ANTIMICROBIAL AGENTs AND CHEMOTHERAY, Aug. 1976, p. 354-362 1 Copyright © 1976 American Society for Microbiology
Vol. 10, No. 2 Printed in U.S.A.
Inhibition of Neisseria gonorrhoeae by a Bacteriocin from Pseudomonas aeruginosa STEPHEN A. MORSE,* PATRICK VAUGHAN, DEANNE JOHNSON, AND BARBARA H. IGLEWSKI Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland,
Oregon 97201 Received for publication 26 March 1976
Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 x g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species ofNeisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most "classical" antibiotics.
Flynn and McEnteggart (11) reported the presence of bacteriocin-like activity in isolates of Neisseria gonorrhoeae. However, Knapp et al. (32) and Walstad et al. (47) were unable to demonstrate bacteriocinogeny in N. gonorrhoeae. The bacteriocin-like activity exhibited by many strains of N. gonorrhoeae was attributed to the production of inhibitory levels of free fatty acids and lysophosphatidylethanolamine (47). Substances from other organisms have also been reported to exhibit bacteriocin-like activity against N. gonorrhoeae. Volk and Kraus (46) reported the in vitro inhibition of N. gonorrhoeae by a meningococcal bacteriocin, and Geizer (12) reported the inhibition of gonococcal growth by unidentified substances produced by strains of Vibrio cholerae, Escherichia coli, Aeromonas hydrophilia, Micrococcus spp., and Pseudomonas aeruginosa. We have also observed a marked inhibition of gonococcal growth by culture supernatants of P. aeruginosa. The isolation, characterization, and identification of this inhibitory factor(s) are the subjects of this investigation. MATERLALS AND METHODS Organisms. Clinical isolates of N. gonorrhoeae were used in these studies. The specific properties of
strains CS-7, JW-31, and 72H870 were previously reported (38, 40). Strains from disseminated gonococcal infections (DGI strains) were obtained from K. Holmes (U.S. Public Health Service Hospital, Seattle, Wash.). Additional clinical isolates from nondisseminated gonococcal infections were obtained from the Multnomah County Health Department, Portland, Ore. Known serological types of N. meningitidis were obtained from H. Schneider (Walter Reed Army Institute of Research, Washington, D.C.). Strains of N. lactamica and other species of Neisseria were obtained from D. Hollis and D. Kellogg, Jr. (Center for Disease Control, Atlanta, Ga.). The identity of all organisms was confirmed by cell morphology in Gram-stained smears, oxidase reaction, and the production of acid from specific carbohydrates (39). T-1 and T-4 colony types (31) from single clinical isolates were subcultured and maintained by selective passage. A nonproteolytic strain of P. aeruginosa (PA-103) (34) was obtained from P. V. Liu (University of Kentucky, Lexington). This strain was used throughout this investigation as a source of R-type pyocin. The titer of the crude and purified pyocin preparations was determined using P. aeruginosa strain PS-7 (10) (obtained from E. Fisher, Portland State University, Portland, Ore.). Medium. The basal medium contained the following per liter: proteose peptone no. 3 (Difco), 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. 354
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When used for the production of R-type pyocins by P. aeruginosa, glycerol (1%, vol/vol) and monosodium glutamate (8.46 g/liter) were added after autoclaving. When used for the growth of Ne'sseria spp., a growth factor supplement (1%, vol/vol), identical in composition to IsoVitaleX enrichment (BBL) but lacking glucose, NaHCO3 (42 mg/liter), and glucose (5 g/liter) were added after autoclaving. GC agar (Difco) plates containing glucose (5 g/liter) and growth factor supplement (1%, vol/vol) were used where indicated. Induction and purification of R-type pyocins. An overnight culture of P. aeruginosa was centrifuged (2,100 x g for 10 min) and resuspended to 1/10 the original volume in a solution containing 0.85% NaCl and 0.1% cysteine hydrochloride at pH 6.5. A 1% (vol/vol) inoculum was used, and the cultures were incubated on a gyratory shaker at 37°C. When the turbidity of the culture reached approximately 150 Klett units, mitomycin C was added at a final concentration of 1 jig/ml. Incubation was continued until extensive lysis of the culture occurred (see Fig. 1). This lysis normally occurred within 3 h after the addition of mitomycin C. The mitomycin C-induced culture was centrifuged at 2,400 x g for 30 min to remove cellular debris, and the resulting supernatant was treated with chloroform (5%, vol/vol). This supernatant fraction was designated crude pyocin. Crude pyocin preparations were further purified by a modification (14) of the method of Kageyama and Egami (29). Briefly, this procedure consisted of the slow addition of 1 M MnCl2 (60 ml/liter of lysate), while stirring, to the crude pyocin preparation. After adjusting the pH to 7.5 with 1 M NaOH, the resulting precipitate was removed by centrifugation (2,400 x g for 15 min). The supernatant was designated partially purified pyocin. Further purification was accomplished lwy the addition of (NH4)2SO4 to 70% saturation and incubation overnight at 4°C. After centrifugation (2,400 x g for 30 min) at 4°C, the pellet containing the pyocin activity was dissolved in 50 ml of 0.01 M tris(hydroxymethyl)aminomethane .(Tris)-hydrochloride (pH 7.5) containing 0.01 M MgCl2 and 0.01 M MgSO4 and dialyzed overnight at 4°C against 2 liters of the same buffer. If necessary, the preparation was clarified by centtifugation (2,400 x g for 15 min at 4°C). The pyocin preparation was then centrifuged at 100,000 x g for 90 min (type 40 rotor, Spinco model L2-65B ultracentrifuge). The gelatinous pellet was gently dissolved in 20 ml of buffer and chromatographed on diethylaminoethyl (DEAE)-cellulose (DE-52, Whatman Biochemicals Ltd., Kent, England) previously washed and equilibrated with the same buffer. An 8-ml sample of pyocin was applied to a column (1.5 by 28 cm) and allowed to adsorb for 1 h. The column was washed with 200 ml of buffer to remove material not adsorbing to the DEAE-cellulose. The pyocins were then eluted with 800 ml of an NaCl gradient (0 to 1.0 M) in 0.01 M Tris buffer containing 0.01 M MgCl2 and MgSO4. Fivemilliliter fractions were collected and analyzed for absorbance at 280 nm and pyocin activity. The fractions exhibiting pyocin activity were pooled, dialyzed against 0.01 M Tris buffer containing 0.01 M
355
MgCl2 and 0.01 M MgSO4 to remove NaCl, and then concentrated by ultracentrifugation (100,000 x g for 90 min). All chromatographic procedures were carried out at 4°C. Pyocin typing. Pyocin typing was performed using the broth method as described by Jones et al. (27). The ALA set of 18 strains of P. aeruginosa (obtained from B. H. Minshew, University of Washington School of Medicine) was used for indicator strains (27). The pyocin type and pattern are reported by the notation described by Farmer and Herman (9). Assay of pyocin activity. Organisms being tested for susceptibility to R-type pyocins were grown overnight on GC agar plates. A suspension of these organisms was prepared in a diluent consisting of 0.85% NaCl and 0.1% HCl (pH 6.4) and adjusted to a Klett reading of 50 to 60. GC agar plates were inoculated by means of a swab dipped into the cell suspension. Undiluted or serially diluted pyocin preparations (5 ul) were applied to the surface of the agar plate. All plates were incubated overnight at 37°C with increased CO2 (5% C02) prior to being read. Pyocin titers are expressed as 200 times the highest dilution that shows complete inhibition. ADP-ribosyl transferase activity. An enzyme mixture containing aminoacyl transferase activity was prepared from crude extracts of rabbit reticulocytes by a modification (6) of the method of Allen and Schweet (1). Adenosine diphosphate (ADP)-ribosyl transferase was measured by the procedure of Collier and Kandel (6). Briefly, the assay mixture in a total volume of 65 ,Ml contained 50 mM Tris-hydrochloride (pH 8.2), 0.1 mM disodium ethylenediaminetetraacetic acid, 40 mM dithiothreitol, 25 Al of the reticulocyte enzyme mixture, 0.367 MM [14Cadenine] nicotinamide adenine dinucleotide (Schwarz/Mann; specific activity, 136 mCi/mmol), and various amounts of purified pyocin preparation, P. aeruginosa exotoxin A, or diphtheria toxin fragment A as indicated. The reaction mixture was incubated at 25°C for 5 min and then stopped by the addition of 65 Ml of 10% trichloroacetic acid. The acid-insoluble precipitates were collected, washed, and counted on a Nuclear-Chicago low background counter as previously described (20). P. aerugirosa exotoxin A was produced and purified by the method of Liu (35) as previously described (19). Diphtheria toxin fragment A was kindly provided by R. J. Collier (Department of Bacteriology, University of California at Los Angeles). Protein was determined by the method of Lowry et al. (36) using bovine serum albumin as a standard. Protease treatment of R-type pyocins. Purified pyocins were diluted to a final concentration of 1 x 104 U/ml in 0.01 M Tris-hydrochloride buffer (pH 7.5) containing 0.01 M MgCl2 and 0.01 M MgSO4. Protease from Streptomyces griseus (type VI, Sigma Chemical Co., St. Louis) or from Bacillus subtilis (type VIII, Sigma Chemical Co.) was added at a final concentration of 1.0 mg/ml. The susceptibility of the pyocin to trypsin was tested by preparing a similar dilution in 0.05 M Tris-hydrochloride buffer (pH 8.1) containing 0.01 M CaCl2; trypsin (type III,
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Sigma Chemical Co.) was added at a final concentration of 0.5 mg/ml. Controls diluted in the appropriate buffer were also included. All samples were incubated at 37°C for 3 h and then titered against P. aeruginosa strain PS-7 andN. gonorrhoeae JW-31. Electron microscopy. Pyocins were prepared for examination in an electron microscope by the negative-staining technique of Brenner and Horne (4). Pyocin preparations were centrifuged at 100,000 x g for 1 h, and the pellet was resuspended in a small volume of 1 M NH4C2H302 (pH 7.0). Formvar-covered copper grids were placed onto a drop of the sample for 1 to 2 min and then blotted dry with filter paper. These grids were then placed onto a drop of 1.5% sodium phosphotungstate (pH 7.0) for 30 s. Excess fluid was removed with filter paper. Samples were examined in a Phillips EM-200 electron microscope at 60 kV. The interaction of pyocins with cells of N. gonorrhoeae was observed by a similar procedure. At 30 min after the addition of pyocin to a liquid culture of N. gonorrhoeae 72H870, a sample was removed and treated as above. The negative-stained preparation was examined in an RCA electron microscope at 50 kV.
RESULTS Purification and characterization of the inhibitory factor. Geizer (12) observed that the growth of N. gonorrhoeae was inhibited by a substance(s) produced during the growth of a strain ofP. aeruginosa. We have confirmed this phenomenon and, further, have sought to isolate and identify the inhibitory substance(s). The inhibitory factor was produced in low concentration during the growth of P. aeruginosa strain PA-103 in a complex medium (Table 1). Addition of mitomycin C (1 Ag/ml) caused extensive lysis of the culture within 3 h (Fig. 1) and resulted in a 16-fold increase in the concentration of the inhibitory factor (Table 1) as de-
termined by titration on P. aeruginosa PS7 and N. gonorrhoeae strains JW-31 and DGI 1947. The titer of the inhibitory factor varied with different strlins ofN. gonorrhoeae (Table 1); no difference was observed with colonial variants of a single strain. The inducible nature of this factor suggested the involvement of a bacteriophage or bacteriophage product. However, no plaques were observed when dilution of supernatants from both induced and noninduced cultures were spotted on N. gonorrhoeae strain JW-31. Instead, the area of growth inhibition increased in turbidity with increasing dilution, suggesting the presence of a nonreplicating bacteriocin. The inhibitory factor was partially purified from supernatants of mitomycin C-induced cultures ofP. aeruginosa strain PA-103 by a procedure used for pyocin purification. Further purification was obtained by DEAE-cellulose chromatography (Fig. 1). The inhibitory factor was TABLE 1. Effect of mitomycin C on the production of gonococcal inhibitory factor by P. aeruginosa PA103a Inhibitory titer
(U/mi)
Organism
Non-
P. aeruginosa PS-7 N. gonorrhoeae JW-31 N. gonorrhoeae DGI 1947 N. gonorrhoeae 1138 (T-1) N. gonorrhoeae 1138 (T-4)
induced
Inue Induced
2560 2560 640 NDb ND
40,960 40,960 10,240 5,120 5,120
a Mitomycin C (1 ,g/ml of medium), when serially diluted, ceased to inhibit the growth of these organisms at a >1:2 dilution (0.5 ,ug/ml). b ND, Not determined.
1.62 A, LI~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~e
as*
so6 6 U U 16 3 12 1
14
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FIG. 1. Purification ofR-type pyocin (611 131) by DEAE-cellulose chromatography. Fractions containing inhibitory activity are indicated by A and B. Insert: induction ofpyocin production in P. aeruginosa PA-103 by mitomycin C (1 Mg/ml). The arrow indicates time of mitomycin C addition.
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eluted with an NaCl gradient of 0 to 1.0 M. Two peaks containing the inhibitory factor were observed. The major peak (A) eluted at an NaCl concentration of 0.06 M and contained more than 90% of the inhibitory activity. A minor peak (B) eluted at an NaCl concentration of 0.91 M and contained less than 10% ofthe activity. The fractions comprising peak A were pooled, dialyzed against 0.01 M Tris-hydrochloride buffer (pH 7.5) to remove the NaCl, and concentrated by ultracentrifugation (100,000 x g for 90 min). This preparation was used for the remainder of these studies. Negative-stained preparations of the purified inhibitory factor were examined in the electron microscope. Particles resembling R-type pyocins were observed in both the uncontracted and contracted states (Fig. 2). In the uncontracted state, these particles measured 111.5 nm in length by 15.3 nm in width. In the contracted state, the particles consisted of an inner core (105 nm in length by 6.5 nm in width) surrounded by a contracted sheath (44.4 nm in length by 18.6 nm in width). Between 20 and 30% of the particles observed in these preparations were in the contracted state. Neither intact bacteriophage nor bacteriophage ghosts ,.
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were seen in any of the negative-stained preparations. The type of the pyocin in both partially purified and purified preparations was determined as described above. The results (data not shown) indicate that the pattern did not change during purification and suggest that one or more pyocin types were not removed during chromatography on DEAE-cellulose. The pyocin pattern is 611 131. R-type pyocins may be differentiated from Stype pyocins by their resistance to proteolytic enzymes (24). Therefore, the effect of various proteolytic enzymes was examined on the inhibition of P. aeruginosa PS-7 and N. gonorrhoeae JW-31 by purified pyocin preparation. The results (not shown) indicate that the concentration did not decrease when compared with an untreated concentration of 1 x 104 U/ ml. These data suggest that the inhibition of N. gonorrhoeae was not due to an S-type pyocin. Many strains ofP. aeruginosa produce a protein toxin (exotoxin A) that inhibits mammalian protein synthesis (20). The toxin catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide to elongation factor-2, resulting in an ADP-ribosyl-elonga-
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FIG. 2. Electron micrograph of a negative-stained preparation of R-type pyocin 611 131. Symbols: uc, uncontracted pyocin; c, contracted pyocin. Bar = 0.1 ,um.
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MORSE ET AL.
tion factor-2 complex which is inactive in peptide chain elongation (19, 20). The possibility that the inhibition of N. gonorrhoeae was due to the presence ofthis toxin in the pyocin preparation was investigated. The results (Table 2) show that neither the crude nor the purified preparation from P. aeruginosa strain PA-103 contains ADP-ribosyl transferase activity. Thus, it seems unlikely that the inhibitory effect of the pyocin preparation is due to exotoxin A. Effect of R-type pyocin on the growth ofN. gonorrhoeae. The effect of R-type pyocin on growing cells of N. gonorrhoeae strain 72H870 was examined by the addition of various concentrations of the purified preparation to exponentially growing cultures. Figure 3 shows a concentration-dependent inhibition of gonococcal growth by the addition of type 611 131 pyocin. At high pyocin concentrations, a complete inhibition of growth occurred within 1 h and was accompanied by extensive lysis of the culture. To ascertain whether a direct interaction occurred between the pyocin and susceptible cells of N. gonorrhoeae, samples were removed from the culture after 30 min and examined by electron microscopy. The results (Fig. 4) demonstrate the binding of pyocins to the surface of the cell. The pyocin-cell interaction appears to result in contraction of the pyocin. No significant association of type 611 131 pyocin occurred with the cell surface of a resistant organism (N. ovis). These data suggest that receptors are present on the cell surface of N. gonorrhoeae. Inhibitory spectrum of pyocin type 611 131. Portions of the purified pyocin preparation were spotted on lawns prepared from clinical isolates of N. gonorrhoeae. Typical patterns of inhibition are shown in Fig. 5. The zone of inhibition was clearly evident in all strains examined. No difference was observed between colony types T-1 and T-4 from the same strain. A negative control of the producer strain, P. aeruginosa PA-103, was also included. TABLE 2. ADP-ribosyl transferase activity in preparations of R-type pyocin (611 131) from P. aeruginosa strain PA-103
ANTIMICROB. AGENTS CHEMOTHER. I
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FIG. 3. Effect of R-type pyocin (611 131) on the growth of N. gonorrhoeae strain 72H870. Purified pyocin was added to exponentially growing cultures (1.4 x 108 colony-forming units/ml) of strain 72H870. Symbols: 0, no additions; 0, 400 U of pyocin per ml; A, 1,000 U of pyocin per ml; *, 4,000 U ofpyocin per ml; *, 20,000 U ofpyocin per ml.
The inhibition of various Neisseria species by this pyocin is shown in Table 3. All isolates of N. gonorrhoeae, from both disseminated and nondisseminated infections, were inhibited. However, only 3 of 20 strains ofN. meningitidis and 5 of 16 strains of N. lactamica were inhibited. No correlation was observed between the serological group and inhibition ofN. meningitidis. None of the other five species tested was inhibited by this pyocin.
DISCUSSION Bacteriocins are antibiotic substances proAcid-insoluAdditions Protein (jug) ble radioacduced by many species of bacteria which are tivity (cpm) thought to be inhibitory for strains of the same or closely related species (42). Bacteriocins are 0 Control', 121 Crude R-type pyocin 7.0 73 protein in nature and range from simple proPurified R-type pyocin 5.0 96 teins (7, 33) to particles resembling bacterioExotoxin A 0.01 2,788 phage components (3, 5, 14, 16, 22, 26). BacterDiphtheria toxin frag0.01 2,839 iocinogenic strains of P. aeruginosa produce ment A several types of bacteriocins (17). The synthesis a The control contained water in place of toxin or of these bacteriocins can be increased by mitopyocin preparation. mycin C treatment (17, 24, 28). These bacterio-
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359
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FIG. 4. Interaction ofR-tyz pyocin 611 131 with cells ofN. gonorrhyae strain 72H870. Bar = 0.1 ym.
cins may be grouped into one of two types (3): the S-type bacteriocins, which are characterized by susceptibility to proteolytic enzymes and a lack of structure in electron microscopy, and the R-type bacteriocins, which are not susceptible to proteolytic enzymes and exhibit the appearance of bacteriophage components. The bacteriocins produced by P. aeruginosa are called pyocins (25). In addition to resistance to proteolytic enzymes, R-type pyocins may be differentiated from S-type pyocins by their adsorption to DEAE-cellulose, molecular weight, rate of diffusion in agar, and neutralization by specific antiserum (24). R-type pyocins bear a close resemblance to the tails of T-even bacteriophage (14, 16, 18, 22, 28). Govan (14) reported that, regardless of the pyocinogenic strain used, the majority of the R-type particles measured approximately 100 by 15 nm in the uncontracted state; contracted particles consisted of a hollow inner core, approximately 100 by 7 nm, par-
tially enclosed in a sheath measuring about 45 by 17 nm. Our findings indicate that the gonococcal inhibitory factor produced by P. aeruginosa PA-103 was an R-type pyocin. Several lines of evidence support this conclusion. The inhibitory factor was resistant to proteolytic enzymes and adsorbed to DEAE-cellulose. Sedimentation of the inhibitory factor by ultracentrifugation (100,000 x g for 90 min) suggests that this substance has a high molecular weight. The same susceptibility pattern (611 131) was always observed when the inhibitory factor was typed on the ALA strains of P. aeruginosa. Furthermore, electron microscopy revealed particles with the same morphology and size as those reported by Govan (14). No tail fibers were observed in negative-stained preparation of 611 131 as previously reported in other R-type pyocins (16, 18). R-type pyocins may be found in both the contracted and uncontracted state (14, 16). Only uncontracted pyocins will adsorb to and kill
360
ANTIMICROB. AGENTS CHEMOTHER.
MORSE ET AL.
-
...
-.
e
FIG. 5. Inhibition of N. gonorrhoeae by an R-type pyocin (611 131). (a) N. gonorrhoeae strain JW-31; (b) N. gonorrhoeae strain 72H870; (c) N. gonorrhoeae strain 1138 (colony type T-1); (d) N. gonorrhoeae strain 1138 (colony type T-4); (e) N. gonorrhoeae strain CS-7; (t) P. aeruginosa strain PA-103.
TABLE 3. Susceptibility of various Neisseria species to purified R-type pyocin (611 131) from P. aeruginosa strain PA-103 SeroSpecies Specsgroup gop
N. gonorrhoeae N. meningitidis
A B C X Y Z
135 N. lactamica N. mucosa N. flava N. subflava N. ovis N. flavescens
No. of strains tested
No. of strains susceptible
56 4
56 0
5 3 3 1 2 2
1 0 0 1 1 & 5 0 0 0 0 0
16 1 1 1 1 1
susceptible cells (14, 16); no adsorption to pyocin-resistant bacteria occurs (14). Lethal effects occur after the contraction of bound pyocins (14). We have demonstrated that R-type pyocin 611 131 adsorbs to the surface of N. gonorrhoeae. The presence of contracted pyocins on the gonococcus suggests that receptor sites on N. gonorrhoeae may be similar to those that exist on the cell surface of certain strains of P. aeruginosa. Of special interest are studies involving shared antigens between heterologous bacterial species. Minden et al. (37) demonstrated that Pseudomonas shared an indeterminate number of antigens with other gramnegative bacteria. The presence of naturally occurring antibodies to N. gonorrhoeae (13) suggests that this organism may also share antigenic determinants with heterologous bacterial species. This possibility is supported by
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INHIBITION OF N. GONORRHOEAE BY R-TYPE PYOCIN
the study of Robbins et al. (43), which showed that N. meningitidis groups A and C share capsular and noncapsular antigenic determinants with strains of E. coli. The lipopolysaccharide (LPS) has been implicated as the receptor for R-type bacteriocins in P. aeruginosa (8, 14, 15, 21, 23) and in some strains of Serratia marcescens (45). The bacteriocin receptor in other strains of S. marcescens is reported to be proteinaceous (45). The receptor for pyocin 611 131 onN. gonorrhoeae has not as yet been identified. However, a receptor located on the 0 polysaccharide portion of the gonococcal LPS appears unlikely. We observed identical titers of 611 131 on T-1 and T-4 colonial variants from the same clinical isolate. Perry et al. (41) determined that LPS from T-1 cells contained a high-molecular-weight 0 polysaccharide that showed considerable variation between strains. This 0 polysaccharide was absent in T-4 cells. To the contrary, Stead et al. (44) were unable to detect 0 polysaccharide side chains in LPS isolated from either T-1 or T-4 cells. In addition, pyocin 611 131 inhibited strains of N. gonorrhoeae isolated during a 5year period from Boston, Mass., Portland, Ore., and Seattle, Wash. Strains from these diverse locales would be expected to show variability in the composition of the 0 polysaccharide side chain as previously reported by Perry et al. (41). Alternatively, the receptor may reside in a common core region of the LPS. This core oligosacchAride may be sufficiently similar in N. gonorrhoeae, N. meningitidis, and N. lactamica to account for the inhibition observed in the latter two species. However, the possibility of a protein receptor cannot be excluded at this time. A capsule may afford some measure of protection against the bactericidal activity of pyocin 611 131. The presence of a capsule in the resistant strains ofN. meningitidis and N. lactamica has not been determined. This capsule could conceivably hinder the binding of the pyocin to its receptor. A similar phenomenon has been observed with respect to the inhibition of bacteriophage binding to encapsulated bacteria (48). Allen and Kelly (2) observed that the titer of a given pyocin frequently varied with different strains of P. aeruginosa. These investigators proposed that the closeness of fit between the reactive portion of the pyocin and its receptor determined the potency of its bactericidal activity. An alternate hypothesis is that the variation in titer reflects the number ofreceptor sites per cell. Strains containing a large number of pyocin receptors per cell would exhibit a lower titer than would strains having fewer receptors
361
per cell. Either hypothesis may explain the variation in titer observed with different strains of N. gonorrhoeae. The mechanism of pyocin-induced killing of N. gonorrhoeae is not known. Kageyama et al. (30) identified a muramidase-like enzyme as a component of R-type pyocins. This enzyme could account for the rapid lysis of gonococcal cultures that were exposed to high concentrations of pyocin. However, other effects, such as an inhibition of macromolecular synthesis or other cell functions that may ultimately lead to cell lysis, cannot be eliminated at this time. In summary, the results of this study indicate that the gonococcal inhibitory factor observed in culture supernatants ofP. aeruginosa PA-103 is the R-type pyocin 611 131. The ability of pyocin 611 131 to inhibit N. gonorrhoeae suggests that either the definition of a bacteriocin with regards to species specificity needs to be reevaluated or that there is a much closer taxonomic relationship between Neisseria and Pseudomonas than heretofore suspected. ACKNOWLEDGMENTS This research was supported by grants from the Medical Research Foundation of Oregon and Phi Beta Psi Sorority. LITERATURE CITED 1. Allen, E. S., and R. S. Schweet. 1962. Synthesis of hemoglobin in a cell-free system. J. Biol. Chem. 237:760-767. 2. Allen, J. C., and P. C. Kelly. 1975. Antigenic heterogeneity among pyocins ofPseudomonas aeruginosa. Infect. Immun. 12:318-323. 3. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 4. Brenner, S., and R. W. Home. 1959. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34:103-110. 5. Coetzee, H. L., H. C. de Klerk, J. N. Koetzee, and J. A. Smit. 1968. Bacteriophage tail-like particles associated with intraspecies killings of Proteus vulgaris. J. Gen. Virol. 2:29-36. 6. Collier, R. J., and J. Kandel. 1971. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J. Biol. Chem. 246:14961503. 7. Dandeau, J. P. 1971. Chemical and immunological study of colicins E,, K, A, and Q. Infect. Immun. 3:19. 8. Dyke, J., and R. S. Berk. 1974. Growth inhibition and pyocin receptor properties of endotoxin from Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 145:1405-1408. 9. Farmer, J. J., III, and L. G. Herman. 1974. Pyocin typing of Pseudomonas aeruginosa. J. Infect. Dis. 130:543-546. 10. Feary, T. W., E. Fisher, and T. N. Fisher. 1963. Lysogeny and phage resistance in Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 113:426-430. 11. Flynn, J., and M. C. McEnteggart. 1971. Bacteriocins from Neisseria gonorrhoeae and their possible role in epidemiological studies. J. Clin. Pathol. 25:60-61.
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12. Geizer, E. 1968. Antibacterial substances produced by different bacteria inhibiting the growth of Neisseria gonorrhoeae. J. Hyg. Epidemiol. Microbiol. Immunol. 12:241-243. 13. Glynn, A. A., and M. E. Ward. 1970. Nature and heterogeneity of the antigens of Neisseria gonorrhoeae involved in the serum bactericidal reaction. Infect. Immun. 2:162-168. 14. Govan, J. R. W. 1974. Studies on the pyocins of Pseudomonas aeruginosa: morphology and mode of action of contractile particles. J. Gen. Microbiol. 80:1-15. 15. Govan, J. R. W. 1974. Studies on the pyocins of Pseudomonas aeruginosa: production of contractile and flexous pyocins in Pseudomonas aeruginosa. J. Gen. Microbiol. 80:17-30. 16. Higerd, T. B., C. A. Baechler, and R. S. Berk. 1969. Morphological studies on relaxed and contracted forms of purified pyocin particles. J. Bacteriol. 98:1378-1389. 17. Holloway, B. W., and V. Krishnapillai. 1975. Bacteriophages and bacteriocins, p. 99-132. In P. H. Clarke and M. H. Richmond (ed.), Genetics and biochemistry of Pseudomonas. John Wiley and Sons, New York. 18. Homma, J. Y., S. Goto, and H. Shionoya. 1967. Relationship between pyocin and temperate phage of Pseudomonas aeruginosa. H. Isolation of pyocins from strain P 1-III and their characteristics. Jpn. J. Exp. Med. 37:373-393. 19. Iglewski, B. H., L. P. Elwell, P. V. Liu, and D. Kabat. 1976. ADP-ribosylation of elongation factor 2 byPseudomonas aeruginosa exotoxin A and by diphtheria toxin, p. 62-69. In S. Shaltiel (ed.), Proceedings of the 4th International Symposium on the Metabolic Interconversion of Enzymes. Springer-Verlag, New York. 20. Iglewski, B. H., and D. Kabat. 1975. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U.S.A.
72:2284-2288. 21. Ikeda, K., and F. Egami. 1969. Receptor substance for pyocin R. I. Partial purification and chemical properties. J. Biochem. 65:603-609. 22. Ishii, S., Y. Nishi, and F. Egami. 1965. The fine structure of a pyocin. J. Mol. Biol. 13:428-431. 23. Ito, S., and M. Kageyama. 1970. Relationship between pyocins and a bacteriophage in Pseudomonas aeruginosa. J. Gen. Appl. Microbiol. 16:231-240. 24. Ito, S., M. Kageyama, and F. Egami. 1970. Isolation and characterization of pyocins from several strains of Pseudomonas aeruginosa. J. Gen. Appl. Microbiol. 16:205-214. 25. Jacob, F. 1954. Biosynthese induite et mode d'action d'une pyocine, antibiotique de Pseudomonas pyocyanea. Ann. Inst. Pasteur Paris 86:149-160. 26. Jayawardene, A., and H. Farkas-Himsley. 1969. Vibriocin; a bacteriocin from Vibrio comma. I. Production, purification, morphology, and immunological studies. Microbios 1:87-98. 27. Jones, L. F., J. P. Zakanycez, E. T. Thomas, and J. J. Farmer III. 1974. Pyocin typing of Pseudomonas aeruginosa: a simplified method. Appl. Microbiol. 27:400-406. 28. Kageyama, M. 1964. Studies of a pyocin. I. Physical and chemical properties. J. Biochem. 55:49-53. 29. Kageyama, M., and F. Egami. 1962. On the purification and some properties of a pyocin, a bacteriocin produced by Pseudomonas aeruginosa. Life Sci. 9:471-
ANTIMICROB. AGENTS CHEMOTHER. 476. 30. Kageyama, M., K. Ikeda, and F. Egami. 1964. Studies of a pyocin. III. Biological properties of the pyocin. J. Biochem. 55:59-64. 31. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274-1279. 32. Knapp, J. S., S. Falkow, and K. K. Holmes. 1975. Reevaluation of bacteriocinogeny in Neisseria gonorrhoeae. J. Clin. Pathol. 28:274-278. 33. Kramer, J., and H. Brandis. 1975. Purification and characterization of two bacteriocins from Streptococcus faecium. J. Gen. Microbiol. 88:93-100. 34. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. II. Effects of lecithinase and protease. J. Infect. Dis. 116:112-116. 35. Liu, P. V. 1973. Exotoxins ofPseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J. Infect. Dis. 128:506-513. 36. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 37. Minden, P., J. K. McClatchy, and R. S. Farr. 1972. Shared antigens between heterologous bacterial species. Infect. Immun. 6:574-582. 38. Morse, S. A., and L. Bartenstein. 1974. Factors affecting autolysis of Neisseria gonorrhoeae. Proc. Soc. Exp. Biol. Med. 145:1418-1421. 39. Morse, S. A., and L. Bartenstein. 1976. Adaptation of the minitek system for the rapid identification of Neisseria gonorrhoeae. J. Clin. Microbiol. 3:8-13. 40. Morse, S. A., S. Stein, and J. Hines. 1974. Glucose metabolism in Neisseria gonorrhoeae. J. Bacteriol. 120:702-714. 41. Perry, M. B., V. Daoust, B. B. Diena, F. E. Ashton, and R. Wallace. 1975. The lipopolysaccharides of Neisseria gonorrhoeae colony types 1 and 4. J. Microbiol. 53:623-629. 42. Reeves, P. 1965. The bacteriocins. Bacteriol. Rev. 29:24-45. 43. Robbins, J. B., R. L. Myerowitz, J. K. Whisnant, M. Argaman, R. Schneerson, Z. T. Handzel, and E. C. Gotschlich. 1972. Enteric bacteria cross-reactive with Neisseria meningitidis groups A and C and Diplococcus pneumoniae types I and III. Infect. Immun. 6:651656. 44. Stead, A., J. S. Main, M. E. Ward, and P. J. Watt. 1975. Studies on lipopolysaccharides from strains of Neisseria gonorrhoeae. J. Gen. Microbiol. 88:123-131. 45. Traub, W. H., and I. Kleber. 1974. Studies on group A (phage tail) bacteriocins of Serratia marcescens. IV. Preliminary characterization of receptors of subgroup II bacteriocins. Zentralbl. Bakteriol. Parasitenkd. In-
fektionskr. Hyg. Abt. I Orig. A 229:490-502. 46. Volk, J., and S. J. Kraus. 1973. Asymptomatic meningococcal urethritis: possible protective value against gonococcal infection by bacteriocin production. Br. J. Vener. Dis. 49:511-512. 47. Waistad, D. L., R. C. Reitz, and P. F. Sparling. 1974. Growth inhibition among strains of Neisseria gonorrhoeae due to production of inhibitory free fatty acids and lysophosphatidylethanolamine: absence of bacte-
riocins. Infect. Immun. 10:481-488. 48. Wilkinson, J. F. 1958. The extracellular polysaccha-
rides of bacteria. Bacteriol. Rev. 22:46-73.
Vol. 10, No. 2 Printed in U.S.A.
Inhibition of Neisseria gonorrhoeae by a Bacteriocin from Pseudomonas aeruginosa STEPHEN A. MORSE,* PATRICK VAUGHAN, DEANNE JOHNSON, AND BARBARA H. IGLEWSKI Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland,
Oregon 97201 Received for publication 26 March 1976
Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 x g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species ofNeisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most "classical" antibiotics.
Flynn and McEnteggart (11) reported the presence of bacteriocin-like activity in isolates of Neisseria gonorrhoeae. However, Knapp et al. (32) and Walstad et al. (47) were unable to demonstrate bacteriocinogeny in N. gonorrhoeae. The bacteriocin-like activity exhibited by many strains of N. gonorrhoeae was attributed to the production of inhibitory levels of free fatty acids and lysophosphatidylethanolamine (47). Substances from other organisms have also been reported to exhibit bacteriocin-like activity against N. gonorrhoeae. Volk and Kraus (46) reported the in vitro inhibition of N. gonorrhoeae by a meningococcal bacteriocin, and Geizer (12) reported the inhibition of gonococcal growth by unidentified substances produced by strains of Vibrio cholerae, Escherichia coli, Aeromonas hydrophilia, Micrococcus spp., and Pseudomonas aeruginosa. We have also observed a marked inhibition of gonococcal growth by culture supernatants of P. aeruginosa. The isolation, characterization, and identification of this inhibitory factor(s) are the subjects of this investigation. MATERLALS AND METHODS Organisms. Clinical isolates of N. gonorrhoeae were used in these studies. The specific properties of
strains CS-7, JW-31, and 72H870 were previously reported (38, 40). Strains from disseminated gonococcal infections (DGI strains) were obtained from K. Holmes (U.S. Public Health Service Hospital, Seattle, Wash.). Additional clinical isolates from nondisseminated gonococcal infections were obtained from the Multnomah County Health Department, Portland, Ore. Known serological types of N. meningitidis were obtained from H. Schneider (Walter Reed Army Institute of Research, Washington, D.C.). Strains of N. lactamica and other species of Neisseria were obtained from D. Hollis and D. Kellogg, Jr. (Center for Disease Control, Atlanta, Ga.). The identity of all organisms was confirmed by cell morphology in Gram-stained smears, oxidase reaction, and the production of acid from specific carbohydrates (39). T-1 and T-4 colony types (31) from single clinical isolates were subcultured and maintained by selective passage. A nonproteolytic strain of P. aeruginosa (PA-103) (34) was obtained from P. V. Liu (University of Kentucky, Lexington). This strain was used throughout this investigation as a source of R-type pyocin. The titer of the crude and purified pyocin preparations was determined using P. aeruginosa strain PS-7 (10) (obtained from E. Fisher, Portland State University, Portland, Ore.). Medium. The basal medium contained the following per liter: proteose peptone no. 3 (Difco), 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. 354
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INHIBITION OF N. GONORRHOEAE BY R-TYPE PYOCIN
When used for the production of R-type pyocins by P. aeruginosa, glycerol (1%, vol/vol) and monosodium glutamate (8.46 g/liter) were added after autoclaving. When used for the growth of Ne'sseria spp., a growth factor supplement (1%, vol/vol), identical in composition to IsoVitaleX enrichment (BBL) but lacking glucose, NaHCO3 (42 mg/liter), and glucose (5 g/liter) were added after autoclaving. GC agar (Difco) plates containing glucose (5 g/liter) and growth factor supplement (1%, vol/vol) were used where indicated. Induction and purification of R-type pyocins. An overnight culture of P. aeruginosa was centrifuged (2,100 x g for 10 min) and resuspended to 1/10 the original volume in a solution containing 0.85% NaCl and 0.1% cysteine hydrochloride at pH 6.5. A 1% (vol/vol) inoculum was used, and the cultures were incubated on a gyratory shaker at 37°C. When the turbidity of the culture reached approximately 150 Klett units, mitomycin C was added at a final concentration of 1 jig/ml. Incubation was continued until extensive lysis of the culture occurred (see Fig. 1). This lysis normally occurred within 3 h after the addition of mitomycin C. The mitomycin C-induced culture was centrifuged at 2,400 x g for 30 min to remove cellular debris, and the resulting supernatant was treated with chloroform (5%, vol/vol). This supernatant fraction was designated crude pyocin. Crude pyocin preparations were further purified by a modification (14) of the method of Kageyama and Egami (29). Briefly, this procedure consisted of the slow addition of 1 M MnCl2 (60 ml/liter of lysate), while stirring, to the crude pyocin preparation. After adjusting the pH to 7.5 with 1 M NaOH, the resulting precipitate was removed by centrifugation (2,400 x g for 15 min). The supernatant was designated partially purified pyocin. Further purification was accomplished lwy the addition of (NH4)2SO4 to 70% saturation and incubation overnight at 4°C. After centrifugation (2,400 x g for 30 min) at 4°C, the pellet containing the pyocin activity was dissolved in 50 ml of 0.01 M tris(hydroxymethyl)aminomethane .(Tris)-hydrochloride (pH 7.5) containing 0.01 M MgCl2 and 0.01 M MgSO4 and dialyzed overnight at 4°C against 2 liters of the same buffer. If necessary, the preparation was clarified by centtifugation (2,400 x g for 15 min at 4°C). The pyocin preparation was then centrifuged at 100,000 x g for 90 min (type 40 rotor, Spinco model L2-65B ultracentrifuge). The gelatinous pellet was gently dissolved in 20 ml of buffer and chromatographed on diethylaminoethyl (DEAE)-cellulose (DE-52, Whatman Biochemicals Ltd., Kent, England) previously washed and equilibrated with the same buffer. An 8-ml sample of pyocin was applied to a column (1.5 by 28 cm) and allowed to adsorb for 1 h. The column was washed with 200 ml of buffer to remove material not adsorbing to the DEAE-cellulose. The pyocins were then eluted with 800 ml of an NaCl gradient (0 to 1.0 M) in 0.01 M Tris buffer containing 0.01 M MgCl2 and MgSO4. Fivemilliliter fractions were collected and analyzed for absorbance at 280 nm and pyocin activity. The fractions exhibiting pyocin activity were pooled, dialyzed against 0.01 M Tris buffer containing 0.01 M
355
MgCl2 and 0.01 M MgSO4 to remove NaCl, and then concentrated by ultracentrifugation (100,000 x g for 90 min). All chromatographic procedures were carried out at 4°C. Pyocin typing. Pyocin typing was performed using the broth method as described by Jones et al. (27). The ALA set of 18 strains of P. aeruginosa (obtained from B. H. Minshew, University of Washington School of Medicine) was used for indicator strains (27). The pyocin type and pattern are reported by the notation described by Farmer and Herman (9). Assay of pyocin activity. Organisms being tested for susceptibility to R-type pyocins were grown overnight on GC agar plates. A suspension of these organisms was prepared in a diluent consisting of 0.85% NaCl and 0.1% HCl (pH 6.4) and adjusted to a Klett reading of 50 to 60. GC agar plates were inoculated by means of a swab dipped into the cell suspension. Undiluted or serially diluted pyocin preparations (5 ul) were applied to the surface of the agar plate. All plates were incubated overnight at 37°C with increased CO2 (5% C02) prior to being read. Pyocin titers are expressed as 200 times the highest dilution that shows complete inhibition. ADP-ribosyl transferase activity. An enzyme mixture containing aminoacyl transferase activity was prepared from crude extracts of rabbit reticulocytes by a modification (6) of the method of Allen and Schweet (1). Adenosine diphosphate (ADP)-ribosyl transferase was measured by the procedure of Collier and Kandel (6). Briefly, the assay mixture in a total volume of 65 ,Ml contained 50 mM Tris-hydrochloride (pH 8.2), 0.1 mM disodium ethylenediaminetetraacetic acid, 40 mM dithiothreitol, 25 Al of the reticulocyte enzyme mixture, 0.367 MM [14Cadenine] nicotinamide adenine dinucleotide (Schwarz/Mann; specific activity, 136 mCi/mmol), and various amounts of purified pyocin preparation, P. aeruginosa exotoxin A, or diphtheria toxin fragment A as indicated. The reaction mixture was incubated at 25°C for 5 min and then stopped by the addition of 65 Ml of 10% trichloroacetic acid. The acid-insoluble precipitates were collected, washed, and counted on a Nuclear-Chicago low background counter as previously described (20). P. aerugirosa exotoxin A was produced and purified by the method of Liu (35) as previously described (19). Diphtheria toxin fragment A was kindly provided by R. J. Collier (Department of Bacteriology, University of California at Los Angeles). Protein was determined by the method of Lowry et al. (36) using bovine serum albumin as a standard. Protease treatment of R-type pyocins. Purified pyocins were diluted to a final concentration of 1 x 104 U/ml in 0.01 M Tris-hydrochloride buffer (pH 7.5) containing 0.01 M MgCl2 and 0.01 M MgSO4. Protease from Streptomyces griseus (type VI, Sigma Chemical Co., St. Louis) or from Bacillus subtilis (type VIII, Sigma Chemical Co.) was added at a final concentration of 1.0 mg/ml. The susceptibility of the pyocin to trypsin was tested by preparing a similar dilution in 0.05 M Tris-hydrochloride buffer (pH 8.1) containing 0.01 M CaCl2; trypsin (type III,
356
ANTiMICROB. AGENTS CHEMOTHER.
MORSE ET AL.
Sigma Chemical Co.) was added at a final concentration of 0.5 mg/ml. Controls diluted in the appropriate buffer were also included. All samples were incubated at 37°C for 3 h and then titered against P. aeruginosa strain PS-7 andN. gonorrhoeae JW-31. Electron microscopy. Pyocins were prepared for examination in an electron microscope by the negative-staining technique of Brenner and Horne (4). Pyocin preparations were centrifuged at 100,000 x g for 1 h, and the pellet was resuspended in a small volume of 1 M NH4C2H302 (pH 7.0). Formvar-covered copper grids were placed onto a drop of the sample for 1 to 2 min and then blotted dry with filter paper. These grids were then placed onto a drop of 1.5% sodium phosphotungstate (pH 7.0) for 30 s. Excess fluid was removed with filter paper. Samples were examined in a Phillips EM-200 electron microscope at 60 kV. The interaction of pyocins with cells of N. gonorrhoeae was observed by a similar procedure. At 30 min after the addition of pyocin to a liquid culture of N. gonorrhoeae 72H870, a sample was removed and treated as above. The negative-stained preparation was examined in an RCA electron microscope at 50 kV.
RESULTS Purification and characterization of the inhibitory factor. Geizer (12) observed that the growth of N. gonorrhoeae was inhibited by a substance(s) produced during the growth of a strain ofP. aeruginosa. We have confirmed this phenomenon and, further, have sought to isolate and identify the inhibitory substance(s). The inhibitory factor was produced in low concentration during the growth of P. aeruginosa strain PA-103 in a complex medium (Table 1). Addition of mitomycin C (1 Ag/ml) caused extensive lysis of the culture within 3 h (Fig. 1) and resulted in a 16-fold increase in the concentration of the inhibitory factor (Table 1) as de-
termined by titration on P. aeruginosa PS7 and N. gonorrhoeae strains JW-31 and DGI 1947. The titer of the inhibitory factor varied with different strlins ofN. gonorrhoeae (Table 1); no difference was observed with colonial variants of a single strain. The inducible nature of this factor suggested the involvement of a bacteriophage or bacteriophage product. However, no plaques were observed when dilution of supernatants from both induced and noninduced cultures were spotted on N. gonorrhoeae strain JW-31. Instead, the area of growth inhibition increased in turbidity with increasing dilution, suggesting the presence of a nonreplicating bacteriocin. The inhibitory factor was partially purified from supernatants of mitomycin C-induced cultures ofP. aeruginosa strain PA-103 by a procedure used for pyocin purification. Further purification was obtained by DEAE-cellulose chromatography (Fig. 1). The inhibitory factor was TABLE 1. Effect of mitomycin C on the production of gonococcal inhibitory factor by P. aeruginosa PA103a Inhibitory titer
(U/mi)
Organism
Non-
P. aeruginosa PS-7 N. gonorrhoeae JW-31 N. gonorrhoeae DGI 1947 N. gonorrhoeae 1138 (T-1) N. gonorrhoeae 1138 (T-4)
induced
Inue Induced
2560 2560 640 NDb ND
40,960 40,960 10,240 5,120 5,120
a Mitomycin C (1 ,g/ml of medium), when serially diluted, ceased to inhibit the growth of these organisms at a >1:2 dilution (0.5 ,ug/ml). b ND, Not determined.
1.62 A, LI~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~e
as*
so6 6 U U 16 3 12 1
14
1
6
7
4
6
iLmirnrnmmi..rn.*.~~~~~~~~~~~~~~~~~~~4
FIG. 1. Purification ofR-type pyocin (611 131) by DEAE-cellulose chromatography. Fractions containing inhibitory activity are indicated by A and B. Insert: induction ofpyocin production in P. aeruginosa PA-103 by mitomycin C (1 Mg/ml). The arrow indicates time of mitomycin C addition.
INHIBITION OF N. GONORRHOEAE BY R-TYPE PYOCIN
VOL. 10, 1976
eluted with an NaCl gradient of 0 to 1.0 M. Two peaks containing the inhibitory factor were observed. The major peak (A) eluted at an NaCl concentration of 0.06 M and contained more than 90% of the inhibitory activity. A minor peak (B) eluted at an NaCl concentration of 0.91 M and contained less than 10% ofthe activity. The fractions comprising peak A were pooled, dialyzed against 0.01 M Tris-hydrochloride buffer (pH 7.5) to remove the NaCl, and concentrated by ultracentrifugation (100,000 x g for 90 min). This preparation was used for the remainder of these studies. Negative-stained preparations of the purified inhibitory factor were examined in the electron microscope. Particles resembling R-type pyocins were observed in both the uncontracted and contracted states (Fig. 2). In the uncontracted state, these particles measured 111.5 nm in length by 15.3 nm in width. In the contracted state, the particles consisted of an inner core (105 nm in length by 6.5 nm in width) surrounded by a contracted sheath (44.4 nm in length by 18.6 nm in width). Between 20 and 30% of the particles observed in these preparations were in the contracted state. Neither intact bacteriophage nor bacteriophage ghosts ,.
357
were seen in any of the negative-stained preparations. The type of the pyocin in both partially purified and purified preparations was determined as described above. The results (data not shown) indicate that the pattern did not change during purification and suggest that one or more pyocin types were not removed during chromatography on DEAE-cellulose. The pyocin pattern is 611 131. R-type pyocins may be differentiated from Stype pyocins by their resistance to proteolytic enzymes (24). Therefore, the effect of various proteolytic enzymes was examined on the inhibition of P. aeruginosa PS-7 and N. gonorrhoeae JW-31 by purified pyocin preparation. The results (not shown) indicate that the concentration did not decrease when compared with an untreated concentration of 1 x 104 U/ ml. These data suggest that the inhibition of N. gonorrhoeae was not due to an S-type pyocin. Many strains ofP. aeruginosa produce a protein toxin (exotoxin A) that inhibits mammalian protein synthesis (20). The toxin catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide to elongation factor-2, resulting in an ADP-ribosyl-elonga-
I^
4
C.
.11
4.fr
IA
.:
FIG. 2. Electron micrograph of a negative-stained preparation of R-type pyocin 611 131. Symbols: uc, uncontracted pyocin; c, contracted pyocin. Bar = 0.1 ,um.
358
MORSE ET AL.
tion factor-2 complex which is inactive in peptide chain elongation (19, 20). The possibility that the inhibition of N. gonorrhoeae was due to the presence ofthis toxin in the pyocin preparation was investigated. The results (Table 2) show that neither the crude nor the purified preparation from P. aeruginosa strain PA-103 contains ADP-ribosyl transferase activity. Thus, it seems unlikely that the inhibitory effect of the pyocin preparation is due to exotoxin A. Effect of R-type pyocin on the growth ofN. gonorrhoeae. The effect of R-type pyocin on growing cells of N. gonorrhoeae strain 72H870 was examined by the addition of various concentrations of the purified preparation to exponentially growing cultures. Figure 3 shows a concentration-dependent inhibition of gonococcal growth by the addition of type 611 131 pyocin. At high pyocin concentrations, a complete inhibition of growth occurred within 1 h and was accompanied by extensive lysis of the culture. To ascertain whether a direct interaction occurred between the pyocin and susceptible cells of N. gonorrhoeae, samples were removed from the culture after 30 min and examined by electron microscopy. The results (Fig. 4) demonstrate the binding of pyocins to the surface of the cell. The pyocin-cell interaction appears to result in contraction of the pyocin. No significant association of type 611 131 pyocin occurred with the cell surface of a resistant organism (N. ovis). These data suggest that receptors are present on the cell surface of N. gonorrhoeae. Inhibitory spectrum of pyocin type 611 131. Portions of the purified pyocin preparation were spotted on lawns prepared from clinical isolates of N. gonorrhoeae. Typical patterns of inhibition are shown in Fig. 5. The zone of inhibition was clearly evident in all strains examined. No difference was observed between colony types T-1 and T-4 from the same strain. A negative control of the producer strain, P. aeruginosa PA-103, was also included. TABLE 2. ADP-ribosyl transferase activity in preparations of R-type pyocin (611 131) from P. aeruginosa strain PA-103
ANTIMICROB. AGENTS CHEMOTHER. I
I
I
I
I
I
I
-1
0
I 1
I 2
I 3
300
200 _
100 _ I-
so
F
0-
lomi
60 -
40
F
20
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FIG. 3. Effect of R-type pyocin (611 131) on the growth of N. gonorrhoeae strain 72H870. Purified pyocin was added to exponentially growing cultures (1.4 x 108 colony-forming units/ml) of strain 72H870. Symbols: 0, no additions; 0, 400 U of pyocin per ml; A, 1,000 U of pyocin per ml; *, 4,000 U ofpyocin per ml; *, 20,000 U ofpyocin per ml.
The inhibition of various Neisseria species by this pyocin is shown in Table 3. All isolates of N. gonorrhoeae, from both disseminated and nondisseminated infections, were inhibited. However, only 3 of 20 strains ofN. meningitidis and 5 of 16 strains of N. lactamica were inhibited. No correlation was observed between the serological group and inhibition ofN. meningitidis. None of the other five species tested was inhibited by this pyocin.
DISCUSSION Bacteriocins are antibiotic substances proAcid-insoluAdditions Protein (jug) ble radioacduced by many species of bacteria which are tivity (cpm) thought to be inhibitory for strains of the same or closely related species (42). Bacteriocins are 0 Control', 121 Crude R-type pyocin 7.0 73 protein in nature and range from simple proPurified R-type pyocin 5.0 96 teins (7, 33) to particles resembling bacterioExotoxin A 0.01 2,788 phage components (3, 5, 14, 16, 22, 26). BacterDiphtheria toxin frag0.01 2,839 iocinogenic strains of P. aeruginosa produce ment A several types of bacteriocins (17). The synthesis a The control contained water in place of toxin or of these bacteriocins can be increased by mitopyocin preparation. mycin C treatment (17, 24, 28). These bacterio-
A5;>_0$ t-A>;,fXl',*f iP,s4 f i INHIBITION OF N. GONORRHOEAE BY R-TYPE PYOCIN
VOL. 10, 1976
359
ift
-J
-
f
I
,.
t
.
4
_
r
t
dS
!
§
S
a
.e
+,*
FIG. 4. Interaction ofR-tyz pyocin 611 131 with cells ofN. gonorrhyae strain 72H870. Bar = 0.1 ym.
cins may be grouped into one of two types (3): the S-type bacteriocins, which are characterized by susceptibility to proteolytic enzymes and a lack of structure in electron microscopy, and the R-type bacteriocins, which are not susceptible to proteolytic enzymes and exhibit the appearance of bacteriophage components. The bacteriocins produced by P. aeruginosa are called pyocins (25). In addition to resistance to proteolytic enzymes, R-type pyocins may be differentiated from S-type pyocins by their adsorption to DEAE-cellulose, molecular weight, rate of diffusion in agar, and neutralization by specific antiserum (24). R-type pyocins bear a close resemblance to the tails of T-even bacteriophage (14, 16, 18, 22, 28). Govan (14) reported that, regardless of the pyocinogenic strain used, the majority of the R-type particles measured approximately 100 by 15 nm in the uncontracted state; contracted particles consisted of a hollow inner core, approximately 100 by 7 nm, par-
tially enclosed in a sheath measuring about 45 by 17 nm. Our findings indicate that the gonococcal inhibitory factor produced by P. aeruginosa PA-103 was an R-type pyocin. Several lines of evidence support this conclusion. The inhibitory factor was resistant to proteolytic enzymes and adsorbed to DEAE-cellulose. Sedimentation of the inhibitory factor by ultracentrifugation (100,000 x g for 90 min) suggests that this substance has a high molecular weight. The same susceptibility pattern (611 131) was always observed when the inhibitory factor was typed on the ALA strains of P. aeruginosa. Furthermore, electron microscopy revealed particles with the same morphology and size as those reported by Govan (14). No tail fibers were observed in negative-stained preparation of 611 131 as previously reported in other R-type pyocins (16, 18). R-type pyocins may be found in both the contracted and uncontracted state (14, 16). Only uncontracted pyocins will adsorb to and kill
360
ANTIMICROB. AGENTS CHEMOTHER.
MORSE ET AL.
-
...
-.
e
FIG. 5. Inhibition of N. gonorrhoeae by an R-type pyocin (611 131). (a) N. gonorrhoeae strain JW-31; (b) N. gonorrhoeae strain 72H870; (c) N. gonorrhoeae strain 1138 (colony type T-1); (d) N. gonorrhoeae strain 1138 (colony type T-4); (e) N. gonorrhoeae strain CS-7; (t) P. aeruginosa strain PA-103.
TABLE 3. Susceptibility of various Neisseria species to purified R-type pyocin (611 131) from P. aeruginosa strain PA-103 SeroSpecies Specsgroup gop
N. gonorrhoeae N. meningitidis
A B C X Y Z
135 N. lactamica N. mucosa N. flava N. subflava N. ovis N. flavescens
No. of strains tested
No. of strains susceptible
56 4
56 0
5 3 3 1 2 2
1 0 0 1 1 & 5 0 0 0 0 0
16 1 1 1 1 1
susceptible cells (14, 16); no adsorption to pyocin-resistant bacteria occurs (14). Lethal effects occur after the contraction of bound pyocins (14). We have demonstrated that R-type pyocin 611 131 adsorbs to the surface of N. gonorrhoeae. The presence of contracted pyocins on the gonococcus suggests that receptor sites on N. gonorrhoeae may be similar to those that exist on the cell surface of certain strains of P. aeruginosa. Of special interest are studies involving shared antigens between heterologous bacterial species. Minden et al. (37) demonstrated that Pseudomonas shared an indeterminate number of antigens with other gramnegative bacteria. The presence of naturally occurring antibodies to N. gonorrhoeae (13) suggests that this organism may also share antigenic determinants with heterologous bacterial species. This possibility is supported by
VOL. 10, 1976
INHIBITION OF N. GONORRHOEAE BY R-TYPE PYOCIN
the study of Robbins et al. (43), which showed that N. meningitidis groups A and C share capsular and noncapsular antigenic determinants with strains of E. coli. The lipopolysaccharide (LPS) has been implicated as the receptor for R-type bacteriocins in P. aeruginosa (8, 14, 15, 21, 23) and in some strains of Serratia marcescens (45). The bacteriocin receptor in other strains of S. marcescens is reported to be proteinaceous (45). The receptor for pyocin 611 131 onN. gonorrhoeae has not as yet been identified. However, a receptor located on the 0 polysaccharide portion of the gonococcal LPS appears unlikely. We observed identical titers of 611 131 on T-1 and T-4 colonial variants from the same clinical isolate. Perry et al. (41) determined that LPS from T-1 cells contained a high-molecular-weight 0 polysaccharide that showed considerable variation between strains. This 0 polysaccharide was absent in T-4 cells. To the contrary, Stead et al. (44) were unable to detect 0 polysaccharide side chains in LPS isolated from either T-1 or T-4 cells. In addition, pyocin 611 131 inhibited strains of N. gonorrhoeae isolated during a 5year period from Boston, Mass., Portland, Ore., and Seattle, Wash. Strains from these diverse locales would be expected to show variability in the composition of the 0 polysaccharide side chain as previously reported by Perry et al. (41). Alternatively, the receptor may reside in a common core region of the LPS. This core oligosacchAride may be sufficiently similar in N. gonorrhoeae, N. meningitidis, and N. lactamica to account for the inhibition observed in the latter two species. However, the possibility of a protein receptor cannot be excluded at this time. A capsule may afford some measure of protection against the bactericidal activity of pyocin 611 131. The presence of a capsule in the resistant strains ofN. meningitidis and N. lactamica has not been determined. This capsule could conceivably hinder the binding of the pyocin to its receptor. A similar phenomenon has been observed with respect to the inhibition of bacteriophage binding to encapsulated bacteria (48). Allen and Kelly (2) observed that the titer of a given pyocin frequently varied with different strains of P. aeruginosa. These investigators proposed that the closeness of fit between the reactive portion of the pyocin and its receptor determined the potency of its bactericidal activity. An alternate hypothesis is that the variation in titer reflects the number ofreceptor sites per cell. Strains containing a large number of pyocin receptors per cell would exhibit a lower titer than would strains having fewer receptors
361
per cell. Either hypothesis may explain the variation in titer observed with different strains of N. gonorrhoeae. The mechanism of pyocin-induced killing of N. gonorrhoeae is not known. Kageyama et al. (30) identified a muramidase-like enzyme as a component of R-type pyocins. This enzyme could account for the rapid lysis of gonococcal cultures that were exposed to high concentrations of pyocin. However, other effects, such as an inhibition of macromolecular synthesis or other cell functions that may ultimately lead to cell lysis, cannot be eliminated at this time. In summary, the results of this study indicate that the gonococcal inhibitory factor observed in culture supernatants ofP. aeruginosa PA-103 is the R-type pyocin 611 131. The ability of pyocin 611 131 to inhibit N. gonorrhoeae suggests that either the definition of a bacteriocin with regards to species specificity needs to be reevaluated or that there is a much closer taxonomic relationship between Neisseria and Pseudomonas than heretofore suspected. ACKNOWLEDGMENTS This research was supported by grants from the Medical Research Foundation of Oregon and Phi Beta Psi Sorority. LITERATURE CITED 1. Allen, E. S., and R. S. Schweet. 1962. Synthesis of hemoglobin in a cell-free system. J. Biol. Chem. 237:760-767. 2. Allen, J. C., and P. C. Kelly. 1975. Antigenic heterogeneity among pyocins ofPseudomonas aeruginosa. Infect. Immun. 12:318-323. 3. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 4. Brenner, S., and R. W. Home. 1959. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34:103-110. 5. Coetzee, H. L., H. C. de Klerk, J. N. Koetzee, and J. A. Smit. 1968. Bacteriophage tail-like particles associated with intraspecies killings of Proteus vulgaris. J. Gen. Virol. 2:29-36. 6. Collier, R. J., and J. Kandel. 1971. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J. Biol. Chem. 246:14961503. 7. Dandeau, J. P. 1971. Chemical and immunological study of colicins E,, K, A, and Q. Infect. Immun. 3:19. 8. Dyke, J., and R. S. Berk. 1974. Growth inhibition and pyocin receptor properties of endotoxin from Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 145:1405-1408. 9. Farmer, J. J., III, and L. G. Herman. 1974. Pyocin typing of Pseudomonas aeruginosa. J. Infect. Dis. 130:543-546. 10. Feary, T. W., E. Fisher, and T. N. Fisher. 1963. Lysogeny and phage resistance in Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 113:426-430. 11. Flynn, J., and M. C. McEnteggart. 1971. Bacteriocins from Neisseria gonorrhoeae and their possible role in epidemiological studies. J. Clin. Pathol. 25:60-61.
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