Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12–15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action. A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12–15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action. The CAMP reaction consists of a distinct zone of hemolysis on blood agar plates produced by Streptococcus agalactiae when grown near the colonies of Staphylococcus aureus (1Christie R. Atkins N.E. Munch-Peterson E. Aust. J. Exp. Biol. Med. Sci. 1944; 22: 197-200Crossref Google Scholar). It has been used in diagnostic microbiology to identify the S. agalactiae strains ever since its discovery in 1944 by Christie et al. (1Christie R. Atkins N.E. Munch-Peterson E. Aust. J. Exp. Biol. Med. Sci. 1944; 22: 197-200Crossref Google Scholar). The proteins responsible for the CAMP reaction are sphingomyelinase from S. aureus and CAMP factor, a protein secreted by S. agalactiae that has a molecular weight of 25 kDa and a pI of 8.9 (2Jurgens D. Shalaby F.Y. Fehrenbach F.J. J. Chromatogr. 1985; 348: 363-370Crossref PubMed Scopus (15) Google Scholar). Sphingomyelinase initially hydrolyzes sphingomyelin to ceramide, which renders the erythrocytes susceptible to the lytic activity of CAMP factor. Erythrocytes from different mammalian species support the CAMP reaction to different extents depending on the sphingomyelin content in their cell membranes (3Sterzik B. Fehrenbach F.J. J. Gen. Microbiol. 1985; 131: 817-820PubMed Google Scholar). Sheep red blood cells are the most susceptible, in keeping with their sphingomyelin content as high as 51% (by moles) (4Ways P. Hanahan D.J. J. Lipid Res. 1964; 5: 318-328Abstract Full Text PDF PubMed Google Scholar). Human red blood cells and rabbit red blood cells, with 26 and 19% mol of sphingomyelin, respectively (4Ways P. Hanahan D.J. J. Lipid Res. 1964; 5: 318-328Abstract Full Text PDF PubMed Google Scholar, 5Nelson G.J. Biochim. Biophys. Acta. 1967; 144: 221-232Crossref PubMed Scopus (229) Google Scholar), were reportedly not sensitive to CAMP factor after sphingomyelinase treatment (3Sterzik B. Fehrenbach F.J. J. Gen. Microbiol. 1985; 131: 817-820PubMed Google Scholar). The latter, however, were rendered susceptible to the CAMP factor after phospholipase C treatment, which converted the glycerophospholipids to diacylglycerol (6Fehrenbach F.J. Schmidt C.M. Sterizik B. Jürgens D. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 317-324Google Scholar), indicating that ceramide is not specifically required for CAMP activity. Previous work with liposomes also suggested that the fraction of cholesterol in the membrane influences the activity of CAMP factor (6Fehrenbach F.J. Schmidt C.M. Sterizik B. Jürgens D. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 317-324Google Scholar, 7Sterzik B. Schmidt C.M. Fehrenbach F.J. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 195-196Google Scholar). CAMP factor has long been known as a pathogenic determinant that exerted lethal effects when administered to rabbits and mice (8Skalka B. Smola J. Zentralbl. Bakteriol. A. 1981; 249: 190-194PubMed Google Scholar). Apart from its membrane damaging activity, CAMP factor was found to bind to the Fc fragments of immunoglobulins, and it was therefore designated as protein B in analogy to protein A of S. aureus (9Jurgens D. Sterzik B. Fehrenbach F.J. J. Exp. Med. 1987; 165: 720-732Crossref PubMed Scopus (61) Google Scholar). The CAMP factor genes of S. agalactiae (10Schneewind O. Friedrich K. Lutticken R. Infect. Immun. 1988; 56: 2174-2179Crossref PubMed Google Scholar), Streptococcus uberis (11Jiang M. Babiuk L.A. Potter A.A. Microb. Pathog. 1996; 20: 297-307Crossref PubMed Scopus (39) Google Scholar), and Streptococcus pyogenes (12Gase K. Ferretti J.J. Primeaux C. McShan W.M. Infect. Immun. 1999; 67: 4725-4731Crossref PubMed Google Scholar) have been cloned in Escherichia coli, and their sequences were found to be highly homologous with each other (12Gase K. Ferretti J.J. Primeaux C. McShan W.M. Infect. Immun. 1999; 67: 4725-4731Crossref PubMed Google Scholar). Co-hemolytic phenomena have also been reported with various other bacterial genera, but the proteins responsible for those, although sometimes referred to by the name “CAMP factor” as well, are not closely related to streptococcal CAMP factor. In this study, we expressed the CAMP factor as a glutathione S-transferase fusion protein in E. coli. The fusion protein was purified to homogeneity and cleaved by thrombin to yield the recombinant CAMP factor, which exhibited hemolytic activity similar to the native protein. We show that CAMP factor forms pores of finite yet heterogeneous size in the membranes of susceptible cells. Additional studies with liposomes show that the process of CAMP factor pore formation is highly cooperative, supporting an oligomeric nature of the membrane lesion. Cloning, Plasmids, and Bacterial Strains—pGEX-KG (13Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1638) Google Scholar) was a generous gift from Jingya Li (National Center for Drug Screening, Shanghai, China). The coding sequence of CAMP factor mature peptide was amplified from S. agalactiae genomic DNA. The following primers were used: forward, 5′-TTT GCC GAT CAA GTG ACA ACT CCA C-3′; reverse, 5′-CAA TCA TGG TAG TAC AAA ATC ACG A-3′. The amplified DNA was cloned into the pET-30a+ plasmid. The insert was then digested with NcoI and XhoI and ligated into NcoI-XhoI-digested pGEX-KG vector. The insert from pET-30a+ was sequenced and matched the published mature peptide sequence for CAMP factor (14Ruhlmann J. Wittmann-Liebold B. Jurgens D. Fehrenbach F.J. FEBS Lett. 1988; 235: 262-266Crossref PubMed Scopus (16) Google Scholar). Expression and Purification of CAMP Factor—The protein was expressed in BL21(DE3) cells. A starter culture (20 ml of Luria Bertani broth containing 100 μg/ml ampicillin) of transformed cells was grown at 37 °C overnight. This overnight culture was then inoculated into 1 liter of Luria Bertani broth containing 100 μg/ml ampicillin. The culture was shaken vigorously at 30 °C until the A 600 reached 0.8. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and the culture was grown for an additional 4 h. Cells were harvested by centrifugation and stored at –20 °C overnight. The frozen cells were thawed on ice for 15 min and resuspended in 20 ml of PBS buffer (16 mm K2HPO4, 150 mm NaCl, pH 7.2) containing protease inhibitor mixture (Sigma). The cells were then lysed by sonication. Triton X-100 was added to the bacterial lysate to a final concentration of 1% (w/v). The lysate was centrifuged at 10,000 × g for 10 min at 4 °C. The fusion protein was purified from the supernatant. The purification procedure was essentially as described by Guan and Dixon (13Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1638) Google Scholar). The supernatant was mixed with 20 ml of 50% (v/v) glutathione-agarose beads and gently shaken at 4 °C for 30 min. The beads were washed four times with 10 ml of PBST buffer (PBS buffer with 1% Triton) at 4 °C, once with PBS buffer, and twice with thrombin cleavage buffer (50 mm Tris, 150 mm NaCl, 2.5 mm CaCl2, pH 8.0) at room temperature. After centrifugation, the agarose beads were mixed with 2 ml of thrombin cleavage buffer containing 12 μg of thrombin. The suspension was incubated with gentle shaking at room temperature for 1 h and centrifuged. The beads were washed with thrombin cleavage buffer twice to retrieve the cleaved protein, and the supernatants were pooled. This procedure typically yielded about 4 mg of purified CAMP factor/liter of liquid culture. Protein concentrations were determined by the Bradford method. Purification of Glutathione S-Transferase-CAMP Fusion Protein— The GST 1The abbreviations used are: GST, glutathione S-transferase; PEG, polyethylene glycol; PC, phosphatidylcholine; C12-ceramide, N-lauroryl-d-erythro-sphingosine; C20-ceramide, N-arachidoyl-d-erythro-sphingosine.-CAMP protein (15Smith D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5036) Google Scholar) bound to the glutathione agarose beads was eluted three times with 2 ml of 50 mm Tris-HCl, 10 mm glutathione (pH 7.5). Circular Dichroism—CD spectra were measured using a Jasco J-715 circular dichroism spectrometer (Jasco, Tokyo, Japan) with a 1-mm path length cuvette. The protein (0.08 mg/ml, 3 μm) was in 8 mm potassium phosphate buffer, pH 7.2. The samples were scanned 10 times from 180 to 250 nm with an 0.5-nm interval. CD spectra were corrected for background and analyzed for protein secondary structure using the CONTIN, CDSSTR and SELCON programs with the 43-protein reference set (cryst.bbk.ac.uk/cdweb/) (16Lobley A. Wallace B.A. Biophys. J. 2001; 80: 373aGoogle Scholar, 17Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (642) Google Scholar, 18Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2486) Google Scholar). The secondary structure was also analyzed with the prediction program GOR IV (npsa-pbil.ibcp.fr). Lysis of Sheep Red Blood Cells—Sheep red blood cells (Cedarlane, Hornby, Ontario, Canada) were washed five times in hemolysis buffer (10 mm Tris-HCl, 150 mm NaCl, pH 7.4) by centrifugation. The erythrocytes were then resuspended in hemolysis buffer to 0.5% (v/v). The cells were incubated at room temperature for 5 min in the presence of 10 mm MgCl2 and with or without 50 milliunits/ml sphingomyelinase from S. aureus (EC 3.1.4.12, Sigma). 180 μl of cell suspension was added to the wells of a microplate containing different amounts of CAMP factor in 20 μl of the former buffer. Hemolysis was measured by the decrease in turbidity (A 650) using a 96-well plate reader (Spectramax 190, Molecular Devices, Sunnyvale, CA). For osmotic protection by polyethylene glycol (PEG), the red cells were made to 5% (v/v) suspension followed by incubation with 500 milliunits/ml sphingomyelinase at room temperature. 20 μl of the erythrocyte suspension was added to 180 μl of hemolysis buffer containing CAMP factor and PEGs of various molecular weights at 30 mm. Hemolysis was assayed by cell turbidity as described above (experiments depicted in Fig. 4A) or by measuring the optical density of the released at nm cells and membranes; experiments in Fig. samples without CAMP factor the of CAMP to erythrocytes were lysed in 5 mm phosphate buffer and washed times until the membrane were in hemolysis buffer with 10 mm MgCl2 and with or without sphingomyelinase for 10 min at 37 °C. CAMP factor protein or fusion protein was After incubation at 37 °C for the were centrifuged at for 10 min and washed twice with the hemolysis buffer. The membrane was in 10 mm Tris, 150 mm NaCl, pH The membrane were analyzed by or by For μg/ml and μg/ml protein were For the concentration of and CAMP factor was 150 and The protein concentration of the membrane was determined by the Bradford and the concentration was by the to protein is by of were from with the of ceramide (Sigma). The in were mixed in various in a and then for an additional 3 h to the The were at room temperature for 1 h in 1 ml of 20 mm 150 mm NaCl, 50 mm pH by For the suspension was to °C The suspension was frozen at –20 °C and thawed at room temperature. The suspension was to liposomes using a Canada) by 10 times a membrane with a pore The was to °C to of the The was by on with 20 mm 150 mm NaCl, pH The concentration was determined by an cholesterol For liposomes without the concentration was from the in Scholar). were on a 3 The liposomes with concentration were mixed with various amounts of CAMP factor in 10 mm Tris-HCl, 150 mm NaCl, 10 mm pH buffer. After incubation at 37 °C for 10 the samples were with the buffer, and was measured The fraction of released was by the of a incubated without CAMP factor. was from a lysed by of membranes and CAMP factor was using a 25 The was with 50 mm Tris-HCl, 150 mm NaCl, mm pH The samples containing erythrocyte membranes were and in 500 μl of buffer (50 mm Tris-HCl, 150 mm NaCl, pH to the and eluted with buffer. of 1 ml were and analyzed by Chemical of CAMP factor concentration was incubated with the liposomes in 10 mm 150 mm NaCl, 10 mm pH at 37 °C for 30 min. The cross-linking was then added to 2.5 mm, and the samples were incubated at 37 °C for 30 min. The reaction was by the of to The samples were then analyzed by red blood cells were with sphingomyelinase as described and lysed with μg/ml CAMP factor, and the membranes were by centrifugation. The membranes were washed three times with 5 mm buffer (pH 7.4) and followed by with pH 7.4) for 5 were then in a electron at 100 Cloning, and Purification of CAMP Factor—The coding sequence for CAMP factor was amplified by and sequenced from a of S. A was The coding sequence was found to at from the published sequence of CAMP factor of S. agalactiae A. O. Lutticken R. Med. Microbiol. PubMed Scopus Google Scholar) at the and the coding sequence of the CAMP factor mature peptide matched the sequence of protein the of CAMP factor of S. agalactiae (14Ruhlmann J. Wittmann-Liebold B. Jurgens D. Fehrenbach F.J. FEBS Lett. 1988; 235: 262-266Crossref PubMed Scopus (16) Google Scholar). from the different of the bacterial We initially to the CAMP factor into a high in E. coli, but to This be related to on the of CAMP factor genes from different bacterial strains (10Schneewind O. Friedrich K. Lutticken R. Infect. Immun. 1988; 56: 2174-2179Crossref PubMed Google Scholar, M. Babiuk L.A. Potter A.A. Microb. Pathog. 1996; 20: 297-307Crossref PubMed Scopus (39) Google Scholar, K. Ferretti J.J. Primeaux C. McShan W.M. Infect. Immun. 1999; 67: 4725-4731Crossref PubMed Google Scholar), which used in in E. coli, or in the This suggests that CAMP factor be to the bacterial cells. We to the CAMP factor, its in that from a cell be similar to the in be by when was by Microbiol. 1991; PubMed Scopus Google Scholar), the and CAMP activity was to to cells as We used the pGEX-KG which a protein expressed as glutathione S-transferase-CAMP factor fusion protein. The fusion protein was purified following a (13Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1638) Google Scholar, D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5036) Google Scholar). The fusion protein to the glutathione-agarose beads was cleaved by and the cleavage was released from the agarose beads The cleaved protein has 20 from the to the of the CAMP factor. This on the activity of CAMP factor. and Circular The GOR IV secondary structure prediction program suggests that the protein consists of an with The of is in the from the CD of the CAMP factor The three CONTIN, and yielded similar which that is about 19% and of CAMP on Sheep found that 50% hemolysis of be in 25 min at 25 °C with CAMP factor The hemolytic activity of the recombinant CAMP factor is comparable with the with CAMP factor, for which of R. Infect. Immun. PubMed Google Scholar) or 1 B. Schmidt C.M. Fehrenbach F.J. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 195-196Google Scholar) have been The CAMP factor the red blood cells without sphingomyelinase a concentration of 12 μg/ml CAMP factor was to an of hemolysis that the is Fig. The cells are about 10,000 times susceptible to CAMP factor the The fusion protein hemolytic activity, about times the protein not This be related to the of of CAMP to Sheep The CAMP protein was incubated with red blood cell membranes with or without sphingomyelinase After the mixture was centrifuged and washed twice to the protein. Fig. 3 that CAMP factor bound to the It also that CAMP factor bind to the cell although the was fusion protein also bound to the red blood cell The but hemolytic activity of suggests that membrane and cell are and that the of the CAMP be in the CAMP a involves of which the membrane structure and to the cell In proteins erythrocytes a form on the cell the of as and as the This to an osmotic and the of into the in the of cells. to the cell suspension this molecular to the the osmotic by the the osmotic protection be used to pores of Previous studies on CAMP factor that after of liposomes with CAMP factor, in that activity was not required for membrane R. Infect. Immun. PubMed Google Scholar). We therefore that CAMP factor activity. Fig. that and a of is with In and have on CAMP cells were incubated with CAMP factor in the presence of washed twice in the buffer, and resuspended in buffer without PEG, hemolysis This that pore formation in the presence of PEG, indicating that by osmotic protection but not by the or lytic of CAMP factor. that CAMP factor forms pores with a diameter on susceptible that the of and are 1.6 and respectively S. J. Chromatogr. 1981; Scopus Google Scholar), experiments suggested that the diameter of CAMP factor pore is by the of pores by electron microscopy Fig. we the osmotic protection experiments with different of CAMP factor. Fig. that the of is and at concentrations of This is in with the of heterogeneous pore size, with the pores at at concentrations of of CAMP red cell membranes with sphingomyelinase and then to high concentrations of CAMP factor, we membrane lesions of various the pores were the reached of 12 nm and pores were and in B and have been reported with S. J. A. Infect. Immun. 1985; PubMed Google Scholar, M. R. C. M. J. S. J. PubMed Scopus Google Scholar). In with the the pores by CAMP factor to be by a of protein, which the molecular of CAMP factor kDa as with kDa for of CAMP on CAMP factor that it was in as it was eluted from a and not the pore are membrane by or other we not formation on membranes by Fig. the pore is in we incubated the CAMP protein with susceptible which were then with and analyzed by was which eluted at the as the that not been incubated with were after membrane with or that CAMP factor not or that the is on the on we cross-linking on CAMP factor incubated with liposomes of ceramide cholesterol and Fig. that oligomeric forms were produced treatment with the of the membranes to CAMP factor the high of cross-linking also suggests the of oligomeric on Liposome the activity of CAMP factor was found to be related to the of diacylglycerol or ceramide and the of cholesterol in and membranes (6Fehrenbach F.J. Schmidt C.M. Sterizik B. Jürgens D. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 317-324Google Scholar, 7Sterzik B. Schmidt C.M. Fehrenbach F.J. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 195-196Google Scholar). membranes containing different concentrations of ceramide or diacylglycerol a for the of CAMP factor on and for the effects of on the activity of CAMP factor. Fig. the from liposomes of various The following were The of permeabilization is not related to the CAMP factor is permeabilization until a concentration is the of then from 20 to by the protein This cooperative is in with an oligomeric mode of of CAMP factor membrane to we ceramide not the activity of CAMP factor on the at as high as this to and to as as ceramide from We however, that cholesterol was the cholesterol concentration was from 25 to the liposomes three times liposomes were The with that the cholesterol content in the membrane the CAMP activity on membranes (6Fehrenbach F.J. Schmidt C.M. Sterizik B. Jürgens D. Alouf J.E. Fehrenbach F.J. Freer J.H. Jeljaszewicz J. Bacterial Protein Toxins. Academic Press, San Diego1984: 317-324Google Scholar). with the most susceptible membranes the concentration of CAMP factor to a permeabilization was a times that required with red blood cells but was similar to the concentration required with erythrocytes Fig. The activity of CAMP factor on the liposomes from membrane or from activity of CAMP factor. we the following the of membrane and CAMP factor was the amounts of were were and at then the of membrane permeabilization be the This however, was not the other were then amounts of membranes not CAMP factor from a of CAMP factor therefore of liposomes to the which was not This suggests that CAMP factor is with membrane The of CAMP factor on the membrane therefore is not to but in to the pore activity of the bound The that CAMP factor forms oligomeric pores in susceptible The size of pores is of S. J. A. Infect. Immun. 1985; PubMed Google Scholar) and the S. J. A. Infect. Immun. 1985; PubMed Google Scholar, L. S. R. Biochem. J. 1991; PubMed Scopus Google Scholar). and pores in diameter S. J. A. Infect. Immun. 1985; PubMed Google Scholar, M. R. C. M. J. S. J. PubMed Scopus Google Scholar, L. S. R. Biochem. J. 1991; PubMed Scopus Google Scholar, S. J. J. Google Scholar), a that also to CAMP factor. In to other proteins and the of CAMP factor are not following membrane This is not with and of oligomerization on membranes has been with as Biochim. Biophys. Acta. PubMed Scopus Google Scholar) or K. O. N. K. 1996; PubMed Scopus Google Scholar) and with M. A. J. A. Microbiol. Lett. 2000; PubMed Scopus Google Scholar), of have not been from the form pores on membrane from were the of the was to be 3 C. L. Microbiol. 1999; PubMed Google CAMP factor sequence to other the which when for 10 in the this not the of P. J. Biol. PubMed Scopus Google Scholar), this not the prediction of the structure of CAMP factor in the oligomeric form. and therefore with other be in the of CAMP membrane and pore which involves sphingomyelinase treatment membrane this not for the in membrane It therefore that the formation is by the in as liposomes were susceptible to CAMP factor to CAMP factor was not on liposomes containing ceramide on This is in to the red cells, which were by sphingomyelinase treatment, and suggests that the of ceramide not in a with the protein. It to be determined or of the red cell membrane for the of is the of CAMP factor The of CAMP factor for bacterial cells, which was in the to to that be as a of CAMP factor in of purified CAMP factor to E. or B. not cell CAMP factor therefore be to bacterial cells from the studies on its mode of to be with different of cells and membranes to a of the of CAMP factor. cholesterol has a on membrane the of the has not yet been in and the of CAMP factor for bacterial cells suggests that it be by in the We and for with electron

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