cholerae Non-Ol and Comparison with a Protease of V. cholerae 01

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1 INFECTION AND IMMUNITY, Sept. 1989, p Vol. 57, No /89/ $2./ Copyright C) 1989, Amerian Soiety for Mirobiology Purifiation and Charaterization of a Protease Produed by Vibrio holerae Non-Ol and Comparison with a Protease of V. holerae 1 TAKESHI HONDA,'* KANCHALEE LERTPOCASOMBAT,l ATSUKO HATA,1 TOSHIO MIWATANI,l AND RICHARD A. FINKELSTEIN2 Researh Institute for Mirobial Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, Japan,' and Department of Mirobiology, Shool of Mediine, University of Missouri, Columbia, Missouri2 Reeived 27 Marh 1989/Aepted 12 June 1989 A protease produed by a linial isolate of Vibrio holerae non-ol was purified to apparent homogeneity by ammonium sulfate frationation and suessive olumn hromatography on DEAE-Sephadex A25, Sephadex G1, Mono Q, and Phenyl Superose. Like the hemagglutinin-protease of V. holerae 1, the purified protease had both hemagglutinating and proteolyti ativities. The protease was heat labile, and in ontrast to rude preparations, no Arrhenius effet was observed with the purified protein. Immunologial analyses indiated that the proteases (or hemagglutinins) derived from V. holerae 1 and non-ol are idential. Vibrio holerae non-o1 strains, formerly alled nonagglutinable or nonholera vibrios, have oasionally been reognized as the ausative agent of small outbreaks and sporadi ases of gastroenteritis (2, 13, 15). Several investigators have reported the prodution of various toxins and toxi substanes by strains of V. holerae non-o1, inluding holera toxin, holera toxin-related enterotoxins (17, 18), Esherihia oli heat-stable enterotoxin-like toxin (1), and hemolysins similar to El Tor hemolysin (16) and V. parahaemolytius thermostable diret hemolysin (19). It has been reported (3, 4, 8, 9) that a soluble zin- and aliumdependent protease from V. holerae 1 auses hemagglutination, hydrolyzes muin, leaves latoferrin, and niks the A subunit of holera enterotoxin. Very reently, Crowther et al. (6) have demonstrated that a zinov-inhibited metalloprotease in V. holerae ulture filtrates, apparently idential to that desribed previously by Booth et al. (3), enhaned the ativity of holera enterotoxin in intestinal loops by eroding the protetive layer of epithelial muus. These ativities of the protease may failitate baterial intestinal olonization or detahment (or both) and enhane toxi ativity. Protease produed by V. holerae non-o1 (5, 11) may also funtion as a virulene fator. Although similarities, and some dissimilarities, between V. holerae non-o1 and 1 proteases have been reported (11), the V. holerae non-o1 protease has not been well haraterized. Thus, in the present work, we desribe the purifiation and some properties of a protease of V. holerae non-o1 and ompare the results with those of V. holerae 1. MATERIALS AND METHODS Baterial strains and ultures. V. holerae non-o1 TH81, an isolate from a patient with traveller's diarrhea, was used (11). Bateria were ultured in 1 liter of trypti soy broth in 5-liter flasks with shaking at 3 C for 2 h. Culture supernatants were obtained by entrifugation (1, x g for 3 min). Protease ativity. Protease ativity was deteted by using a single-diffusion tehnique in agar gel ontaining skim milk as a substrate (8, 11). For more quantitative analysis, a test tube method using azoasein (azoasein assay; Sigma Chem- * Corresponding author ial Co., St. Louis, Mo.) as a substrate was performed by the proedure previously desribed (1). The optial density at 42 nm was determined with a Titertek Multiskan apparatus (Flow Laboratories, In., MLean, Va.). One protease unit (PU) was defined as the smallest dose that digests 5% of.5 mg of the substrate in 1 h of inubation. Purifiation of protease. The ell-free ulture supernatants were frationated with ammonium sulfate, and the 4 to 55% ammonium sulfate-insoluble material was suspended in.2 M Tris buffer (ph 8.) and dialyzed to the same buffer. Insoluble material was removed by entrifugation at 13, x g for 2 min. The dialyzed materials were applied to a DEAE-Sephadex A25 olumn (2.2 by 3 m) equilibrated with.2 M Tris buffer (ph 8.). The olumn was washed with 5 ml of the appliation buffer, and bound material was then eluted with 1, ml of a linear gradient of to 1. M NaCl in the same buffer. Frations with protease ativity were onentrated to about 5 ml on a membrane (PM-1; Amion Corp., Lexington, Mass.) and then applied further to a Sephadex G1 olumn (2.2 by 1 m) equilibrated with.2 M Tris buffer. The frations ontaining protease ativity were then applied to a Mono Q olumn in a fast-protein liquid hromatographi system. A linear gradient of to.3 M NaCl in.2 M aeti aid buffer (ph 6.) was used for elution. The frations ontaining protease ativity were finally applied to a Phenyl Superose (Pharmaia Fine Chemials, Uppsala, Sweden) fast-protein liquid hromatography olumn equilibrated with.5 M phosphate buffer (ph 7.) ontaining 3 M NaCl. Material was eluted with 2 ml of a linear gradient of 3. to M NaCl in the phosphate buffer, and then the olumn was further washed with distilled water without NaCl. Protein determination. Protein amounts were determined by a protein assay kit (Bio-Rad Laboratories, Rihmond, Calif.). Bovine serum albumin was used as the standard. Hemagglutination assay. Mirotiter quantitation of ativity on hiken erythroytes suspended in Krebs-Ringer buffer was performed as previously desribed (11). The titer was defined as the reiproal of the highest dilution whih aused hemagglutination. To test for inhibitory effets, various amounts of the following ompounds were added to 3 PU of purified protease in azoasein assay and hemagglutination assay systems: Downloaded from on August 24, 218 by guest

2 28 HONDA ET AL. E I A E C m 1. 4'.5 o 2 rj - Fration Number I-.-j I/ I t, C.3 I.@ E Z 6 2 E s o - ozd.25 z Fration Number ) II- %" lb Retention time (min) Retention time (min) FIG. 1. Typial elution profiles of V. holerae non-1 protease from DEAE-Sephadex A25 (A), Sephadex G1 (B), Mono Q (C), and Phenyl-Superose (D) olumns, respetively. Eah bars represent the fration for the next step. The broken lines indiate gradient elution profiles. B D r X INFECT. IMMUN. Downloaded from on August 24, 218 by guest EDTA, o-phenanthroline, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor (type 11-S), and %x2-maroglobulin (all from Sigma). After inubation of protease with these ompounds for 2 min at 37 C, residual ativity with azoasein was measured in a Titertek Multiskan apparatus. Purified hemagglutinin-protease of V. holerae 1 and its antiserum. Purified hemagglutinin-protease of V. holerae 1 and its antiserum were prepared as desribed previously (9) Ȧntisera. Antiserum against the purified protease of V. holerae non-1 was raised in rabbits by a proedure previously desribed (12) using 2,ug of purified protease as the antigen for eah immunization. PAGE. Sodium dodeyl sulfate-polyarylamide slab gel eletrophoresis (PAGE) and onventional PAGE were performed by the methods of Laemmli (14) and Davis (7), respetively. RESULTS Purifiation of protease. The protease was purified from a large-sale ulture (about 5.8 liters) by sequential steps of preipitation with ammonium sulfate (4 to 55%), DEAE- Sephadex A25 ion-exhange olumn hromatography, Sephadex G1 gel filtration olumn hromatography, Mono Q fast-protein liquid hromatography, and Phenyl Superose

3 VOL. 57, 1989 V. CHOLERAE 1 AND NON-O1 PROTEASES 281 TABLE 1. Purifiation of V. holerae non-o1 protease Total protein Total ativity Sp at Relative. b Step Vol (ml) (mg) (PU) (PU/mg of protein) ativity" Yield (%) Culture supernatant 5, x x Ammonium sulfate (4-55%) x X DEAE-Sephadex A X x Sephadex G x x Mono Q x x Phenyl-Superose x x "The speifi ativity of the ulture supernatant was taken as the standard. b The total protease ativity reovered from eah step was ompared with that of the ulture supernatant. hydrophobi fast-protein liquid hromatography. Most of the protease was eluted from the DEAE-Sephadex A25 olumn (Fig. 1A) with about.1 M NaCl. The first peak from a Sephadex G1 olumn (Fig. 1B) was found to ontain protease ativity. The protease was then further purified by Mono Q (Fig. 1C) and Phenyl-Superose (Fig. 1D) olumns, from whih the protease was eluted with about.1 M NaCl and M NaCl, respetively. Typial results for purifiation of the protease are summarized in Table 1. By this proedure, the final reovery was about 1.6% and the relative ativity was inreased about 25 times. PAGE of purified protease. Conventional PAGE showed that purified protease of V. holerae non-1 migrated as a single band with protease ativity. The major protease ativity of V. holerae 1 was seen in the same position as the protease ativity of V. holerae non-o1 (Fig. 2). The purified protease of V. holerae non-1 gave one protein band on sodium dodeyl sulfate-page (Fig. 3) in almost the same position as that of V. holerae 1, although the latter gave another weaker protein band. The moleular weight of the protease (subunit) of V. holerae non-1 was estimated to be about 32, on the basis of its mobility on sodium dodeyl sulfate-page. Biologial ativity of purified protease. The purified protease was shown to possess hemagglutination ativity; the minimum amounts of protease neessary for protease and hemagglutinating ativities were 1.5 and 4.5 p.giml, respetively. The optimal ph for expressing protease ativity was about 8 to 9 (Fig. 4). Physiohemial harateristis. The heat stability of the protease and hemagglutinating ativities of V. holerae non-1 protease were studied. With the purified protease, both ativities were ompletely inativated at 75 C or higher temperatures for 15 min. The reativation phenomenon, whih was previously observed with rude preparations (11), was not demonstrated with the pure preparation (Table 2). The protease ativity of purified V. holerae non-1 protease, like the protease of V. holerae 1(3), was inhibited by EDTA (final onentration, 1 mm), zinov (16 jim), o- phenanthroline (1 mm), and 2 maroglobulin (16 mg/ml) but not by phenylmethyl sulfonyl fluoride (1 mm) or trypsin inhibitor (4,ug/ml). Immunologial analysis. Polylonal antisera to the purified proteases of V. holerae 1 and non-1 formed fused preipitin lines with both of the purified proteases. In various onfigurations of double immunopreipitation assays (Fig. 5), no spurs indiative of nonidentity were observed. Crude non-1 protease behaved identially to purified non-1 protease in these assays. The protease ativities of V. holerae 1 and non-1 were ompletely inhibited by the homologous and heterologous antisera. Neutralization of hemagglutinating ativities of the purified proteases by the antisera was not obvious beause of nonspeifi inhibition of normal preimmune serum. Inorporation of mannose or fuose did not inhibit the protease or hemagglutinating ativities of the purified proteases. DISCUSSION Purifiation of a protease produed by V. holerae non-1 to apparent homogeneity was ahieved in this study. The purified protease had bifuntional ativities of both protease and hemagglutination. Both ativities were labile on heat treatment for 15 min at 75 C or higher temperatures. Peuliar I 2..C..;.s 3 Downloaded from on August 24, 218 by guest FIG. 2. Eletrophoreti analyses of proteases by onventional PAGE. After eletrophoresis, the gel was overlaid on an agar plate ontaining skim milk to observe the protease ativity (1). The gel was removed from the agar plate before the piture was taken. Lanes 1 and 2 ontained the proteases (1 p.g/ml) of V. holerae 1 and non-o1, respetively. FIG. 3. Sodium dodeyl sulfate-page of proteases of V. holerae 1 and non-o1. Lanes: 1, marker proteins (moleular weights, 2,1, 3,, 43,, and 67,); 2 and 3, purified proteases from V. holerae 1 and non-o1, respetively.

4 282 HONDA ET AL. INFECT. IMMUN. E.2 :2 3 4 C A ph FIG. 4. Effet of ph on the protease ativity of V. holerae non-o1. The buffers HCl-glyine (.1 M), aeti aid-sodium aetate (.1 M), sodium-potassium phosphate (.1 M), and Tris hydrohloride (.2 M) were used for phs of 3. to 4., 5. to 6., 7., and 8. to 9., respetively. phenomena, suh as the Arrhenius effet seen previously (1.1) with rude preparations of V. holerae non-1 but not with those of V. holerae 1, were not seen with the purified protease preparation of V. holerae non-1. This suggests that the unusual results, different from those obtained with V. holerae 1, obtained with the rude preparation of V. holerae non-o1 were probably due to some fator(s) present in the rude preparation of V. holerae non-1. The present data, obtained by physiohemial and immunologial analyses, suggest that the V. holerae non-1 and 1 proteases are idential. Although we did not examine the possible role(s) of the V. holerae non-o1 protease in baterial attahment to gut epithelium in this study, by analogy to the V. holerae 1 protease (4, 8, 9) we ould postulate that the V. holerae non-1 protease serves as an attahment fator(s) whih enhanes toxi ativity by niking holera toxin (4) or degrading intestinal muin (6). Beause both V. holerae 1 and non-1 produe idential or similar proteases (or hemagglutinins), prodution of the proteases, however, annot fully explain why V. holerae 1 auses epidemis and TABLE 2. Effet of heat treatment on hemagglutinating and protease ativities of rude and purified V. holerae non-o1 protease Hemagglutinating Heating temp(s) ativityb Protease ativity' (oc)a Crude Pure Crude Pure Unheated , , a Applied for 15 min eah time. b Reiproal of the highest dilution that aused hemagglutination. A42 in the azoasein assay. FIG. 5. Ouhterlony double gel immunodiffusion test. Wells: 1 and 5, antisera to V. holerae non-o1 and 1 proteases, respetively; 2 and 3, rude and purified protease preparations of V. holerae non-o1; 4 purified protease of V. holerae 1. pandemis, whereas V. holerae non-1 does not (2, 13, 15). This is also indiated by a previous analysis of a large number of V. holerae strains for the presene of the hemagglutinin-protease (5). The preise role of the protease produed by V. holerae non-o1 in the pathogenesis of V. holerae non-o1 gastroenteritis requires further study. ACKNOWLEDGMENTS This work was supported by a grant-in-aid for sientifi researh from the Ministry of Eduation, Siene, and Culture of Japan. R.A.F. was supported in part by U.S. Publi Health Servie grant AI from the National Institute of Allergy and Infetious Diseases. LITERATURE CITED 1. Arita, M., T. Takeda, T. Honda, and T. Miwatani Purifiation and haraterization of Vibrio holerae non-o1 heat-stable enterotoxin. lnfet. Immun. 52: Blake, P. A., R. E. Weaver, and D. G. Hollis Diseases of humans (other than holera) aused by vibrios. Annu. Rev. Mirobiol. 34: Booth, B. A., M. Boesman-Finkelstein, and R. A. Finkelstein Vibrio holerae soluble hemagglutinin/protease is a metalloenzyme. Infet. Immun. 42: Booth, B. A., M. Boesman-Finkelstein, and R. A. Finkelstein Vibrio holerae hemagglutinin/protease niks holera enterotoxin. Infet. lmmun. 45: Booth, B. A., and R. A. Finkelstein Presene of hemagglutinin/protease and other potential virulene fators in 1 and non-o1 Vibrio holerae. J. Infet. Dis. 154: Crowther, R. S., N. W. Roomi, R. E. F. Fahim, and J. F. Forstner Vibrio holerae metalloproteinase degrades intestinal muin and failitates enterotoxin-indued seretion from rat intestine. Biohim. Biophys. Ata 924: Davis, B. J Dis eletrophoresis. II. Method and appliation to human serum proteins. Ann. N.Y. Aad. Si. 121: Finkelstein, R. A., M. Boesman-Finkelstein, and P. Holt Vibrio holerae hemagglutinin/letin/protease hydrolyzes fibronetin and ovomuin: F. M. Burnet revisited. Pro. Natl. Aad. Si. USA 8: Finkelstein, R. A., and L. F. Hanne Purifiation and haraterization of the soluble hemagglutinin (holera letin) produed by Vibrio holerae. Infet. Immun. 36: Hingley, S. T., A. T. Hastie, F. Kueppers, and M. Higgins Disruption of respiratory ilia by proteases inluding those of Pseudomonas aeruginosa. Infet. Immun. 54: Honda, T., B. A. Booth, M. Boesman-Finkelstein, and R. A. Finkelstein Comparative study of Vibrio holerae non-o1 protease and soluble hemagglutinin with those of Vibrio holerae 1. Infet. Immun. 55: Downloaded from on August 24, 218 by guest

5 VOL. 57, 1989 V. CHOLERAE 1 AND NON-O1 PROTEASES Honda, T., Y. Ni, and T. Miwatani Purifiation and haraterization of a hemolysin produed by a linial isolate of Kanagawa phenomenon-negative Vibrio parahaemolytius and related to the thermostable diret hemolysin. Infet. Immun. 56: Janda, J. M., C. Powers, R. G. Bryant, and S. L. Abbott Current perspetives on epidemiology and pathogenesis of linially signifiant Vibrio spp. Clin. Mirobiol. Rev. 1: Laemmli, U. K Cleavage of strutural proteins during the assembly of the head of bateriophage T4. Nature (London) 227: Morris, J. G., Jr., and R. E. Blak Cholera and other vibrioses in the United States. N. Engl. J. Med. 312: Yamamoto, K., M. Al-omani, T. Honda, Y. Takeda, and T. Miwatani Non-O1 Vibrio holerae hemolysin: purifiation, partial haraterization, and immunologial relatedness to El Tor hemolysin. Infet. Immun. 45: Yamamoto, K., Y. Takeda, T. Miwatani, and J. P. Craig Purifiation and some properties of a non-o1 Vibrio holerae enterotoxin that is idential to holera enterotoxin. Infet. Immun. 39: Yamamoto, K., Y. Takeda, T. Miwatani, and J. P. Craig Evidene that a non-o1 Vibrio holerae produes enterotoxin that is similar but not idential to holera enterotoxin. Infet. Immun. 41: Yoh, M., T. Honda, and T. Miwatani Purifiation and partial haraterization of a non-o1 Vibrio holerae hemolysin that ross-reats with thermostable diret hemolysin of Vibrio parahaemolytius. Infet. Immun. 52: Downloaded from on August 24, 218 by guest