- Email: [email protected]
Identification of three porins in the outer membrane of Bacteroides fragilis
ELSEVIER
FEMS Microbiology Letters 127 (1995) 181-186
Identification of three porins in the outer membrane of Bacteroides fragilis *, Yoriko
Kobayashi, Masayuki Nakano, Maki Sakurai, Naomasa Gotoh, Takeshi Nishino
Katsunori Kanazawa
Department of Microbiology, Kyoto Pharmaceutical University Yamashina, Kyoto, Japan
Received 19 December 1994; revised 1 February 1995; accepted 6 February 1995
Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides frugilis; three (A4, 51000, 92 000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M, 5.5 000 outer membrane protein showed no detectable pore-forming activity. The three B. fiagilis pork formed pores that allowed the penetration of uncharged saccharides of h4, lower than 340-400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins. Keywords:
Porin; Outer membrane
protein; Transmembrane
diffusion;
1. Introduction
The penetration of small hydrophilic molecules across the outer membrane of Gram-negative bacteria is governed by diffusion pores made of pore-forming proteins, or porins. Many porins have been purified from aerobic Gram-negative bacteria and studied in detail [l]. However, little is known about porins of anaerobic Gram-negative bacteria, except Pelobacter venetianus [2]. Bacteroides fragilis has been of particular clinical
interest as an anaerobic opportunistic pathogen which
* Corresponding author. Present address: Sumitomo Pharmaceuticals Research Center, Discovery Research Laboratories III, 3-1-98 Kasugade Naka, Konohana, Osaka, 554, Japan. Tel: (+ 816) 466 5271; Fax: (+ 81-6)466 5491. 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(95)00059-3
of European
Microbiological
Bacteroides
fragilis
is resistant to a number of structurally unrelated antibiotics. The permeability barrier function of the outer membrane has been considered as one of the reasons for this antibiotic resistance. It has been reported that the limited diffusion of /3-lactams through the outer membrane of B. fragilis caused its resistance to these antibiotics, this acting in synergy with a periplasmic /3-lactamase [3-71. Previous studies on the intact outer membrane of B. fragilis revealed the presence of rather small hydrophilic pores [8,9], suggesting the limited outer membrane permeability of this bacteria. However, porins of B. fragilis have not previously been characterised, nor their precise role in solute diffusion been determined. Experiments were conducted to purify and to characterise porins in the outer membrane of B. fragilis. This study reports that the three outer memSocieties.
AII rights reserved
182
K. Kanazawa et al. /FEMS Microbiology Letters 127 (1995) 181-186
brane proteins were found to form the relatively small diffusion pores in the reconstituted liposomes.
2. Materials and methods Bacteroides fragilis ATCC25285 was grown as previously described [S]. The outer membrane was isolated according to the method of Mizushima and Yamada [lo], except that 15 mM EDTA final concentration was added to the cell suspension (since B. fragilis is more resistant to EDTA than E. coli), and that isopycnic sucrose density gradient centrifugation was not performed, since the purity of the outer membranes was not improved by this treatment (data not shown). The outer membrane proteins were extracted from the purified outer membrane with a solution containing 2% (w/v> nonanoyl-N-methylglucamide (MEGA-91, 5 mM EDTA and 10 mM Tris-HCl, pH 8.0 (buffer A) at room temparature. The centrifuged supematant (200000 X g for 1 h at 25“ C) was subjected to DEAE ion-exchange chromatography. The elution profile was shown in Fig. 1. The fractions (III, IV and V) showed a porin activity as examined by the liposome swelling assay. Fraction III, IV and V contained 92K, 125K and 55K,
0.7-
and 51K proteins, respectively, as major proteins and a small amount of other proteins. These three fractions were then subjected to a further column chromatography. Fraction III was applied onto a Phenyl TOYOPEARL650S (TOSOH) column equilibrated with 0.01% (w/v) C,zE,, 2 M ammonium sulfate, 20% (w/v) glycerol, 100 mM Tris-HCl, pH 7.5. The column was eluted with 3% (w/v> sodium cholate, 10 mM Tris-HCl, pH 8.2, and then with 2% (w/v> MEGA-9, 100 mM CaC12, 10 mM Tris-HCI, pH 8.5 (buffer B). A homogeneous preparation of 92K protein was obtained in buffer B eluates. Fraction IV was applied onto a MonoQ HR5/5 FPLC column (Pharmacia) equilibrated with buffer A and the column was eluted with a O-O.5 M linear gradient of NaCl in the same buffer. The 125K protein was obtained in the flow-through fraction and the 55K protein was eluted at a NaCl concentration around 0.2 M. Fraction V was subjected again to DEAE ion-exchange HPLC chromatography under identical conditions to the first chromatography. The 51K protein was eluted at a NaCl concentration of around 0.4 M. The purified proteins were reconstituted into liposome membranes as [ll], except that SM-2 Bio-beads were not added. Preparation of proteoliposomes con-
1 H
0.6 -
E gasz zo.4
-
E) 2:0.3ho.2Ii
a
O.l-
Fraction
Number
Fig. 1. Elution profile of the outer membrane proteins of B. fragilis by the DEAF ion-exchange HPLC. The purified outer membrane (40 mg of protein) was mixed with 5 ml of a solution containing 2% MEGA-9, 5 mM EDTA and 10 mM Tris-HCl, pH 8.0, and the supematant was applied on a TSKgel DEAE-SPW column (7.5 mm id. X 7.5 cm) equilibrated with buffer A (see Materials and methods). The column was eluted with a O-O.5 M linear gradient of sodium chloride in buffer A at a flow rate of 0.5 ml/min. Eluates were collected of a rate of 1 ml per fraction. An absorbance at 280 run was recorded with a flow-type UV detector.
K. Kamzawa
et al./FEMS
rate of glucose through the purified outer membrane appeared to be roughly one third of that through the MEGA- extracts at 5 and 10 pg of protein per pmol of phospholipid (data not shown), indicating that the outer membrane porins were extracted efficiently with 2% (w/v> MEGA-9. The four outer membrane proteins (51K, 55K, 92K and 125K protein) were purified to apparent homogeneity using SDS-PAGE (Fig. 2, c-f) by the procedures described above, and in the legend to Fig. 1. The purification of the proteins, other than the 92K protein, was achieved by anion-exchange chromatography. The 92K protein was purified by hydrophobic interaction chromatography. Using the procedure described above, we could separate 92K protein from other proteins which were readily eluted from the hydrophobic adsorbent. Three proteins (51K, 92K and 125K protein) produced glucose-permeable pores in liposome membranes, as examined by the liposome swelling assay, but 55K protein showed little pore-forming activity
taining the purified outer membrane was done by the procedure described earlier [l], except that a lipid mixture consisting of phosphatidylcholine and dicetylphosphate at a molar ratio of 97:3 was used. The liposome swelling assay and calculation of the diffusion rate were performed as reported previously [l]. The porin-free liposome was impermeable to the test solutes used.
3. Results and discussion 3.1. Purification and identification of porins The outer membrane of B. fragilis was successively separated from the inner membrane by the method of Mizushima and Yamada [lo] with some modifications. From the purified outer membrane, 95% of the outer membrane proteins were extracted with nonionic surfactant, MEGA- (Fig. 2. a,b). The diffusion
Mr Mr
a
183
Microbiology Letters 127 (1995) 181-186
C
d
e
f
Mr
b
Fig. 2. SDS-PAGE of the purified outer membrane, 2% MEGA-9-extracted outer membrane proteins and the purified outer membrane proteins. Samples were heated in sample buffer 1141 at 95” C for 5 min. Lanes CM,): M, markers: myosin (200000), /3-galactosidase (1162501, phosphorylase b (97400), bovine serum albumin (662001, ovalbumin (45000), carbonic anhydrase (31000), soybean trypsin inhibitor (21500), lysozyme (14400). (a): outer membrane; (b):2% MEGA-9-extracted outer membrane proteins; cc): 55K protein; Cd): 51K protein; (e): 125K protein; (0: 92K protein.
184
K. Kanazawa et al. / FEMS Microbiology Letters 127 (1995) 181-186
(data not shown).
e
0.3
d 2 2 k 8
0.2
0.1
0 0
5
10
PROTEIN / LIPID (jig/p
15 mole)
Fig. 3. Effect of protein concentration on the diffusion rate of glucose. Proteoliposomes were reconstituted from 3 pmol of phosphatidylcholine, 0.093 pmol of dicetyl phosphate and the specified amount of outer membrane or porins in 0.5 ml of 40 mosM stachyose-1 mM MOPS buffer (pH 7.2) as described in Materials and methods. The test solute was 40 mosM glucose in 1 mM MOPS buffer (pH 7.2). The liposome swelling assay was done as described in Materials and methods. Symbols: 0, B. fragilis outer membrane; n , 51K porin; 0, 125K porin; 0, 92K porin; A, E. coli OmpF porin; A, E. coli PhoE porin.
These results indicate that 51K, 92K and 125K protein are porins in the outer membrane of B. fiagilis. To our knowledge this is the first report of purification of Bacteroides porins. Previously, it was reported that the deficiency of outer membrane proteins of Zt4, 49-50K were found in some clinical isolates of B. fragilis, which are resistant to cefoxitin, one of the @-lactam antibiotics which is thought to penetrate into the cell via porins [7]. This report suggested the presence of porin proteins of M, 49-50K in the outer membrane of B. fiagilis, but it is not clear at present whether the newly purified 51K protein is related to the reported proteins. 3.2. Characterization of permeability properties of porins
The effect of the amount of porin proteins on the diffusion
rate of glucose was determined.
The results
C
01.2. 1
2
1
3456
2
3456
Mr, SACCHARIDE ( x 102) Fig. 4. Permeability properties of the liposome membranes reconstituted from the purified outer membrane and porins of B. fragilis. Proteoliposomes were reconstituted from 3 pmol of phosphatidylcholine, 0.093 pmol of dicetyl phosphate and the purified outer membrane (60 /lg of protein), 30 pg of 92K porin and 125K porin, or 6 /.~g of 51K porin in 0.5 ml of 40 mosM stachyose-1 mM MOPS buffer (pH 7.2) as described in Materials and methods. The test solutes were 40 mosM saccharides in 1 mM MOPS buffer (pH 7.2). The liposome swelling assay was done as described in Materials and methods. The diffusion rates are presented as relative to the diffusion rate of arabinose for the purified outer membrane (A>, 125K porin (B), 92K porin (C) and 51K porin (D). Saccharides used were: 1, arabmose; 2, glucose; 3, mannose; 4, galactose; 5, a-methyl glucopiranoside; 6, a-methyl mannopiranoside; 7, a-methyl galactopiranoside; 8, sucrose; 9, maltose; 10, treharose; 11, melezitose; 12, maltotriose; and 13, raffinose.
K. Kanazawa et al. / FEMS Microbiology
showed that the diffusion rates increased as the amount of purified porins was raised, indicating that the diffusion rate depends on the amount of porin added (Fig. 3). However, the diffusion rates were not linearly related to the amount of the protein added as observed before [12]. The reason for this is not clear, though the diffusion of glucose was linearly related to the amount of purified outer membrane added. As shown in Fig. 3, the solute diffusion through B. fragilis porin appeared to be inefficient compared with E. coli porin, with 92K porin of B. fragilis demonstrating the highest pore-forming activity. Differences in the diffusion rate of uncharged saccharides, e.g. glucose, may be due to the difference of pore size. However, a possibility of artifacts, such as the influence of surfactants, cannot be ignored. In fact, purified porins of B. fragilis appeared to be more unstable in the surfactant solution than E. coli porins. The pore-forming activity of 51K and 125K porin was gradually reduced during storage over several weeks in buffer A. Compared with these two porins, 92K porin seemed to be a little more stable. In order to further characterise the pores formed by these identified porins, the diffusion rates of uncharged saccharides with different M, through these porins were determined by the liposome swelling assay and compared with those through the purified outer membrane. The diffusion rates were plotted as described by Nakae et al. [13]. The results in Fig. 4 showed that the size of pores formed by 51K and 92K porin are similar and they are slightly smaller than that formed by 125K porin. The three purified porins allowed the diffusion of pentoses and hexoses in the manner of a molecular sieve, but the diffusion rates of the saccharides larger than disaccharides were low (Fig. 4, B-D). A similar result was obtained for the purified outer membrane (Fig. 4, A), indicating that the pore-forming activity of the outer membrane porins was well preserved in the purified porins under this experimental condition. As shown in Fig. 4, the diffusion rates of disaccharides were low but varied. The reason for this is not clear, but it may be due to the difference of pore structure in addition to the difference of pore size. In this paper we present the possibility that the purified 51K, 92K and 125K outer membrane proteins are porins in the outer membrane of B. fragilis. The data also indicate that these porins form relatively small
Letters 127 (1995) 181-186
185
pores which allow the diffusion of saccharides of M, smaller than 340-400. Furthermore, the permeability properties of these porins are consistent with those of intact B. fragilis [8,9]. These results suggest that the identified pot-ins play an important part in the solutes diffusion through the outer membrane of B. fragilis. Therefore, it is likely that an intrinsic resistance of B. fiugilis to multiple antibiotics is probably due to the low antibiotics diffusion through the outer membrane into the cells. In order to confirm the involvement of the identified porins in drug resistance of B. fiagilis, the diffusion of /?-lactam antibiotics through the B. fragilis porin pores in vitro are being determined.
Acknowledgements We thank Prof. Taiji Nakae for his valuable vice and helpful discussions.
ad-
References [l] Nakae, T. (1986) Outer membrane permeability of bacteria. CRC Crit. Rev. Microbial. 13, l-62. [2] Schmid, A., Benz, R. and Schink, B. (1991) Identification of two porins in Pelobacter uenetianus fermenting high-molecular-mass polyethylene glycols. J. Bacterial. 173,4909-4913. [3] Cuchural, G.J., Hurlbut, S., Malamy, M.H. and Tally, F.P. (1988) Permeability to p-lactams in Bacteroides fiagilis. J. Antimicrob. Chemother. 22, 785-790. [4] Olsson, B., Dombusch, K. and Nord, C.E. (1979) Factors contributing to /3-lactam antibiotics in Bacteroides fragilis. Antimicrob. Agents Chemother. 15, 263-268. [S] Cuchural, G.J., Tally, F.P., Jacobus, N.V., Marsh, P.K. and Mayhew, J.W. (19831 Cefoxitin inactivation by Bacteroides fragilis. Antimicrob. Agents Chemother. 24, 936-940. [6] Cuchural, G.J., Malamy, M.H. and Tally, F.P. (1986) /3Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob. Agents Chemother. 30, 645-648. [7] Piddock, L.J.V. and Wise, R. (19871 Cefoxitin resistance in Bacteroides species: Evidence indicating two mechanisms causing decreased susceptibility. J. Antimicrob. Chemother. 19, 161-170. [8] Kobayashi, Y. and Nakae, T. (1986) The permeability property of the outer membrane of Bacteroides fragilis, a strictly anaerobic opportunistic pathogen. Biochem. Biophys. Res. Commun. 141, 292-298. [9] Kobayashi, Y., Akatsuka, A. and Nakae, T. (1987) Electron microscopic visualization of the outer membrane permeabil-
186
K. Kanazawa et al. / FEMS Microbiology
ity of Bacteroides fragilis. FEMS Microbial. Len. 48, 325329. [lo] Mizushima, S. and Yamada, H. (1975) Isolation and characterization of two outer membrane preparations from Escherichia coli. Biochim. Biophys. Acta 375, 44-53. [ll] Yoshihara, E. and Nakae, T. (1989) Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores. J. Biol. Chem. 264, 6297-6301. [12] Ishii, J. and Nakae, T. (1988) Size of diffusion pore of
Letters 127 (199.5) 181-186
Alcaligenes faecalis. Antimicrob. Agents Chemother. 32, 378-384. [13] Nakae, R. and Nakae, T. (1982) Diffusion of aminoglycoside antibiotics across the outer membrane of Escherichia coli. Antimicrob. Agents Chemother. 22, 554-559. [14] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature London 227, 680-685.
FEMS Microbiology Letters 127 (1995) 181-186
Identification of three porins in the outer membrane of Bacteroides fragilis *, Yoriko
Kobayashi, Masayuki Nakano, Maki Sakurai, Naomasa Gotoh, Takeshi Nishino
Katsunori Kanazawa
Department of Microbiology, Kyoto Pharmaceutical University Yamashina, Kyoto, Japan
Received 19 December 1994; revised 1 February 1995; accepted 6 February 1995
Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides frugilis; three (A4, 51000, 92 000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M, 5.5 000 outer membrane protein showed no detectable pore-forming activity. The three B. fiagilis pork formed pores that allowed the penetration of uncharged saccharides of h4, lower than 340-400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins. Keywords:
Porin; Outer membrane
protein; Transmembrane
diffusion;
1. Introduction
The penetration of small hydrophilic molecules across the outer membrane of Gram-negative bacteria is governed by diffusion pores made of pore-forming proteins, or porins. Many porins have been purified from aerobic Gram-negative bacteria and studied in detail [l]. However, little is known about porins of anaerobic Gram-negative bacteria, except Pelobacter venetianus [2]. Bacteroides fragilis has been of particular clinical
interest as an anaerobic opportunistic pathogen which
* Corresponding author. Present address: Sumitomo Pharmaceuticals Research Center, Discovery Research Laboratories III, 3-1-98 Kasugade Naka, Konohana, Osaka, 554, Japan. Tel: (+ 816) 466 5271; Fax: (+ 81-6)466 5491. 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(95)00059-3
of European
Microbiological
Bacteroides
fragilis
is resistant to a number of structurally unrelated antibiotics. The permeability barrier function of the outer membrane has been considered as one of the reasons for this antibiotic resistance. It has been reported that the limited diffusion of /3-lactams through the outer membrane of B. fragilis caused its resistance to these antibiotics, this acting in synergy with a periplasmic /3-lactamase [3-71. Previous studies on the intact outer membrane of B. fragilis revealed the presence of rather small hydrophilic pores [8,9], suggesting the limited outer membrane permeability of this bacteria. However, porins of B. fragilis have not previously been characterised, nor their precise role in solute diffusion been determined. Experiments were conducted to purify and to characterise porins in the outer membrane of B. fragilis. This study reports that the three outer memSocieties.
AII rights reserved
182
K. Kanazawa et al. /FEMS Microbiology Letters 127 (1995) 181-186
brane proteins were found to form the relatively small diffusion pores in the reconstituted liposomes.
2. Materials and methods Bacteroides fragilis ATCC25285 was grown as previously described [S]. The outer membrane was isolated according to the method of Mizushima and Yamada [lo], except that 15 mM EDTA final concentration was added to the cell suspension (since B. fragilis is more resistant to EDTA than E. coli), and that isopycnic sucrose density gradient centrifugation was not performed, since the purity of the outer membranes was not improved by this treatment (data not shown). The outer membrane proteins were extracted from the purified outer membrane with a solution containing 2% (w/v> nonanoyl-N-methylglucamide (MEGA-91, 5 mM EDTA and 10 mM Tris-HCl, pH 8.0 (buffer A) at room temparature. The centrifuged supematant (200000 X g for 1 h at 25“ C) was subjected to DEAE ion-exchange chromatography. The elution profile was shown in Fig. 1. The fractions (III, IV and V) showed a porin activity as examined by the liposome swelling assay. Fraction III, IV and V contained 92K, 125K and 55K,
0.7-
and 51K proteins, respectively, as major proteins and a small amount of other proteins. These three fractions were then subjected to a further column chromatography. Fraction III was applied onto a Phenyl TOYOPEARL650S (TOSOH) column equilibrated with 0.01% (w/v) C,zE,, 2 M ammonium sulfate, 20% (w/v) glycerol, 100 mM Tris-HCl, pH 7.5. The column was eluted with 3% (w/v> sodium cholate, 10 mM Tris-HCl, pH 8.2, and then with 2% (w/v> MEGA-9, 100 mM CaC12, 10 mM Tris-HCI, pH 8.5 (buffer B). A homogeneous preparation of 92K protein was obtained in buffer B eluates. Fraction IV was applied onto a MonoQ HR5/5 FPLC column (Pharmacia) equilibrated with buffer A and the column was eluted with a O-O.5 M linear gradient of NaCl in the same buffer. The 125K protein was obtained in the flow-through fraction and the 55K protein was eluted at a NaCl concentration around 0.2 M. Fraction V was subjected again to DEAE ion-exchange HPLC chromatography under identical conditions to the first chromatography. The 51K protein was eluted at a NaCl concentration of around 0.4 M. The purified proteins were reconstituted into liposome membranes as [ll], except that SM-2 Bio-beads were not added. Preparation of proteoliposomes con-
1 H
0.6 -
E gasz zo.4
-
E) 2:0.3ho.2Ii
a
O.l-
Fraction
Number
Fig. 1. Elution profile of the outer membrane proteins of B. fragilis by the DEAF ion-exchange HPLC. The purified outer membrane (40 mg of protein) was mixed with 5 ml of a solution containing 2% MEGA-9, 5 mM EDTA and 10 mM Tris-HCl, pH 8.0, and the supematant was applied on a TSKgel DEAE-SPW column (7.5 mm id. X 7.5 cm) equilibrated with buffer A (see Materials and methods). The column was eluted with a O-O.5 M linear gradient of sodium chloride in buffer A at a flow rate of 0.5 ml/min. Eluates were collected of a rate of 1 ml per fraction. An absorbance at 280 run was recorded with a flow-type UV detector.
K. Kamzawa
et al./FEMS
rate of glucose through the purified outer membrane appeared to be roughly one third of that through the MEGA- extracts at 5 and 10 pg of protein per pmol of phospholipid (data not shown), indicating that the outer membrane porins were extracted efficiently with 2% (w/v> MEGA-9. The four outer membrane proteins (51K, 55K, 92K and 125K protein) were purified to apparent homogeneity using SDS-PAGE (Fig. 2, c-f) by the procedures described above, and in the legend to Fig. 1. The purification of the proteins, other than the 92K protein, was achieved by anion-exchange chromatography. The 92K protein was purified by hydrophobic interaction chromatography. Using the procedure described above, we could separate 92K protein from other proteins which were readily eluted from the hydrophobic adsorbent. Three proteins (51K, 92K and 125K protein) produced glucose-permeable pores in liposome membranes, as examined by the liposome swelling assay, but 55K protein showed little pore-forming activity
taining the purified outer membrane was done by the procedure described earlier [l], except that a lipid mixture consisting of phosphatidylcholine and dicetylphosphate at a molar ratio of 97:3 was used. The liposome swelling assay and calculation of the diffusion rate were performed as reported previously [l]. The porin-free liposome was impermeable to the test solutes used.
3. Results and discussion 3.1. Purification and identification of porins The outer membrane of B. fragilis was successively separated from the inner membrane by the method of Mizushima and Yamada [lo] with some modifications. From the purified outer membrane, 95% of the outer membrane proteins were extracted with nonionic surfactant, MEGA- (Fig. 2. a,b). The diffusion
Mr Mr
a
183
Microbiology Letters 127 (1995) 181-186
C
d
e
f
Mr
b
Fig. 2. SDS-PAGE of the purified outer membrane, 2% MEGA-9-extracted outer membrane proteins and the purified outer membrane proteins. Samples were heated in sample buffer 1141 at 95” C for 5 min. Lanes CM,): M, markers: myosin (200000), /3-galactosidase (1162501, phosphorylase b (97400), bovine serum albumin (662001, ovalbumin (45000), carbonic anhydrase (31000), soybean trypsin inhibitor (21500), lysozyme (14400). (a): outer membrane; (b):2% MEGA-9-extracted outer membrane proteins; cc): 55K protein; Cd): 51K protein; (e): 125K protein; (0: 92K protein.
184
K. Kanazawa et al. / FEMS Microbiology Letters 127 (1995) 181-186
(data not shown).
e
0.3
d 2 2 k 8
0.2
0.1
0 0
5
10
PROTEIN / LIPID (jig/p
15 mole)
Fig. 3. Effect of protein concentration on the diffusion rate of glucose. Proteoliposomes were reconstituted from 3 pmol of phosphatidylcholine, 0.093 pmol of dicetyl phosphate and the specified amount of outer membrane or porins in 0.5 ml of 40 mosM stachyose-1 mM MOPS buffer (pH 7.2) as described in Materials and methods. The test solute was 40 mosM glucose in 1 mM MOPS buffer (pH 7.2). The liposome swelling assay was done as described in Materials and methods. Symbols: 0, B. fragilis outer membrane; n , 51K porin; 0, 125K porin; 0, 92K porin; A, E. coli OmpF porin; A, E. coli PhoE porin.
These results indicate that 51K, 92K and 125K protein are porins in the outer membrane of B. fiagilis. To our knowledge this is the first report of purification of Bacteroides porins. Previously, it was reported that the deficiency of outer membrane proteins of Zt4, 49-50K were found in some clinical isolates of B. fragilis, which are resistant to cefoxitin, one of the @-lactam antibiotics which is thought to penetrate into the cell via porins [7]. This report suggested the presence of porin proteins of M, 49-50K in the outer membrane of B. fiagilis, but it is not clear at present whether the newly purified 51K protein is related to the reported proteins. 3.2. Characterization of permeability properties of porins
The effect of the amount of porin proteins on the diffusion
rate of glucose was determined.
The results
C
01.2. 1
2
1
3456
2
3456
Mr, SACCHARIDE ( x 102) Fig. 4. Permeability properties of the liposome membranes reconstituted from the purified outer membrane and porins of B. fragilis. Proteoliposomes were reconstituted from 3 pmol of phosphatidylcholine, 0.093 pmol of dicetyl phosphate and the purified outer membrane (60 /lg of protein), 30 pg of 92K porin and 125K porin, or 6 /.~g of 51K porin in 0.5 ml of 40 mosM stachyose-1 mM MOPS buffer (pH 7.2) as described in Materials and methods. The test solutes were 40 mosM saccharides in 1 mM MOPS buffer (pH 7.2). The liposome swelling assay was done as described in Materials and methods. The diffusion rates are presented as relative to the diffusion rate of arabinose for the purified outer membrane (A>, 125K porin (B), 92K porin (C) and 51K porin (D). Saccharides used were: 1, arabmose; 2, glucose; 3, mannose; 4, galactose; 5, a-methyl glucopiranoside; 6, a-methyl mannopiranoside; 7, a-methyl galactopiranoside; 8, sucrose; 9, maltose; 10, treharose; 11, melezitose; 12, maltotriose; and 13, raffinose.
K. Kanazawa et al. / FEMS Microbiology
showed that the diffusion rates increased as the amount of purified porins was raised, indicating that the diffusion rate depends on the amount of porin added (Fig. 3). However, the diffusion rates were not linearly related to the amount of the protein added as observed before [12]. The reason for this is not clear, though the diffusion of glucose was linearly related to the amount of purified outer membrane added. As shown in Fig. 3, the solute diffusion through B. fragilis porin appeared to be inefficient compared with E. coli porin, with 92K porin of B. fragilis demonstrating the highest pore-forming activity. Differences in the diffusion rate of uncharged saccharides, e.g. glucose, may be due to the difference of pore size. However, a possibility of artifacts, such as the influence of surfactants, cannot be ignored. In fact, purified porins of B. fragilis appeared to be more unstable in the surfactant solution than E. coli porins. The pore-forming activity of 51K and 125K porin was gradually reduced during storage over several weeks in buffer A. Compared with these two porins, 92K porin seemed to be a little more stable. In order to further characterise the pores formed by these identified porins, the diffusion rates of uncharged saccharides with different M, through these porins were determined by the liposome swelling assay and compared with those through the purified outer membrane. The diffusion rates were plotted as described by Nakae et al. [13]. The results in Fig. 4 showed that the size of pores formed by 51K and 92K porin are similar and they are slightly smaller than that formed by 125K porin. The three purified porins allowed the diffusion of pentoses and hexoses in the manner of a molecular sieve, but the diffusion rates of the saccharides larger than disaccharides were low (Fig. 4, B-D). A similar result was obtained for the purified outer membrane (Fig. 4, A), indicating that the pore-forming activity of the outer membrane porins was well preserved in the purified porins under this experimental condition. As shown in Fig. 4, the diffusion rates of disaccharides were low but varied. The reason for this is not clear, but it may be due to the difference of pore structure in addition to the difference of pore size. In this paper we present the possibility that the purified 51K, 92K and 125K outer membrane proteins are porins in the outer membrane of B. fragilis. The data also indicate that these porins form relatively small
Letters 127 (1995) 181-186
185
pores which allow the diffusion of saccharides of M, smaller than 340-400. Furthermore, the permeability properties of these porins are consistent with those of intact B. fragilis [8,9]. These results suggest that the identified pot-ins play an important part in the solutes diffusion through the outer membrane of B. fragilis. Therefore, it is likely that an intrinsic resistance of B. fiugilis to multiple antibiotics is probably due to the low antibiotics diffusion through the outer membrane into the cells. In order to confirm the involvement of the identified porins in drug resistance of B. fiagilis, the diffusion of /?-lactam antibiotics through the B. fragilis porin pores in vitro are being determined.
Acknowledgements We thank Prof. Taiji Nakae for his valuable vice and helpful discussions.
ad-
References [l] Nakae, T. (1986) Outer membrane permeability of bacteria. CRC Crit. Rev. Microbial. 13, l-62. [2] Schmid, A., Benz, R. and Schink, B. (1991) Identification of two porins in Pelobacter uenetianus fermenting high-molecular-mass polyethylene glycols. J. Bacterial. 173,4909-4913. [3] Cuchural, G.J., Hurlbut, S., Malamy, M.H. and Tally, F.P. (1988) Permeability to p-lactams in Bacteroides fiagilis. J. Antimicrob. Chemother. 22, 785-790. [4] Olsson, B., Dombusch, K. and Nord, C.E. (1979) Factors contributing to /3-lactam antibiotics in Bacteroides fragilis. Antimicrob. Agents Chemother. 15, 263-268. [S] Cuchural, G.J., Tally, F.P., Jacobus, N.V., Marsh, P.K. and Mayhew, J.W. (19831 Cefoxitin inactivation by Bacteroides fragilis. Antimicrob. Agents Chemother. 24, 936-940. [6] Cuchural, G.J., Malamy, M.H. and Tally, F.P. (1986) /3Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob. Agents Chemother. 30, 645-648. [7] Piddock, L.J.V. and Wise, R. (19871 Cefoxitin resistance in Bacteroides species: Evidence indicating two mechanisms causing decreased susceptibility. J. Antimicrob. Chemother. 19, 161-170. [8] Kobayashi, Y. and Nakae, T. (1986) The permeability property of the outer membrane of Bacteroides fragilis, a strictly anaerobic opportunistic pathogen. Biochem. Biophys. Res. Commun. 141, 292-298. [9] Kobayashi, Y., Akatsuka, A. and Nakae, T. (1987) Electron microscopic visualization of the outer membrane permeabil-
186
K. Kanazawa et al. / FEMS Microbiology
ity of Bacteroides fragilis. FEMS Microbial. Len. 48, 325329. [lo] Mizushima, S. and Yamada, H. (1975) Isolation and characterization of two outer membrane preparations from Escherichia coli. Biochim. Biophys. Acta 375, 44-53. [ll] Yoshihara, E. and Nakae, T. (1989) Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores. J. Biol. Chem. 264, 6297-6301. [12] Ishii, J. and Nakae, T. (1988) Size of diffusion pore of
Letters 127 (199.5) 181-186
Alcaligenes faecalis. Antimicrob. Agents Chemother. 32, 378-384. [13] Nakae, R. and Nakae, T. (1982) Diffusion of aminoglycoside antibiotics across the outer membrane of Escherichia coli. Antimicrob. Agents Chemother. 22, 554-559. [14] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature London 227, 680-685.