FEMS Microbiology Letters 98 (1992) 197-200 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
197
FEMSLE 05136
Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1 Atsuko Naka, Koichiro Yamamoto, Toshio Miwatani and Takeshi H o n d a Research b~stitute for Microbial Diseases, Osaka Unil ersity, Osaka, Japan
Received 3 June 1992 Revision received 10 August 1992 Accepted 19 August 1992
Key words: Vibrio cholerae non-O1; Protease; Hemagglutinin; Vibrio cholerae O1
1. SUMMARY
2. INTRODUCTION
Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae nonO1 were characterized. The hemagglutinin/ protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37°C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the Cterminal region of the 34-kDa hemagglutinin/ protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutihating activity decreased, suggesting that the Cterminal region of the hemagglutinin/protease is related to hemagglutinating activity.
Some isolates (or strains) of Vibrio cholerae non-O1 are important causative agents of human diarrhoeal disease as well as of extraintestinal infections [1-3]. V. cholerae non-O1, a bacterium closely related to V. cholerae O1, produces various virulence factors such as cholera toxin (CT), CT-like toxin [4,5], thermostable direct haemolysin (TDH)-like toxin (NAG-rTDH) [6] heat stable (ST)-like toxin (NAG-ST) [7] and hemagglutinin/ protease (NAG-HA/P) [8,9]. The NAG-HA/P is indistinguishable from the hemagglutinin/protease (Vc-HA/P) of V. cholerae O1 [9], which is believed to play an important role in the pathogenesis of cholera by adhering to, and also detaching from, the intestinal epithelium [10,11], activating CT by nicking the CT-A subunit [12], and cleaving physiologically important substances including secretory IgA [13]. By analogy to VcH A / P , NAG-HA/P may also play an important role in the pathogenesis of V. cholerae non-O1 [8,9,12]. We found two forms of NAG-HA/P after purification by means of an improved rapid pro-
Correspondence to: A. Naka, Research Institute for Microbial Diseases, Osaka University, Yamada-oka, Suita, Osaka 565, Japan.
198
cedure using a monoclonal antibody [14]. The present paper describes the characterization of the two forms of N A G - H A / P .
was more accurately quantified using the azocasein assay as described previously [9].
3.4. Hemagglutination assay Hemagglutination was quantified as previously described [9] using 1.5% chicken erythrocytcs suspended in Krebs-Ringer buffer. The titer was defined as the reciprocal of the highest dilution which caused hemagglutination as described previously [9].
3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains and culture conditions The strain used was V. cholerae non-O1 strain TH81 isolated from a patient with traveller's diarrhea at Osaka Airport Quarantine Station. The strain was cultured in tryptic soy broth in a flask at 30°C for 20 h as described previously [9].
3.5. Electrophoresis Sodium dodecyl sulfate (SDS) slab polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli [15]. A molecular-mass marker kit was used (Daiichi Pure Chemical, Tokyo, Japan).
3, 2. Purification of NA G-HA / P Culture supernatant was fractionated with ammonium sulfate (40-55%) and N A G - H A / P was further purified by successive column chromatographies (Method I) [9] and by immunoaffinity chromatography using a monoclonal antibody as described previously [14].
3.6. lmmunoblotting (Western blot) After transfer to nitrocellulose sheets, samples were allowed to react overnight at 4°C react with 5% (v/v) rabbit polyclonal antiserum against the purified N A G - H A / P in a buffer (phosphate-buffered saline:PBS) containing 0.05% Tween 20, followed by reaction with anti-rabbit immunoglobulin G conjugated with peroxidase (Organon
3.3. Assay of protease actiuity Protease activity was qualitatively detected by single radial diffusion in a 1.5% agar gel containing 1.5% skim milk as the substrate. The activity
A
B
97.4 ' ~ 66.3 42.4 -'~ 30.0 20.1 •. ~
1
2
3
4
5
14.4
1
2
3
4
Fig. 1. Time course of degradation of the 34-kDa N A G - H A / P incubated at 37°C, analyzed by SDS-PAGE (A) and Western blotting using a polyclonal antibody (B). Lanes 1, 2, 3 and 4 were incubated for 0, 30, 60 min and 24 h, respectively. Lane 5, molecular mass markers (97.7, 66.3, 42.4, 30.0, 20.1 and 14.4-kDa, respectively (A).
199 Teknika, West Chester). The enzyme activity was detected by exposure to enzyme substrate as described previously [16].
3. Z N-terminal amino acid sequencing The N-terminal amino acid sequences of peptides blotted from SDS-polyacrylamide gels to P V D F (polyvinylidene difluoride) membranes (ProBlott TM Applied Biosystems) were determined using an Applied Biosystems 473 A gasliquid-phase sequencer with detection using P T H (phenylthiohydantoin) derivatives. The P T H derivatives were sequenced and analyzed by standard procedures. Twenty cycles were done, and the amino acid residues were identified by comparison to a j3-1actoglobulin standard (Applied Biosystems).
4. R E S U L T S A N D D I S C U S S I O N When the purified 34-kDa N A G - H A / P protein was incubated at 37°C, a shift to a 32-kDa form occurred (Fig. 1A), suggesting that the 32kDa peptide is an autodigestive version of 34-kDa N A G - H A / P . Further degradation of the 32-kDa N A G - H A / P was not evident within 24 h incubation. This 32-kDa protein was the major product we previously obtained using a complex purification procedure (Method I) [9]. Immunological cross-reactivity of these two N A G - H A / P was demonstrated by Western blotting (Fig. 1B) and a double-gel diffusion test, suggesting antigenic identity between the two forms (data not shown). Relatedness of the 34- and 32-kDa forms of N A G - H A / P was also confirmed by determining their N-terminal amino acid sequences. The first 20 amino acids in the sequences of the two forms of N A G - H A / P were identical ( A Q A T G T G P GGNQKTGRYEYG), suggesting that the shift from 34- to 32-kDa occurred by autodigestion of C-terminal amino acids of the larger form. The above amino acid sequences of N A G - H A / P were identical with those reported for V c - H A / P [17], confirming our previous finding that N A G H A / P and V c - H A / P are physicochemically and immunologically indistinguishable. It was reported [17] that the V c - H A / P consisted of 414
Table 1 Comparison of the activityof NAG-HA/P after shift from 34to 32-kDa form Incubation period e (h) 0(34kDa) l(34+32kDa) Protease activity ~' 0.56 [1] Hemagglutinatingactivity t, 2 5
24 (32kDa)
0.85 [ 1.5]
1.7 [2.5]
2 ~
2 i
~' Protease activity was measured using an azocasein assay (PU/~zg). [ ]: Relative activity to untreated control (time 0 h). b Reciprocal titer of highest dilution that causes hemagglutination of chicken erythrocytes. " 34-kDa NAG-HA/P was incubated at 37°C for the indicated periods.
amino acids of 46.7 kDa and is subsequently processed to the 32-kDa product, the form in which it is usually purified. In the present study, the 45-47-kDa form was not found, perhaps because the monoclonal antibody used here for immunoaffinity column chromatography does not recognize the 45-47-kDa form because it was selected using a partially processed form rather than the native 45-47-kDa form. We further characterized the difference between the 34- and 32-kDa forms regarding their biological activities. As summarized in Table 1, the proteolytic activity of the 32-kDa protein was 2.5-times more active than that of the larger form. On the other hand, the hemagglutination titer of the 32-kDa form was less than that of the 34-kDa form. These results suggest that the Cterminal amino acid(s) have important roles in hemagglutination and also for reducing proteolytic activity. The benefits and role(s) of this shift require further study.
ACKNOWLEDGEMENTS This study was supported by a Grant-in-Aid for Scientific Reseach from the Ministry of Education, Science and Culture of Japan.
200 REFERENCES [l] Blake, P.A., Weaver, R.E. and Hollis, D.G. (1980) Annu. Rev. Microbiol. 34, 341-367. [2] Hughes, R.E., Hollis, D.G., Gangarosa, E.J. and Weaver, R.E. (1978) Ann. Intern. Med. 88, 602-606. [3] Morris, J.G., Jr. and Black, R.E. (1985) N. Engl. J. Med. 312, 343-350. [4] Yamamoto, K., Takeda, Y., Miwatani, T. and Craig, J.P. (1983) Infect. lmmun. 39, 1128-1135. [5] Yamamoto, K., Takeda, Y., Miwatani, T. and Craig, J.P. (1983) Infect. Immun. 41,896-901. [6] Yoh, M., Honda, T. and Miwatani, T. (1986) Infect. Immun. 52, 319-322. [7] Arita, M., Takeda, T., Honda, T. and Miwatani, T. (1986) Infect. Immun. 52, 45-49. [8] Honda, T., Booth, B.A., Boesman-Finkelstein, M. and Finkelstein, R.A. (1987) Infect. Immun. 55,451-454.
[9] Honda, T., Lertpocasombat, K., Hata, A., Miwatani, T. and Finkelstein, R.A. (1989) Infect. Immun. 57, 27992803. [10] Finkelstein, R.A. and Hanne, L.F. (1982) Infect. Immun. 36, 1199-1208. [11] Finkelstein, R.A., Boesman-Finkelstein, M., Chang, Y. and Hase, C.C. (1992) Infect. Immun. 60, 472-478. [12] Booth, B.A., Boesmann- Finkelstein, M. and Finkelstein, R.A. (1984) Infect. Immun. 45, 558-560. [13] Finkelstein, R.A., Boesman-Finkelstein, M. and Holt, P. (1983) Proc. Natl. Acad. Sci. USA 80, 1092-1095. [14] Naka, A., Honda, T. and Miwatani, T. (1992) Microbiol. Immunol. 36, 419-423. [15] Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. [16] Honda, T., Ni, Y., Yoh, M. and Miwatani, T. (1989) Med. Microbiol. Immunol. 178, 245-253. [17] Hase, C.C. and Finkelstein, R.A. (1991) J. Bacteriol. 173, 3311-3317.
197
FEMSLE 05136
Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1 Atsuko Naka, Koichiro Yamamoto, Toshio Miwatani and Takeshi H o n d a Research b~stitute for Microbial Diseases, Osaka Unil ersity, Osaka, Japan
Received 3 June 1992 Revision received 10 August 1992 Accepted 19 August 1992
Key words: Vibrio cholerae non-O1; Protease; Hemagglutinin; Vibrio cholerae O1
1. SUMMARY
2. INTRODUCTION
Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae nonO1 were characterized. The hemagglutinin/ protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37°C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the Cterminal region of the 34-kDa hemagglutinin/ protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutihating activity decreased, suggesting that the Cterminal region of the hemagglutinin/protease is related to hemagglutinating activity.
Some isolates (or strains) of Vibrio cholerae non-O1 are important causative agents of human diarrhoeal disease as well as of extraintestinal infections [1-3]. V. cholerae non-O1, a bacterium closely related to V. cholerae O1, produces various virulence factors such as cholera toxin (CT), CT-like toxin [4,5], thermostable direct haemolysin (TDH)-like toxin (NAG-rTDH) [6] heat stable (ST)-like toxin (NAG-ST) [7] and hemagglutinin/ protease (NAG-HA/P) [8,9]. The NAG-HA/P is indistinguishable from the hemagglutinin/protease (Vc-HA/P) of V. cholerae O1 [9], which is believed to play an important role in the pathogenesis of cholera by adhering to, and also detaching from, the intestinal epithelium [10,11], activating CT by nicking the CT-A subunit [12], and cleaving physiologically important substances including secretory IgA [13]. By analogy to VcH A / P , NAG-HA/P may also play an important role in the pathogenesis of V. cholerae non-O1 [8,9,12]. We found two forms of NAG-HA/P after purification by means of an improved rapid pro-
Correspondence to: A. Naka, Research Institute for Microbial Diseases, Osaka University, Yamada-oka, Suita, Osaka 565, Japan.
198
cedure using a monoclonal antibody [14]. The present paper describes the characterization of the two forms of N A G - H A / P .
was more accurately quantified using the azocasein assay as described previously [9].
3.4. Hemagglutination assay Hemagglutination was quantified as previously described [9] using 1.5% chicken erythrocytcs suspended in Krebs-Ringer buffer. The titer was defined as the reciprocal of the highest dilution which caused hemagglutination as described previously [9].
3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains and culture conditions The strain used was V. cholerae non-O1 strain TH81 isolated from a patient with traveller's diarrhea at Osaka Airport Quarantine Station. The strain was cultured in tryptic soy broth in a flask at 30°C for 20 h as described previously [9].
3.5. Electrophoresis Sodium dodecyl sulfate (SDS) slab polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli [15]. A molecular-mass marker kit was used (Daiichi Pure Chemical, Tokyo, Japan).
3, 2. Purification of NA G-HA / P Culture supernatant was fractionated with ammonium sulfate (40-55%) and N A G - H A / P was further purified by successive column chromatographies (Method I) [9] and by immunoaffinity chromatography using a monoclonal antibody as described previously [14].
3.6. lmmunoblotting (Western blot) After transfer to nitrocellulose sheets, samples were allowed to react overnight at 4°C react with 5% (v/v) rabbit polyclonal antiserum against the purified N A G - H A / P in a buffer (phosphate-buffered saline:PBS) containing 0.05% Tween 20, followed by reaction with anti-rabbit immunoglobulin G conjugated with peroxidase (Organon
3.3. Assay of protease actiuity Protease activity was qualitatively detected by single radial diffusion in a 1.5% agar gel containing 1.5% skim milk as the substrate. The activity
A
B
97.4 ' ~ 66.3 42.4 -'~ 30.0 20.1 •. ~
1
2
3
4
5
14.4
1
2
3
4
Fig. 1. Time course of degradation of the 34-kDa N A G - H A / P incubated at 37°C, analyzed by SDS-PAGE (A) and Western blotting using a polyclonal antibody (B). Lanes 1, 2, 3 and 4 were incubated for 0, 30, 60 min and 24 h, respectively. Lane 5, molecular mass markers (97.7, 66.3, 42.4, 30.0, 20.1 and 14.4-kDa, respectively (A).
199 Teknika, West Chester). The enzyme activity was detected by exposure to enzyme substrate as described previously [16].
3. Z N-terminal amino acid sequencing The N-terminal amino acid sequences of peptides blotted from SDS-polyacrylamide gels to P V D F (polyvinylidene difluoride) membranes (ProBlott TM Applied Biosystems) were determined using an Applied Biosystems 473 A gasliquid-phase sequencer with detection using P T H (phenylthiohydantoin) derivatives. The P T H derivatives were sequenced and analyzed by standard procedures. Twenty cycles were done, and the amino acid residues were identified by comparison to a j3-1actoglobulin standard (Applied Biosystems).
4. R E S U L T S A N D D I S C U S S I O N When the purified 34-kDa N A G - H A / P protein was incubated at 37°C, a shift to a 32-kDa form occurred (Fig. 1A), suggesting that the 32kDa peptide is an autodigestive version of 34-kDa N A G - H A / P . Further degradation of the 32-kDa N A G - H A / P was not evident within 24 h incubation. This 32-kDa protein was the major product we previously obtained using a complex purification procedure (Method I) [9]. Immunological cross-reactivity of these two N A G - H A / P was demonstrated by Western blotting (Fig. 1B) and a double-gel diffusion test, suggesting antigenic identity between the two forms (data not shown). Relatedness of the 34- and 32-kDa forms of N A G - H A / P was also confirmed by determining their N-terminal amino acid sequences. The first 20 amino acids in the sequences of the two forms of N A G - H A / P were identical ( A Q A T G T G P GGNQKTGRYEYG), suggesting that the shift from 34- to 32-kDa occurred by autodigestion of C-terminal amino acids of the larger form. The above amino acid sequences of N A G - H A / P were identical with those reported for V c - H A / P [17], confirming our previous finding that N A G H A / P and V c - H A / P are physicochemically and immunologically indistinguishable. It was reported [17] that the V c - H A / P consisted of 414
Table 1 Comparison of the activityof NAG-HA/P after shift from 34to 32-kDa form Incubation period e (h) 0(34kDa) l(34+32kDa) Protease activity ~' 0.56 [1] Hemagglutinatingactivity t, 2 5
24 (32kDa)
0.85 [ 1.5]
1.7 [2.5]
2 ~
2 i
~' Protease activity was measured using an azocasein assay (PU/~zg). [ ]: Relative activity to untreated control (time 0 h). b Reciprocal titer of highest dilution that causes hemagglutination of chicken erythrocytes. " 34-kDa NAG-HA/P was incubated at 37°C for the indicated periods.
amino acids of 46.7 kDa and is subsequently processed to the 32-kDa product, the form in which it is usually purified. In the present study, the 45-47-kDa form was not found, perhaps because the monoclonal antibody used here for immunoaffinity column chromatography does not recognize the 45-47-kDa form because it was selected using a partially processed form rather than the native 45-47-kDa form. We further characterized the difference between the 34- and 32-kDa forms regarding their biological activities. As summarized in Table 1, the proteolytic activity of the 32-kDa protein was 2.5-times more active than that of the larger form. On the other hand, the hemagglutination titer of the 32-kDa form was less than that of the 34-kDa form. These results suggest that the Cterminal amino acid(s) have important roles in hemagglutination and also for reducing proteolytic activity. The benefits and role(s) of this shift require further study.
ACKNOWLEDGEMENTS This study was supported by a Grant-in-Aid for Scientific Reseach from the Ministry of Education, Science and Culture of Japan.
200 REFERENCES [l] Blake, P.A., Weaver, R.E. and Hollis, D.G. (1980) Annu. Rev. Microbiol. 34, 341-367. [2] Hughes, R.E., Hollis, D.G., Gangarosa, E.J. and Weaver, R.E. (1978) Ann. Intern. Med. 88, 602-606. [3] Morris, J.G., Jr. and Black, R.E. (1985) N. Engl. J. Med. 312, 343-350. [4] Yamamoto, K., Takeda, Y., Miwatani, T. and Craig, J.P. (1983) Infect. lmmun. 39, 1128-1135. [5] Yamamoto, K., Takeda, Y., Miwatani, T. and Craig, J.P. (1983) Infect. Immun. 41,896-901. [6] Yoh, M., Honda, T. and Miwatani, T. (1986) Infect. Immun. 52, 319-322. [7] Arita, M., Takeda, T., Honda, T. and Miwatani, T. (1986) Infect. Immun. 52, 45-49. [8] Honda, T., Booth, B.A., Boesman-Finkelstein, M. and Finkelstein, R.A. (1987) Infect. Immun. 55,451-454.
[9] Honda, T., Lertpocasombat, K., Hata, A., Miwatani, T. and Finkelstein, R.A. (1989) Infect. Immun. 57, 27992803. [10] Finkelstein, R.A. and Hanne, L.F. (1982) Infect. Immun. 36, 1199-1208. [11] Finkelstein, R.A., Boesman-Finkelstein, M., Chang, Y. and Hase, C.C. (1992) Infect. Immun. 60, 472-478. [12] Booth, B.A., Boesmann- Finkelstein, M. and Finkelstein, R.A. (1984) Infect. Immun. 45, 558-560. [13] Finkelstein, R.A., Boesman-Finkelstein, M. and Holt, P. (1983) Proc. Natl. Acad. Sci. USA 80, 1092-1095. [14] Naka, A., Honda, T. and Miwatani, T. (1992) Microbiol. Immunol. 36, 419-423. [15] Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. [16] Honda, T., Ni, Y., Yoh, M. and Miwatani, T. (1989) Med. Microbiol. Immunol. 178, 245-253. [17] Hase, C.C. and Finkelstein, R.A. (1991) J. Bacteriol. 173, 3311-3317.
