Protein I of Neisseria gonorrhoeae outer membrane is a porin

FEMS Microbiology Letters 12 (1981) 305-309 Published by Elsevier/North-Holland Biomedical Press

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Protein I of Neisseria gonorrhoeae outer membrane is a porin James T. Douglas, Michael D. Lee * and Hiroshi N i k a i d o

*

Department of Microbiology, Unit:ersiO, of Hawaii. Manoa Campus, ttonolulu, Hawaii 96822, and * Department of Microbiologv and Immunology, Universi(v of California, Berkeley, CA 94720, U.S.A.

Received and accepted 10 August 1981

1. I N T R O D U C T I O N

2. MATERIALS A N D M E T H O D S

The outer membrane of Gram-negative bacteria serves as a selective permeability barrier that allows the penetration of small, hydrophilic solutes [1]. In Escherichia coli [2], Salmonella typhimurium [3], and Pseudomonas aeruginosa [4], this hydrophilic penetration pathway was shown to be produced by major outer membrane proteins called porins [2,3], which have apparent Mrs in the range of 34000-38000. The proteins of the ohter membrane of Neisseria gonorrhoeae have received much attention. The membrane contains only a few major proteins, and it has been proposed [5] to call the most prominent protein with an apparent M~ of 32000 to 37000, "protein I". This protein has been called major (or principal) outer membrane protein in the past [6,7]. Although minor proteins with lower apparent Mrs have been found to play roles in the association of gonococci with leukocytes or in their attachment to host cell surfaces and their resistance to proteases [8-11], the function of protein I has not been known. In this communication, using reconstituted vesicles, we show that protein I is gonococcal porin. During the course of this work, an abstract [12] reporting the protein-Iinduced increase in conductance of planar lipid bilayers has appeared.

2.1. The preparation of outer membrane N. gonorrhoeae strain F62 (type P + + / T ) , a gift of J. James, was grown on a clear typing agar medium. The medium is a modification (J. James, personal communication) of that described in [9], and contained, per liter, Difco Proteose Peptone No. 3, 7.5 g; BBL Thiotone peptone, 7.5 g; NaC1, 5g; KH2PO4, lg; KzHPO4, 4g; BBL soluble starch, 1 g; and Difco Noble Agar, 12 g, and 10 ml of IsoVitale X (BBL) was added after cooling. The bacteria were harvested from 25 plates (15 cm in diameter) after 20h, washed in 10 mM N-2hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-NaOH buffer, pH 7.4, resuspended in this buffer and ruptured through two passages, at 15000 p.s.i., in a Ribi cell fractionator. (This and all subsequent steps were carried out at 0-4°C.) The unbroken cells and large fragments were removed by centrifuging at 12000 × g for 20 min. Then the membranes were sedimented by centrifuging at 170000 × g for 1 h, and the resuspended membranes were fractionated on a linear, 20% to 70% (w/v), gradient of sucrose in 10 mM HEPESNaOH, pH 7.4. After 17 h of centrifugation in a Spinco SW27 rotor, the membranes were separated into two diffuse zones; the lower, whitish zone was saved as the outer membrane fraction.

0378-1097/81/0000-0000/$02.75 't 1981 Federation of European Microbiological Societies

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2.2. Polyacrylamide gel electrophoresis P o l y a c r y l a m i d e gel electrophoresis in slab gels was carried out in the presence of 0.2% SDS [13].

strength t h a n most other m e m b r a n e proteins. Thus a m a t e r i a l enriched in the outer m e m b r a n e was o b t a i n e d b y b l e n d i n g the cells in 0.9% N a C 1 / I % e t h a n o l a m i n e for 1 min in a Sorvall O m n i m i x e r ,

2.3. Reconstitution of vesicles and assay of permeability P h o s p h o l i p i d vesicles c o n t a i n i n g various protein fractions were r e c o n s t i t u t e d as follows. A m i x t u r e of 3.3 /~mol of egg p h o s p h a t i d y l c h o l i n e (Sigma, T y p e I X - E ) a n d 0.09 /~mol of dic e t y l p h o s p h a t e (Sigma) in c h l o r o f o r m - m e t h a n o l (2: 1) was d r i e d up on the b o t t o m of a test tube, then an aqueous solution of the test p r o t e i n (total volume, 0.2 ml) was a d d e d a n d the suspension was sonicated until it b e c a m e translucent. The suspension was d r i e d again a n d r e s u s p e n d e d in 0.3 ml of 17% ( w / v ) D e x t r a n T-20 as described a l r e a d y [14], except that l i p o s o m e s were filtered through a 3/~m M i l l i p o r e filter in o r d e r to r e m o v e large aggregates of lipids. The p e r m e a b i l i t y of vesicle m e m b r a n e s was tested b y diluting the vesicles into isotonic solutions of various sugars, a n d b y observing the rates of initial swelling of vesicles caused b y water influx following the influx of sugar molecules t h r o u g h the p r o t e i n channel [15]. A l t h o u g h the p r o t e i n fractions used for r e c o n s t i t u t i o n c o n t a i n e d up to 40 /~g of s o d i u m cholate, control experiments showed that this a m o u n t of cholate d i d not p r o duce any interference with the f o r m a t i o n a n d swelling of the liposomes.

2.4. Other methods Protein in the m e m b r a n e p r e p a r a t i o n s a n d their extracts was d e t e r m i n e d b y the L o w r y p r o c e d u r e [16].

3. R E S U L T S A N D D I S C U S S I O N

3.1. Prefiminary studies A p a r t i a l p u r i f i c a t i o n of p r o t e i n I was achieved b y taking a d v a n t a g e of the fact that it is less r e a d i l y solubilized b y m i l d detergents at low ionic

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Fig. 1. HPLC gel filtration pattern of outer membrane proteins from N. gonorrhoeae. Outer membrane (containing 600 /~g protein) prepared as described in METHODS was extracted in a mixture containing 2% sodium cholate, 1 M NaC1, and 0.05 M Tris-HC1 buffer, pH 8.0, with a total volume of 0.2 ml, with sonication at room temperature. After centrifugation at 210 000 ×g for 50 min at 4°C, 150/~1 of the supernatant was injected into an HPLC system equipped with a 0.75 ×60 cm column of TSK SW3000 and a Perkin-Elmer LC-75 variable wavelength detector. The column was eluted with a solution containing 2% sodium cholate, 0.5 m Na2SO4, and 0.01 M sodium phosphate buffer, pH 8.0, at room temperature at a flow rate of 0.5 ml/min. Fractions were collected and numbered as shown in the figure. Protein concentrations were determined from the peak area, by using, as standard, the peak area of bovine serum albumin chromatographed under similar conditions.

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and by recovering a fraction that was not sedimented at 1 7 0 0 0 X g for 20 min, but was sedimented after 90 min at 45 000 x g. Extraction of this material with 50 mM glycine-NaOH, pH 9.0/10 mM E D T A / 1 . 5 % Na-deoxycholate left behind a material that could be sedimented by centrifugation at 100000 X g for 3 h at 4°C. This "pellet" material was strongly enriched in protein I (and also presumably in peptidoglycan), and showed strong porin activity in the liposome swelling assay. However, since the preparation was always contaminated by a few minor proteins, a

more systematic purification approach, described below, was initiated.

3.2. Purification of protein I After using several detergents under a limited set of conditions, we found that cholate at high ionic strength produces efficient extraction of protein I from outer membranes, prepared as described in METHODS. The solubilized proteins were then separated by gel filtration using high pressure liquid chromatography (HPLC); HPLC was ad-

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Fig. 2. SDS polyacrylamide gel electrophoresis of fractions separated by gel filtration. Fractions obtained in the experiment of Fig. 1 were dialyzed against a solution containing 0.05% sodium cholate and 5 m M Tris-HC1 buffer, pH 7.4, overnight at 4°C. A portion of each dialyzed fraction containing 2 - 6 ~g of protein (depending on the number of protein components present) was applied to the slab gel analysis as described in METHODS. Lanes b, c, d, e, and f contain fractions 3, 4, 5, 7, and 8 of Fig. 1, respectively. The original outer membrane sample (g) and its cholate-NaC1 extract (h) have also been included in the analysis. Lane a contained M r standards at 92 500, 66 200, 45 000, 31000, 21 500, and 14400. The electropherogram also shows the presence of traces of a contaminant with an apparent M r of 18000. The position of protein I, which migrated as an M r 37000 protein, is also shown by an arrow.

308 vantageous because of high resolution, rapidity, and less extensive dilution of the sample. We found that conditions were less critical for the separation of analytical scale samples (up to 2/~g) of the cholate extract, but with preparative scale samples (up to 600/~g) proteins tended to aggregate together and become eluted at the void volume even in the presence of cholate, unless very high concentrations of both sodium cholate and Na 2SO4 were present in the elution buffer. The elution profile of a typical preparative run is shown in Fig. 1. The fractions obtained in this separation gave the SDS polyacrylamide gel patterns shown in Fig. 2; clearly a satisfactory separation of protein I from other proteins has been achieved in fraction 3.

3.3. Channel-forming activity of protein I When a portion of each fraction containing 0.8 /~g protein was reconstituted with phospholipids, and the permeability of the liposomes to glucose assayed by their rates of swelling, only the liposomes containing fraction 3 showed strong permeability (Fig. 3). This shows that only protein I exhibits porin activity under our conditions of assay. Liposomes reconstituted with fraction 3 were also tested for permeability to several sugars with varying sizes. In the penetration of solutes through a channel, the wider the channel is, the less strongly affected are the rates of solute diffusion by the size of the solute [17]. We found that solute-size dependence with gonococcal porin channel was somewhat less than that seen with the E. coli porin channel [18], and was in this sense reminiscent of that seen with Pseudomonas aeruginosa porin (H. Nikaido, to be published; see also [19]). Thus N. gonorrhoeae porin may produce a channel somewhat larger than the E. eoli porin channel; the properties of the gonococcal porin channel will be reported elsewhere. It is noted that protein I is eluted from the gel filtration column far ahead of other proteins of comparable or only slightly lower MrS (Fig. 1).

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Minutes Fig. 3. Swellingof liposomescontaining various fractions. Liposomes were made by adding portions (each containing 0.8 /zg protein) of dialyzed eluate fractions from the experiment of Fig. 1 (see the legend for Fig. 2), to phospholipids as described in METHODS. The liposome suspension (15 ~1) was then diluted into an isotonic solution of glucose (0.6 ml), and the swelling due to the influx of glucose through porin channels was monitored by the decrease of absorbance. The curves labeled 3, 4, 5, and 8 show experiments with liposomes reconstituted with dialyzed fractions 3, 4, 5, and 8, respectively,of Fig. 1. The control without any proteins added showed no swelling (not shown).

Although it has not been possible to run a regular set of soluble proteins of known Mrs under the same conditions, the gel filtration pattern of these proteins under somewhat different conditions suggest that protein I was eluted from the column as an oligomer. It is known that E. coli and S. typhimurium porins exist as stable trimers [20,21], and cross-linking studies suggest that protein I exists as a trimer in intact cells [22].

ACKNOWLEDGEMENTS This research has been supported in part by a USPHS Grant AI-09644 and an American Cancer Society Grant BC-20. We thank Dr. N. Vedros for

309 t h e u s e o f R i b i f r a c t i o n a t o r , a n d D r s . J. J a m e s a n d G e o . F. B r o o k s for t h e i r h e l p i n t h e c u l t i v a t i o n o f t h e cells.

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