Identification and Characterization of Peptidoglycan-Associated Proteins in Neisseria-Gonorrhoeae

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11-1989

Stuart A. Hill

Ralph C. Judd University of Montana - Missoula, [email protected]

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Recommended Citation Hill, Stuart A. and Judd, Ralph C., "Identification and Characterization of Peptidoglycan-Associated Proteins in Neisseria-Gonorrhoeae" (1989). Biological Sciences Faculty Publications. 106. https://scholarworks.umt.edu/biosci_pubs/106

This Article is brought to you for free and open access by the Biological Sciences at ScholarWorks at University of Montana. It has been accepted for inclusion in Biological Sciences Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. INFECTION AND IMMUNITY, Nov. 1989, p. 3612-3618 Vol. 57, No. 11 0019-9567/89/113612-07$02.00/0 Copyright X 1989, American Society for Microbiology Identification and Characterization of Peptidoglycan-Associated Proteins in Neisseria gonorrhoeae STUART A. HILLt* AND RALPH C. JUDD Division ofBiological Sciences, University of Montana, Missoula, Montana 59812 Received 18 April 1989/Accepted 14 July 1989

The principal proteins associated with Neisseria gonorrhoeae peptidoglycan (PG), as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, are the following: two proteins at approximately 90 kilodaltons (kDa), single major species at both 60 and 44 kDa, a 34- to 36-kDa protein, and three proteins between 28 and 32 kDa. A protein analogous to Escherichia coli Braun lipoprotein was not detected with gonococcal cell wall preparations. The identity of the PG-associated proteins was confirmed immunologically with antibody generated against purified cell wails. Two types of protein species, dithiothreitol extractable (the majority) and alkylation dependent (primarily the 34- to 36-kDa protein), appeared to be associated with the N. gonorrhoeae cell wall fraction. It was found that a crucial step in the extraction of the proteins from the PG fraction was the inclusion of an acetone-water wash of the purified PG pellet. Studies with cell wall preparations obtained from N. gonorrhoeae intrinsically labeled with 32P revealed that the acetone wash was removing phospholipid from the cell wall fraction and thus facilitating protein extraction. Autoradiographic analysis with PG material derived from 125j-surface-labeled cells indicated that the 44-kDa protein is exposed on the surface of the organism even when associated with the PG layer. Radioimmunoprecipitation with anti-PG antibody confirmed these findings. Lectin analysis (wheat germ agglutinin conjugated to horseradish peroxidase) suggested that the 34- to 36-kDa protein is covalently attached to the PG layer.

Gonorrhea is a disease solely of human beings and is suppressed when cells were grown at pH 6 in the presence of caused by the gram-negative diplococcus Neisseria gonor- Mg2+ ions or reduced 50% at pH 6 in the absence of the rhoeae. The biology of N. gonorrhoeae is complex, probably cation (32). Similarly, it was found that penicillin-induced reflecting the need of the organism to survive in various lysis could also be prevented by growth at a low pH in the anatomical niches while withstanding a variety of host presence of Mg2+ (33). These results suggest that the de- defenses within the human body. The organism is a typical crease in the autolytic activity of the PG at a low pH was due gram-negative bacterium, with an inner cytoplasmic mem- in part to an increased stability of the cell, possibly through brane, a thin peptidoglycan (PG) layer, and an outer mem- the association of protein with the PG (13, 14). brane (OM) containing protein, lipopolysaccharide, and We looked for the PG-associated protein(s) in N. gonor- phospholipid. Various OM proteins, including the major OM rhoeae for another reason. PG isolated from 1251-surface- protein P.1 (2, 19), the opacity-associated protein(s) P.11 labeled cells contained significant amounts of radioisotope (30), and several less-well-defined protein components that which remained with the PG even after extensive solubiliza- include P.III (18), H.8 antigen (6, 29), and a 44-kilodalton tion. This result suggested that surface-exposed proteins (kDa) protein (35), have been characterized. In contrast to were associated with the PG and suggested a means by the situation with other gram-negative organisms (4), a which we could purify surface-exposed OM proteins from PG-associated protein analogous to Braun lipoprotein (BLP) crude OM-protein-PG mixtures by selectively hydrolyzing has not been found in N. gonorrhoeae (15, 34). Nonetheless, the proteins away from the PG in a relatively pure state. In gonococci are believed to possess a PG-associated protein(s) this study we identify the proteins which are associated with after in vitro growth in acidic medium (pH 6) (13, 14). gonococcal PG; at least one of these proteins is surface However, the identity of this protein(s) is complicated exposed. The removal of phospholipid from the cell wall because of the extreme insolubility of the N. gonorrhoeae fraction was found to greatly facilitate the solubilization and PG-protein complex. extraction of these PG-associated proteins. These findings The association of protein with the cell wall fraction (i.e., constitute preliminary evidence for the covalent association the fraction remaining after exhaustive extraction with de- of a 34- to 36-kDa protein with the PG. tergent) under acidic conditions may represent an important structural modification that is relevant to the survival of MATERIALS AND METHODS gonococci in the human body (8). Several studies have Strains. N. gonorrhoeae CS7 (13, 14), a gift from Tom addressed the effects of pH on the biology of gonococci Dougherty (Rockefeller University, New York, N.Y.), was (12-14, 22, 25, 32, 33). One found that the organism had an primarily used in this study. Strains JS1, JS3, FA635, and enhanced ability to adhere to host cells at a low pH (25). FA638 have been described elsewhere (19). Growth on solid Also, structural analysis of gonococcal PG (12, 32, 33) medium utilized the clear medium described demonstrated that the maximum autolytic activity occurred typing by Swanson (30). Single colonies were passaged daily and when cells were grown in vitro at pH 9. This effect could be grown at 36.5°C in a 5% CO2 atmosphere. In all cases only transparent colonies were selected. For the majority of the * Corresponding author. experiments described, cells were grown in liquid medium. t Present address: Laboratory of Microbial Structure and Func- In this case, bacteria were swabbed from a 14-h plate and tion, Rocky Mountain Laboratories, Hamilton, MT 59840. suspended in prewarmed medium to an optical density at 600 3612 VOL. 57, 1989 N. GONORRHOEAE PEPTIDOGLYCAN-ASSOCIATED PROTEIN 3613 nm of 0.05. The liquid medium was identical to the clear blots were washed extensively with phosphate-buffered sa- typing medium except for the omission of the agar and the line-Tween and developed by the same method as that used inclusion of NaHCO3 (420 ng/ml) as the CO2 source. The for antibody detection. To assess competitive inhibition of liquid medium was buffered to pH 6 (conditions at which WGA, we preincubated lectin preparations overnight at optimum protein association occurs with the PG fraction [13, room temperature in various sugar solutions. Each lectin- 14]) by adjusting the potassium salt ratios. The cells were sugar solution was used to probe proteins immobilized on grown in conical flasks at 37°C by using a wrist-action nitrocellulose. The following sugar solutions were used: 50 shaker. Growth was monitored by measuring the optical mM glucose, 50 mM N-acetylglucosamine, or the soluble density in a Coleman 2 spectrophotometer. fraction resulting from a 2-day lysozyme (derived from Radiolabeling of gonococci. Surface iodination of N. gon- Streptomyces globisporus [Miles Scientific, Div. Miles Lab- orrhoeae with 1251 was done by the lodogen (Sigma Chemi- oratories, Inc., Naperville, Ill.]) hydrolysis at 37°C of puri- cal Co., St. Louis, Mo.) procedure as described by Judd fied gonococcal PG (a [3H]GlcNH2-labeled PG sample was (18). For the intrinsic labeling of phospholipid with H332P04, used as an internal control to monitor the extent of hydrol- bacteria were grown in liquid medium containing 2 ,uCi of ysis that was >90%). H332P04 per ml. The cells were labeled continuously Radioimmunoprecipitation. Radioimmunoprecipitation with throughout growth. Labeling was terminated by pouring anti-CS7 cell wall antiserum was performed exactly as late-log-phase cultures over ice, with excess radiolabel being described by Swanson and Barrera (31). Whole, intact removed by washing the cells with phosphate-buffered sa- gonococci were surface iodinated and reacted with antibody line. before solubilization with Zwittergent 3-14. The surface- Preparation of purified cell wails. Purified cell walls were labeled proteins were precipitated with Sepharose 4B-CLB prepared as described by Gomez-Miguel and Moriyon (11) beads coated with protein A (Sigma). for the preparation of Brucella abortus PG. For large-scale Phospholipid analysis. 32P-labeled phospholipids were ex- isolation of cell walls, 1-liter cultures were used. The cells tracted from whole cells by the procedure described by Bligh were harvested by centrifugation and refluxed at 100°C for 20 and Dyer (3). 32P-labeled phospholipids were also isolated min in 10 ml of 4% sodium dodecyl sulfate (SDS)-50 mM from the pooled acetone washes of the cell wall material. sodium acetate (pH 5.6) with stirring. The pellet was col- Phospholipids were prepared for paper chromatography with lected by centrifugation (10,000 x g) and extracted two more Whatman SG81 paper by drying the acetone samples in times under identical conditions. The resulting pellet was vacuo and suspending the anhydrous phospholipids in a washed six times with 50 mM sodium acetate (pH 5.6). The small volume of chloroform. The chromatography solvent washed pellet was extracted three times with acetone-water systems used were chloroform-methanol-2,6-dimethyl-4- (6:1 [vol/vol]). The acetone-washed residue was collected by heptanone-acetic acid-water (35:10:50:25:4) in the first di- centrifugation at 10,000 x g for 20 min. mension and chloroform-methanol-2,6-dimethyl-4-hepta- Extraction of acetone-washed cell walls. Detergent extrac- none-pyridine-0.5 M NH4Cl (pH 9.8) (60:30:45:70:12) in the tion of cell walls was carried out overnight at 37°C on a second dimension. The phospholipids were visualized by rocking platform. Cell walls were extracted with 4% SDS in autoradiography of the chromatograms as described above. the presence of either 8% 2-mercaptoethanol (Sigma) or 5 mM dithiothreitol (Sigma). In some cases reduced sulfydryl RESULTS groups were alkylated with iodoacetamide (Sigma) before detergent extraction. Generally, alkylation of a 50-,ul sample Identification of PG-associated proteins in N. gonorrhoeae. (approximately 250 ,ug of protein) was done with 10 ,u of 100 From purified acetone-extracted PG, we were unable to mM iodoacetamide in the dark at room temperature for 3 h. extract protein by strictly nondetergent means (NaCl, Alkylated cell walls were pelleted and suspended for further EDTA, and urea extraction). However, extraction of ace- extraction. tone-washed PG at 37°C overnight with 4% SDS containing Preparation of anti-CS7 cell wall antibody. Antiserum was either 8% 2-mercaptoethanol or 5 mM dithiothreitol resulted generated in a rabbit by inguinal and axillary injections of in the liberation of several protein species from the PG- purified cell walls in complete Freund adjuvant. These protein complex (Fig. 1A). Principal proteins associated with injections were followed 1 week later with intramuscular and CS7 cell walls were two at approximately 90 kDa, one each subcutaneous injections in incomplete Freund adjuvant and at 60 and 44 kDa, and three between 28 and 32 kDa. Little subsequent intravenous inoculations at approximately 1- protein could be extracted when the acetone wash was week intervals. omitted before detergent extraction (Fig. 1A, lane B) or in Electrophoresis. SDS-polyacrylamide gel electrophoresis the absence of a reducing agent. These results suggest the (PAGE) was done by the method of Laemmli (20) at a involvement of disulfide linkages in the PG-protein complex. constant power of 10 W. The gels were fixed and stained in Tryptic digestion of N. gonorrhoeae cell wall material with 25% isopropanol-5% acetic acid containing 0.05% Coomas- the same conditions as those described by Braun and Rehn sie brilliant blue. Autoradiography of gels was done with (5) failed to liberate a protein analogous to the Braun XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) with lipoprotein of Escherichia coli cell walls, while E. coli high-speed intensifying screens at -70°C. treated in an identical fashion yielded a single major species Immunoblotting. Immunoblotting was performed exactly at approximately 14 kDa (Fig. 1A, lane E). In contrast, as described by Judd (19) with the low-phosphate buffer lysozyme digestion of N. gonorrhoeae PG facilitated the system (pH 8) of Batteiger et al. (1). liberation of all of the PG-associated protein from the Protein immobilized on nitrocellulose after transfer was PG-protein complex (data not shown). The Western blot also probed with the lectin wheat germ agglutinin (WGA) (immunoblot) in Fig. 1B shows the association of H.8 conjugated to horseradish peroxidase. The blots were ini- antigen with gonococcal PG. The blot also demonstrates that tially blocked overnight in 0.05% Tween in phosphate- H.8 antigen tended to accumulate with the PG fraction as buffered saline. The lectin conjugate (5 [lI; 1 mg/ml; Sigma) cells entered the stationary phase of growth (this occurred was reacted with the paper for 2 h at room temperature. The after approximately 10 h). H.8 antigen was not released by 3614 HILL AND JUDD INFECT. IMMUN.

with CS7 PG (Fig. 3B). Identical results were also found for A. MW A B C D E strain JS1. A comparison of Fig. 3B with the phospholipid profile obtained from the extraction of whole cells (Fig. 3A) tentatively identified this lipid as phosphatidylglycerol. 92 Trace amounts of several other lipids were also found. The 66 phospholipid(s) associated with the PG fraction was nonco- valently bound to protein found in the PG complex, judging 45 from the lack of a signal from any protein in this fraction when 32P-labeled cell wall material was separated by SDS- PAGE and then autoradiographed (Fig. 3C). 31 - Immunological confirmation of the identity of gonococcal PG-associated protein. Antiserum was generated in a single rabbit to purified CS7 PG preparations that had not under- 21 - gone acetone extraction and was screened against various whole-cell lysates by immunoblotting (Fig. 4B). The pre- 14 - dominant antigens recognized by this antiserum were a 30-kDa protein, lesser bands at approximately 36, 40, and 56 kDa, and multiple bands upward from 60 kDa. The spectrum resembled the SDS-PAGE profile presented in Fig. 1A, except for the lack of recognition of the 90-kDa proteins. The same antiserum shown in Fig. 4B was used to radioimmu- B. 8 10 12 14 16 (hrsXl noprecipitate CS7 surface-labeled proteins. The autoradio-

WC W S R s R S R S R s WC graph in Fig. 4C demonstrates that the antiserum recognized and precipitated surface-labeled 44-, 60-, and 90-kDa proteins from intact gonococci. Lectin characterization of PG-associated protein. If protein is covalently bound to the PG-protein complex, as for BLP FIG. 1. SDS-PAGE analysis of cell wall fractiotns. (A) Coomas- in E. coli (4), then a PG disaccharide subunit might associate sie-stained gel containing equivalent purified PG saamples as judged with a specific N. gonorrhoeae protein, and this band should by volume. Lanes: A, whole-cell lysate of strain CS,7; B, purified PG react with WGA (10). A 34- to 36-kDa protein in whole-cell sample (not washed with acetone-water) extracted with detergent in lysate fractions reacted with the WGA conjugate when the presence of 2-mercaptoethanol; C, same as laine B except that electroblotted onto nitrocellulose (Fig. 5). No other compo- the PG sample was washed with acetone-water Ibefore detergent nents in the whole-cell lysates were recognized by the WGA extraction; D, effect of trypsin (5) on gonococcal under the conditions described. The results of the trypsin on E. coli PG prepared under the same cond[itions as those in conjugate lanes B, C, and D; MW, molecular weight markers Numbers at left competitive inhibition studies (Fig. SA, C, and D) of WGA indicate molecular weights in thousands. The re;adily identifiable demonstrated that the hydrolysate obtained from lysozyme proteins are indicated by bars. (B) Immunoblot oiIf gonococcal PG digestion of gonococcal PG was a more potent inhibitor than preparations probed with H.8 monoclonal antibo(dy. PG was pre- that resulting from treatment with 50 mM N-acetylglucos- pared from identical culture volumes of strain FAi638 at the times amine (apparently reflecting the lower equilibrium constant indicated in hours. After detergent extraction, the solution was of the former [10] for WGA). The inclusion of50 mM glucose centrifuged and the pellet (R) and supernatant (S') fractions were in the reaction mixture caused no inhibition of WGA bind- isolated. WC, Whole-cell lysate. ing, as expected; likewise, concanavalin A conjugated to peroxidase (specific for glucose) failed to react. When ace- the action of trypsin, as assessed by Westeri n blot analysis. tone-washed PG fractions were alkylated with iodoaceta- PG-protein material probed with a P.III-speciific monoclonal mide before detergent extraction, a 36-kDa protein that antibody (MC01) proved to be negative. reacted with the WGA conjugate was extracted (Fig. SE). When purified PG fractions were obtainedI from surface- This protein failed to react with a P.I-specific monoclonal labeled bacteria, a single protein was founcJ to be readily antibody (4G5; Genetic Systems, Seattle, Wash.). The above iodinated, even though closely associated with the PG frac- data suggest that a protein with a variable molecular mass tion; this was the 44-kDa OM protein (Fig. 2.A to D). Strain (between 34 and 36 kDa, depending upon the strain) is FA638 was unusual in that the 44-kDa OM pprotein was not covalently attached to the PG fraction. surface exposed when associated with the P'G fraction; for this strain it was found that a 60-kDa prote,in was surface DSUSO labeled (Fig. 2E). For strain FA635, both the 44- and 60-kDa DISCUSSION proteins were found to be accessible to surfac:e labeling (Fig. Previous work (13, 14) had demonstrated that protein 2D). associated with the N. gonorrhoeae PG fraction when cells Analysis of acetone-soluble material. The crucial feature of were grown under acidic conditions (pH 6) in vitro. From the this rigorous extraction procedure was foiund to be the data presented in this report, it is clear that N. gonorrhoeae acetone-water washing step, suggesting thatt this step was possesses PG-associated proteins, as defined in the literature extracting a lipidlike substance-possibly phc)spholipid. Iso- for other gram-negative organisms (4, 5, 9, 11, 24). The lation of purified PG from cells intrinsically l abeled with 32p multiple species associated with the gonococcal PG fraction revealed that approximately 1% of the toital 32p counts consisted of several OM proteins (e.g., the 44-kDa protein) remained with the cell wall fraction after extensive 4% SDS and several other proteins, presumably periplasmic in origin. treatment. Analysis of the acetone-water Nwashes for the Identical results were found for all strains that we tested, presence of phospholipids by two-dimensiotnal paper chro- regardless of the culture conditions. However, as previously matography demonstrated a single phosphollipid associated described (13, 14), there was considerable strain variation VOL. 57, 1989 N. GONORRHOEAE PEPTIDOGLYCAN-ASSOCIATED PROTEIN 3615

A. B. C. D. E.

WC P WC P WC P WC P WC P . MW MW .. 92 - ni: - 92 66 - ^_t t v -66

_t ._- _L.t. _E.i w -45 45 - k XF s XF' F...... w - 31 - 31

21 - -21

- 14

FIG. 2. SDS-PAGE analysis of cell wall fractions isolated from surface-labeled strain JS1 (A), strain JS3 (B), strain CS7 (C), strain FA635 (D), and strain FA638 (E). PG preparations were obtained from surface-labeled cells as described previously (18) and extracted with detergent in the presence of a reducing agent. WC, Whole-cell lysate; P, PG fraction. Arrows indicate surface-labeled PG-associated proteins. MW, Molecular weights in thousands.

with regard to the amount of protein associated with the PG These results are in contrast to those reported in the fraction. A direct assessment of protein concentration by mid-1970s by two groups (15, 34) who were unable to detect colorimetric determination or determination of specific ac- proteinaceous material in association with their purified PG tivities with a radiolabel was found to be impractical, pri- preparations by amino acid analysis. Several reasons may marily because of the extreme insolubility of the complex in account for this disparity. (i) In the unsuccessful studies, the that complete solubilization was never achieved. This can be bacteria were grown in media buffered at pH 7. Hebeler et al. best appreciated in Fig. 2, in which the surface-labeled (13, 14) have since demonstrated that the optimum culture samples still emitted a considerable signal at the interface of conditions for protein association occur at pH 6, the pH at the stacking and separating gels. However, we believe that which the cells were grown for the majority of the experi- complete solubilization of the OM was achieved during the ments reported here. (ii) In the unsuccessful studies, the preparation of the PG fraction because lipopolysaccharide bacteria were harvested during exponential growth. The could not be demonstrated by silver staining, immunological immunoblot in Fig. 1B indicates that a greater degree of probing, or autoradiography of samples intrinsically labeled protein association occurs upon entry into the stationary with 32p (Fig. 3C). However, this may reflect the greater phase of growth (beginning at 10 h in Fig. 1B). Similar results degree of solubility of certain components than of others. have also been reported for another gram-negative bacte-

A. VWhole cell32P PhospholiPid B. 32P Phospholipids Isolated C. SDS Page Extraction from CS7 Cell Walls 1 2 3

I^r ci

Ak. _1 I

OLS lit L2nd L2nd

FIG. 3. Analysis of 32P-labeled phospholipids isolated from a purified cell wall fraction of strain CS7. Strain CS7 was labeled continuously with 32P until the late log phase. Cell walls were isolated and washed with acetone-water (6:1 [vollvol]). The acetone-water fractions were pooled and dried in vacuo, and the phospholipids were analyzed by two-dimensional paper chromatography. PE, Phosphatidylethanolamine; PGI, phosphatidylglycerol; CL, cardiolipin; LPS, lipopolysaccharide; PI, OMP protein P.I. (A) Analysis of phospholipids derived from whole cells (3) (trace amounts of an additional phospholipid were also seen close to the origin but were omitted from this particular figure). (B) Analysis of phospholipids contained in the acetone-water wash of strain CS7. Trace phospholipids are indicated by the arrows. (C) Autoradiograph of an SDS-PAGE gel of the 32P-labeled cell wall material (Coomassie staining of this fraction revealed the presence of proteins). Lanes: 1, '25I-surface-labeled whole-cell preparation; 2, whole-cell preparation intrinsically labeled with 32p; 3, 32P-labeled PG preparation. 3616 HILL AND JUDD INFECT. IMMUN.

A. B. C. MAW 1 2 3 4 5 MW 12 3 4 5 I u ITI.

92- -z :-92 66- _b -66 '..... 45- -- -45 ,.... P.|I.` 31- _-u -31 .w1

.: ::: .: :: 14- _ 1 4 ...... :: ...... FIG. 4. Immunological analysis of gonococci with antibody raised against purified CS7 PG. (A) Whole-cell lysates of strains JS1 (lane 1), JS3 (lane 2), CS7 (lane 3), FA635 (lane 4), and FA638 (lane 5) electroblotted onto nitrocellulose and stained with India ink for comparison. MW, Molecular weight markers; numbers at left and right indicate molecular weights in thousands. PI, OMP protein P.I. (B) Immunoblot with anti-CS7 cell wall antibody (1:50 dilution). Strains were as in panel A. Preimmune serum was negative under identical conditions. (C) Radioimmunoprecipitation of CS7 1251-surface-labeled proteins (31) with the anti-CS7 PG antibody. Lanes: I, whole-cell lysate preparation of surface-labeled bacteria; II, surface-labeled proteins precipitated with the anti-CS7 PG antibody; III, preimmune serum control. P.I., OMP protein P.I. rium, Proteus mirabilis (9). (iii) They could possibly have extreme insolubility of the complex. After considerable been using a strain that did not possess much PG-associated effort, we found that a crucial step in the solubilization, protein. Hebeler et al. (13, 14) have also demonstrated that extraction, and manipulation of these proteins was the the amount of protein association appears to be somewhat inclusion of an acetone-water wash of the cell wall fraction strain specific. Indeed, one of the strains that was reported before extraction with detergent (compare lane B with lane C to lack PG-associated proteins (RUG 40) was later shown to in Fig. 1A). A possible explanation for the insolubility possess these proteins. Thus, the use of an inappropriate problem of the PG-associated proteins could well be the strain, exacerbated by the use of unfavorable conditions, presence of phospholipid in the purified cell walls (Fig. 3). may account for the previously unsuccessful demonstrations Proteins that are tightly complexed with phospholipids are of PG-associated proteins in N. gonorrhoeae. not unknown, an example being the membrane penicillinase Initial attempts (13, 14) to identify N. gonorrhoeae PG- found in Bacillus licheniformis (21). The association of associated proteins were probably thwarted because of the phospholipid with the cell wall fraction is no doubt enhanced

B. D. 1 2 3 1 2 3 1 2 3 i 2 3

..... t ...... :: ... .. :.: .. :. .. F. .. .. S :: .... ;. *.:: **.<4:R>.. ::..ig ,.t:... a...... --... _F

::...... *: ..... *..122 E...... No2ME 2ME lodoacetamide ...... " WC P We P ::... WC:.;.: P.

FIG. 5. Lectin analysis of gonococcal proteins probed with WGA conjugated to horseradish peroxidase. Whole-cell lysates of N. gonorrhoeae JS1 (lanes 1), CS7 (lanes 2), and FA638 (lanes 3) and various cell wall fractions obtained from strain CS7 were electroblotted onto nitrocellulose and probed for the presence of PG subunits associated with GC proteins by using the PG-specific probe WGA (10). (A) WGA in the presence of 50 mM glucose. (B) Concanavalin A conjugated to horseradish peroxidase (specific for glucose). (C) WGA in the presence of 50 mM N-acetylglucosamine (50% inhibitory concentration [10]). (D) WGA in the presence of the supernatant fraction resulting from a 2-day lysozyme hydrolysis of gonococcal PG (percentage of hydrolysis that was >90%, as determined with a labeled internal control). (E) WGA probing of various CS7 cell wall fractions. WC, whole-cell lysate; P, cell wall fraction; 2ME, 2-mercaptoethanol. VOL. 57, 1989 N. GONORRHOEAE PEPTIDOGLYCAN-ASSOCIATED PROTEIN 3617 by the 0 acetylation of the gonococcal PG sugar backbone an antigen recognized by convalescent-phase antisera (35), a itself (26, 27). Interestingly, we also found an increase in the protein of similar molecular weight has been implicated in percentage of 0 acetylation of PG with growth under acidic serum resistance (17), and a 44-kDa surface-exposed protein conditions (data not shown). 0 acetylation is also known to is also believed to bind the lysosomal enzyme cathepsin G increase upon entry into the stationary phase (27). Both of (28). Consequently, N. gonorrhoeae PG-associated proteins these conditions correlate with elevated levels of proteins may also represent important surface antigens, similar to the associated with the cell wall fraction, as determined by situation with H. influenzae. SDS-PAGE analysis. Consequently, one can envision that gonococcal cell walls are particularly hydrophobic in char- LITERATURE CITED acter, and this may enhance the hydrophobic barriers found 1. Batteiger, B., W. J. Newhall V, and R. B. Jones. 1982. The use in the OM or provide additional ones (22). of Tween-20 as a blocking agent in the immunological detection Our attempts to demonstrate in N. gonorrhoeae a protein of proteins transferred to nitrocellulose membranes. J. Immu- that is equivalent to the bound form of BLP were unsuccess- nol. Methods 55:297-307. ful (Fig. 1A). In addition, we were unable to demonstrate the 2. Blake, M. S., and E. C. Gotschlich. 1982. Purification and partial association of lipoprotein with gonococcal cell walls, based characterization of the major outer membrane protein of Neis- on of cell wall material isolated from seria gonorrhoeae. Infect. Immun. 36:277-283. fluorographic analysis 3. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid [3H]palmitate-labeled cells. However, a recent study (29) of extraction and purification. Can. J. Biochem. Physiol. 379: H.8 antigen demonstrated that this protein is a lipoprotein. 911-917. Other work analyzing [3H]palmitate-labeled whole-cell ly- 4. Braun, V. 1975. Covalent lipoprotein from the outer membrane sates (7) suggested that a 44-kDa protein and several proteins of Escherichia coli. Biochim. Biophys. Acta 415:335-377. between 28 and 32 kDa are lipoproteins (results that we were 5. Braun, V., and K. Rehn. 1969. Chemical characterization, able to confirm). Whether the proteins demonstrated in this spatial distribution and function of a lipoprotein of the Esche- study correspond to those believed to possess fatty acid richia coli cell walls. The specific effect of trypsin on the must also await further clarification. membrane structure. Eur. J. Biochem. 10:426-434. The data in SE that the of 6. Cannon, J. G., W. J. Black, I. Nachamkin, and P. W. Stewart. presented Fig. suggest integrity 1984. Monoclonal antibody that recognizes an outer membrane the PG-protein complex is maintained by disulfide interac- antigen common to the pathogenic Neisseria species but not to tions in that iodoacetamide treatment is required to release a most nonpathogenic Neisseria species. Infect. Immun. 43:994- protein from the PG cell wall fraction that contains a PG 999. subunit. The lack of recognition by the P.I-specific mono- 7. Chen, C. Y., C. S. Parsons, and S. A. Morse. 1984. Membrane clonal antibody 4G5 argues that this protein is not P.1 but is proteolipids of Neisseria gonorrhoeae, p. 360-365. In G. K. a protein of similar molecular weight. The fact that the lectin Schoolnik, G. F. Brooks, S. Falkow, C. E. Frasch, J. S. reacts with the 34- to 36-kDa protein in the whole-cell lysate Knapp, J. A. McCutchan, and S. A. Morse (ed.), The patho- fractions (interestingly, in a stairlike pattern reminiscent of genic neisseriae. American Society for Microbiology, Washing- P.I) suggests that a pool of these molecules attached to a PG ton, D.C. 8. Cohen, L. 1969. The influence of pH on vaginal discharges. Br. subunit but unattached to the murein sacculus may exist. An J. Vener. Dis. 45:241-245. analogous situation has also been found for BLP in E. coli 9. Gmeiner, J., H.-P. Kroll, and H. H. Martin. 1978. The covalent (23), in which nascent BLP attached to a PG disaccharide rigid-layer lipoprotein in cell walls of Proteus mirabilis. Eur. J. subunit has been isolated away from the PG layer in the OM Biochem. 83:227-233. fraction. Consequently, this may provide gonococci with a 10. Goldstein, I. J., and C. E. Hayes. 1978. The lectins: carbohy- mechanism with which they can associate a protein with the drate-binding proteins of plants and animals. Adv. Carbohydr. PG layer when conditions dictate (such as growth under Chem. Biochem. 35:214-224. acidic conditions). Such a protein is most likely a periplas- 11. Gomez-Miguel, M. J., and I. Moriyon. 1986. Demonstration of a mic from the lack of in the peptidoglycan-linked lipoprotein and characterization of its protein, judging recognition trypsin fragment in the outer membrane ofBrucella spp. Infect. radioimmunoprecipitation experiment. Immun. 53:678-684. An initial thrust of this study was to identify the surface- 12. Goodell, E. W., M. Fazio, and A. Tomasz. 1978. Effect of exposed proteins associated with the PG layer in the hope of benzylpenicillin on the synthesis and structure of the cell possibly using this association as a means to rapidly purify envelope of Neisseria gonorrhoeae. Antimicrob. Agents Che- OM proteins of interest. With regard to this point, we believe mother. 13:514-526. that we have been successful in identifying at least one major 13. Hebeler, B. H., S. A. Morse, W. Wong, and F. E. Young. 1978. surface-exposed OM protein, this being the 44-kDa protein Evidence for peptidoglycan-associated protein(s) in Neisseria (Fig. 2 and 4C). Additional proteins (60 and 90 kDa) may also gonorrhoeae. Biochem. Biophys. Res. Commun. 81:1011-1017. be surface from the data in 14. Hebeler, B. H., W. Wong, S. A. Morse, and F. E. Young. 1979. exposed, judging precipitation Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan Fig. 4C. However, the 60-kDa protein is not present in protein complex. Infect. Immun. 23:353-359. purified OM preparations (16), suggesting that it may be a 15. Hebeler, B. H., and F. E. Young. 1976. Chemical composition periplasmic protein that has been accidentally labeled, pos- and turnover of peptidoglycan in Neisseria gonorrhoeae. J. sibly by iodine gaining access through the porin molecule Bacteriol. 126:1180-1185. during the labeling process. Recently, a surface-exposed 16. Johnston, K. H., and E. C. Gotschlich. 1974. Isolation and PG-associated protein was demonstrated in Haemophilus characterization of the outer membrane of Neisseria gonor- influenzae (24) and is now believed to be an important rhoeae. J. Bacteriol. 119:250-257. surface antigen for the organism. The original observations 17. Joiner, K. A., K. A. Warren, C. Hammer, and M. M. Frank. demonstrating the presence of this protein in H. influenzae 1985. Bactericidal but not nonbactericidal C5b-9 is associated with distinct outer membrane proteins in Neisseria gonor- resulted from radioimmunoprecipitation experiments with rhoeae. J. Immunol. 134:1920-1925. antisera generated against purified cell walls. Whether the 18. Judd, R. C. 1982. '25I-peptide mapping of protein III isolated 44-kDa protein or any other PG-associated protein in gono- from four strains of Neisseria gonorrhoeae. Infect. Immun. cocci is an important surface antigen must await further 37:622-631. study. However, evidence suggests that a 44-kDa protein is 19. Judd, R. C. 1986. Evidence for N-terminal exposure of the 3618 HILL AND JUDD INFECT. IMMUN.

prqtein IA subclass of Neisseria gonorrhoeae protein I. Infect. 49:7-13. Immun. 54:408-414. 28. Shafer, W. M. 1988. Lipopolysaccharide masking of gonococcal 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the outer membrane proteins modulates binding of bactericidal assembly of the head of bacteriophage T4. Nature (London) cathepsin G to gonococci. J. Gen. Microbiol. 134:539-545. 227:680-685. 29. Strittmatter, W., and P. J. Hitchcock. 1986. Isolation and 21. Lampen, J. O., and J. B. K. Neilsen. 1984. N-terminal glyceride- preliminary biochemical characterization of the gonococcal H.8 cysteine modification of membrane penicillinases in gram-posi- antigen. J. Exp. Med. 164:2038-2048. tive bacteria. Methods Enzymol. 106:365-368. 30. Swanson, J. 1982. Colony opacity and protein II compositions of 22. Magnusson, K.-E., E. Kihlstrom, L. Norlander, A. Norqvist, J. gonococci. Infect. Immun. 37:359-368. Davies, and S. Normark. 1979. Effect of colony type and pH on 31. Swanson, J., and 0. Barrera. 1983. Immunological characteris- the surface charge and hydrophobicity of Neisseria gonor- tics of gonococcal outer membrane protein II assessed by rhoeae. Infect. Immun. 26:397-401. and coagglutination. J. 23. Mett, H., R. Bracha, and D. Mirelman. 1980. Soluble nascent immunoprecipitation, immunoblotting, peptidoglycan in growing Escherichia coli cells. J. Biol. Chem. Exp. Med. 157:1405-1420. 255:9884-9890. 32. Wegener, W. S., B. H. Hebeler, and S. A. Morse. 1977. Cell 24. Munson, R. S., and D. M. Granoff. 1985. Purification and partial envelope of Neisseria gonorrhoeae: relationship between auto- characterization of outer membrane proteins P5 and P6 from lysis in buffer and the hydrolysis of peptidoglycan. Infect. Haemophilus influenzae type b. Infect. Immun. 49:544-549. Immun. 18:210-219. 25. Pearce, W. A., and T. M. Buchanan. 1978. Attachment role of 33. Wegener, W. S., B. H. Hebeler, and S. A. Morse. 1977. Cell gonococcal pili. Optimum conditions and quantification of ad- envelope of Neisseria gonorrhoeae: penicillin enhancement of herence of isolated pili to human cells in vitro. J. Clin. Invest. peptidoglycan hydrolysis. Infect. Immun. 18:717-725. 61:931-943. 34. Wolf-Watz, H., T. Elmros, S. Normark, and G. D. Bloom. 1975. 26. Rosenthal, R. S., J. K. Blundeli, and H. R. Perkins. 1982. Cell envelope of Neisseria gonorrhoeae: outer membrane and Strain-related differences in lysozyme sensitivity and extent of peptidoglycan composition of penicillin-sensitive and -resistant O-acetylation of gonococcal peptidoglycan. Infect. Immun. 37: strains. Infect. Immun. 11:1332-1341. 826-829. 35. Zak, K., J.-L. Diaz, D. Jackson, and J. E. Heckels. 1984. 27. Rosenthal, R. S., M. A. Gfell, and J. W. Folkening. 1985. Antigenic variation during infection with Neisseria gonorrhoe- Influence of protein synthesis inhibitors on regulation of extent ae: detection of antibodies to surface proteins in sera of patients of O-acetylation of gonococcal peptidoglycan. Infect. Immun. with gonorrhea. J. Infect. Dis. 149:166-174.