to 37°C. An equal volume of labeled cholesterol (0.1 ,uCi/ml in the same buffer) was then added to each tube, and incubation was continued at 37°C. At 30-min intervals, tubes were removed and centrifuged at 30,000 x g for 30 min to remove membranes. The radioactivity remaining in the supernatant was determined by liquid scintillation spectrometry. Binding of cholesterol to BSA. Samples (0.5 ml) of BSA in 25 mM phosphate buffer (pH 7.2) containing 10 mM MgCl2, 85 mM NaCl, and 0.01% (vol/vol) Tween 80 were transferred to glass centrifuge tubes and warmed to 37°C. An equal volume of labeled cholesterol (0.1 ftCi/ml in the same buffer) was then added to each tube. After 60 min, 1 ml of cold 10% (wt/vol) trichloroacetic acid was added, and the tubes were incubated at 0°C for 30 min. The precipitates were removed by centrifugation at 12,000 x g for 10 min, washed once with cold 5% (wt/vol) trichloroacetic acid, and then dissolved in ethanol. The radioactivity of the samples was determined by liquid scintillation spectrometry.

RESULTS

Binding of [1,2-3H]cholesterol to cell suspensions of N. gonorrhoeae. The effect of cell concentration on the kinetics of cholesterol binding at 37°C is shown in Fig. 1. Binding was linear during the first hour of incubation and then gradually decreased. Extended incubation (Fig. 1, insert) indicated that the rate of binding continued to decrease slowly, with maximum binding occurring by 10 h (53.4% of the added label). The kinetics of cholesterol binding were similar at all four cell concentrations tested, although the decrease in the rate of binding after 1 h was more pronounced at higher cell concentrations. Therefore, a cell concentration of ca. 2 mg (dry weight)/ml (470 Klett units) was used in all subsequent studies. It should be noted that although the rate of cholesterol binding was con-

700

MILLER ET AL.

INFECT. IMMUN.

0.3

-

0.1

0

t10

15

20

Hours of Incubation

' L2

-

C.3

0 0

12

Hours of Incubation FIG. 1. Effect of cell concentration on the binding of cholesterol by N. gonorrhoeae CS-7. Symbols: (0) original suspension of 630 Klett units (ca. 5.7 mg [dry weight]/ml); (A) 2.8 mg (dry weight)/ml; ([) 1.4 mg (dry weight)/ml; (V) 0.7mg (dry weight)/ml. Insert shows cholesterol bound as a function of time to N. gonorrhoeae CS- 7 at a cell concentration of ca. 2 mg (dry weight)/ml (470 Klett units).

siderably slower than the rate of progesterone binding to N. gonorrhoeae, the total amount of cholesterol bound (53.4%) was >2.5 times the total amount of progesterone bound (20%) under similar conditions (8). The studies reported above used N. gonorrhoeae CS-7; however, similar kinetics of cholesterol binding were observed with other strains of N. gonorrhoeae, including several fresh clinical isolates. The effect of varying the concentration of labeled cholesterol is shown in Fig. 2. Typical binding kinetics were observed at each of the four cholesterol concentrations employed (Fig. 2, insert); however, the decrease in the rate of binding after 1 h was more pronounced at higher cholesterol concentrations. When the amount of

cholesterol bound after 2 h of incubation was plotted as a function of the cholesterol added (0 to 3.5 ng/mg [dry weight]), a linear relationship was observed. In each case, approximately 20% of the added cholesterol was bound. Comparison of [1,2-3Hlcholesterol binding by N. gonorrhoeae and other gram-negative bacteria. It was previously reported (8) that the binding of cholesterol by N. gonorrhoeae differed from that of Neisseria mucosa and P. aeruginosa. We have expanded this observation employing a wild-type (strain DB21) and a deep rough outer membrane mutant (strain TA1535) of S. typhimurium. The results (Fig. 3) demonstrate that gonococci bound significantly more cholesterol than the other gram-

CHOLESTEROL BINDING

VOL. 22, 1978

701

0.8

3 0.6

-

z 0 0

0.4

Hi

1.0.2.03 O

U)0 Ci)~~~~~~~~~~C

C~~~~~~~~

0

0) %-' -i

0

00 0

1

HOURS

0

5.0 1.0 2.0 CHOLESTEROL ADDED (ng/mg dry wt.)

~~~~~2 4.0

FIG. 2. Effect of cholesterol concentration on the binding of cholesterol to N. gonorrhoeae CS-7. Large graph depicts cholesterol bound as a function of cholesterol added. Insert: cholesterol bound as a function of time. Symbols: (E) 3.4; (A) 1.7; (0) 0.8; (V) 0.4 ng of cholesterol per mg (dry weight), respectively.

negative bacteria examined. S. typhimurium TA1535 and P. aeruginosa bound small amounts of cholesterol, whereas S. typhimurium DB21 bound minimal amounts of this steroid. The differences in the binding between the wildtype and deep rough strains of S. typhimurium suggested a relationship between cholesterol binding and outer-membrane architecture or permeability. Effect of environmental conditions on cholesterol binding. The binding of [1,2-3H]cholesterol to cells of N. gonorrhoeae CS-7 was proportional to the incubation temperature (Fig. 4). Although the rate of binding was markedly slower at lower temperatures (negligible binding at 0C), typical kinetics were observed at all temperatures (0 to 4600) examined. The rate of binding (k = slope) was not directly proportional to the temperature, but a linear relationship was observed when the negative log of k was plotted as a function of the temperature (00) (Fig. 4, insert).

The effect of pH on cholesterol binding was also examined. The amount of cholesterol bound after 2 h of incubation in binding buffer at pH 6, 7, and 8 was 0.068, 0.077, and 0.086 ng/mg (dry weight), respectively. N. gonorrhoeae CS-7 does not undergo extensive lysis when suspended in buffer, however, the slow release of ['4C]adenine from these cells (pH 8 > 7 > 6; data not shown) suggests that the increased binding at high pH may be due to an increase in cell permeability. That cell lysis could affect the binding of cholesterol by exposing more binding sites was examined by measuring the binding of [1,2-3H]cholesterol by N. gonorrhoeae JW 31 (autolytic strain) under conditions optimum for autolysis (pH 8.0, without Mg2+) (20). The results indicate that cells incubated in the absence of Mg2e bound significantly more cholesterol than did control cells in the presence of 10 mM Mg2+ (Fig. 5A). Release of ['4C]adenine (Fig. 5A) and the decrease in turbidity (Fig. 5B) confirmed that the rate of autolysis was greater in the absence than

702

INFECT. IMMUN.

MILLER ET AL.

0.15

_

0.10

E

S1.

C.V

0.05-

0~~~~~~~ 0

1 Hours of Incubation

FIG. 3. Comparison of cholesterol binding by N. gonorrhoeae CS-7 and other gram-negative bacteria. Symbols: (0) N. gonorrhoeae CS-7; (L) S. typhimurium TA1535 (deep rough strain); (A) P. aeruginosa; (0) S. typhimurium DB21 (wild-type).

in the presence of Mg2". These results indicate fractions (ethanol-soluble and ethanol ether-solthat, although significant binding occurred in uble cell fractions). Negligible amounts of chothe absence of autolysis, binding was signifi- lesterol were associated with the cold or hot trichloroacetic acid- or papain-soluble cell fraccantly increased during cell lysis. on tions. The high concentration of cellular lipid activity Effect of cellular metabolic cholesterol binding. Cholesterol did not in- associated with the cell membrane suggested hibit the growth of N. gonorrhoeae (8). In fact, that cholesterol was binding to these structures. Binding of [1,2-3Hlcholesterol to isolated cholesterol was bound by actively growing culrole membranes. In contrast to the binding of the examined cell We data). tures (unpublished of cellular metabolic activity on the binding of cholesterol exhibited by whole cells (Fig. 3), the [1,2-3H]cholesterol to cell suspensions of N. gon- cell envelopes of all bacteria tested bound the orrhoeae CS-7. The results (Table 1) indicated labeled cholesterol equally well (Fig. 6). The that metabolic poisons (Hg2+, azide) and incu- binding kinetics differed considerably from those bation under anaerobic conditions (N2 atmos- observed with whole cells. Binding to cell envephere) had no effect on binding although they lopes was rapid, with maximum amounts of choseverely inhibited oxygen uptake by the cells. lesterol bound after 1 h of incubation. Over 88% Addition of glucose, which markedly stimulated of the added cholesterol was bound to memthe rate of oxygen uptake, also had no effect on brane preparations containing 1.3 mg of protein per ml. cholesterol binding. The incubation temperature did not have a Location of bound cholesterol. Cells of N. gonorrhoeae CS-7 were chemically fractionated dramatic effect on the binding of cholesterol to after 2 h of incubation in the presence of [1,2- cell envelopes of N. gonorrhoeae CS-7 (data not 3H]cholesterol. The results (data not shown) shown). Although the binding of cholesterol at showed that ca. 98% of the labeled cholesterol 00C was somewhat slower than at 370C, there was little difference in the total amount of chowas associated with the lipid-containing cell

CHOLESTEROL BINDING

VOL. 22, 1978

703

39

0 1lw~ ~ ~ ~ k0.11S

t o.

0.011

=

O

1 Hours of Incubation

~~~~~~~~~~2

FIG. 4. Effect of temperature on the binding of cholesterol by N. gonorrhoeae CS-7. Symbols: (0) 46°C; (O) 35oC; (A) 24°C; (0) 10°C; (V) O°C. The k value represents the slope of the corresponding line. Insert: negative log of k as a function of temperature SAQ.

lesterol bound after 2.5 h of incubation. These results are in marked contrast to the effect of temperature on the binding of cholesterol to whole cells (Fig. 4). The binding of cholesterol to whole-cell envelopes (Fig. 6) did not distinguish between outer-membrane and inner (cytoplasmic)-membrane binding. To determine the location of the bound cholesterol, the inner and outer mem-

branes were isolated from cells of N. gonorrhoeae CS-7 previously labeled by incubation

with [1,2-3H]cholesterol. Cell membranes were centrifuged to equilibrium on linear sucrose density gradients (15 to 65%), and the radioactivity associated with each of the membranes was counted. Although cholesterol was associated with both membranes, the cytoplasmic membrane (buoyant density = 1.185 g/cm3) bound

704

MILLER ET AL.

INFECT. IMMUN.

0.2 O-

3

E CP

w

0'

4

z 0

U)

-J

0.1

C -i

0

w

z

U) w -J

0 a

x C.)

C.

Lu

0

0

HOURS 100 B 801

601 i-

I-

40_ 20 2

0

HOURS

FIG. 5. Effect of autolysis on the binding of [1,2-3H~cholesterol to N. gonorrhoeae JW-31. (A) Cholesterol bound (0, without Mg2"; O. with Mg2") and [4C]adenine release (0, without Mg2"; , with 10 mM Mg2") as a function of time. (B) Decrease in turbidity (Klett units) as a function of time in the presence (l) and absence (0) of 10 mM Mg2+.

TABLE 1. Effect of cellular metabolic activity on the binding of [1,2-3Hicholesterol by N. gonorrhoeae CS-7a Cholesterol Conditionsbound (%)b

Conditions

Control (aerobic) Control (N2)c Hg-treated cells Azide (10 mM) Glucose (28 mM) Azide (10 mM) + glucose (28 mM)

100.0 107.0 104.0 97.8 105.0 102.0

min) (ti/

02uptake

1.3 ND 0 0 27.5 0.6

a Values represent the average of three experiments. ND, Not determined. b Percent cholesterol bound to control cells after 120 min of incubation at 370C. eCells were incubated in sealed tubes and flushed with N2 periodically before and during the binding assay. d Cells (470 Klett units) were treated with HgC12 (10 mM) for 10 min, washed once, and suspended to the original volume as described for control cells.

nearly twice as much [1,2-3H]cholesterol as the outer membrane (buoyant density = 1.255 g/cm3) when compared on the basis of protein concentration (0.66 and 0.36 ng/mg of protein,

respectively). Characterization of bound cholesterol. To determine whether the bound cholesterol was chemically modified by esterification, cells were incubated in the presence of [1,2-3H]cholesterol for 2 h, followed by isolation of the inner and outer membranes. The cell membranes were extracted with chloroform-methanol (2:1) for 5 h, and the extract was analyzed by thin-layer chromatography. The results (data not shown) indicated that ca. 99% of the bound radioactivity in both membrane preparations migrated with an Rf value corresponding to free cholesterol (Rf = 0.25). Negligible radioactivity was associated with areas corresponding to the Rf values of cholesterol acetate (Rf = 0.80), cholesterol palmitate (Rf = 0.95), or cholesterol oleate (Rf= 0.95).

CHOLESTEROL BINDING

VOL. 22, 1978

705

0.5

C

0

06

z

0

0.2

w -J

0

0.

0.

0

HOURS 6. cholesterol cell envelopes of N. gonorrhoeae CS-7 and other gramFIG. Comparison of binding by negative bacteria. Symbols: (0) N. gonorrhoeae CS-7; (5) S. typhimurium TA1535 (deep rough strain); (-) P.

aeruginosa; (A) S. typhimurium DB21 (wild-type).

TABLE 2. Effect of unlabeled steroids on the Inhibition of [1,2-3H]cholesterol binding binding of [1,2-3Hcholesterol to N. gonorrhoeae by unlabeled steroids. Additional information CS-7 concerning the binding of cholesterol was obtained by competition studies with other stebound (%)b Unlabeled steroids roids. Inhibition of [1,2-3H]cholesterol binding by a 60-min preincubation with various concen- None ............. 100.0 trations of unlabeled cholesterol demonstrated Cholesterol ............ 67.1 that the inhibition was concentration dependent Cholesterol acetate ...... 87.8 ...... between 1 and 8 ,ug of cholesterol per ml. Below Progesterone .......................... 121.0 100. 1 ,ug/ml no inhibition was observed; maximum Testosterone ..................0........ ....... 97.6 (32%) inhibition was observed at concentrations Cortisone Unlabeled steroids were added to the cells 60 min 28,ug/ml. To insure maximum inhibition a concentration of 20 ,ug/ml was used for all other before addition of the [3H]cholesterol at a final consteroids tested. Only cholesterol, and to a lesser centration of 20 ,tg/ml. b Percentage of the label bound to the control cells extent cholesterol acetate, inhibited [1,2-3H]cholesterol binding (Table 2). Testosterone and cor- after 120 min of incubation at 370C. Values represent tisone had little effect, and progesterone actually the average of three experiments. increased the binding of the labeled cholesterol slightly. Therefore, it appeared that progester- binding of cholesterol by microorganisms in vivo one and cholesterol did not compete for the might be influenced by the presence of serum same binding site on the cell surface. albumin. Therefore, we examined the binding of Effect of BSA on cholesterol binding. The [1,2-3H]cholesterol by BSA and the effect of ............

a

706

INFECT. IMMUN.

MILLER ET AL.

BSA on the binding of cholesterol by N. gonorrhoeae CS-7. The results (data not shown) indicated that the binding of [1,2-3H]cholesterol by BSA was dependent on the BSA concentration. Maximum binding (63% of added cholesterol) was achieved with a BSA concentration of 5 mg/ml, with no significant increase in cholesterol binding at higher concentrations of BSA. The effect of BSA on the binding of cholesterol by N. gonorrhoeae CS-7 is shown in Table 3. Concentrations of BSA

Binding of cholesterol by Neisseria gonorrhoeae.

Vol. 22, No. 3 INFECTION AND IMMUNITY, Dec. 1978, p. 698-708 0019-.9567/78/0022-0698$02.00/0 Copyright i 1978 American Society for Microbiology Prin...
1MB Sizes 0 Downloads 0 Views