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An immunologically active cell-wall peptide polymer obtained from the culture filtrates of Staphylococcus aureus
210
BBA
PRELIMINARY
NOTES
21130
An immunologically active cell-wall peptide polymer obtained from the culture filtrates of Staphylococcus aureus Cell-walt peptide is one of the two elemental structural components of the "mucopeptide" which is considered to exist in the cell walls of all bacterial species as their "basal structure 'u. Immunological activity of the bacterial cell-wall peptide (polymer) has not hitherto been demonstrated, although immunological activity has recently been reported for streptococcal mucopeptide 2. In the present study, an immunologically active cell-wall peptide polymer has been obtained and purified from culture filtrate a of a viscid wound strain of Staphylococcus 4-6. The cultivation of cells was based on WILEY'S original method. Hyperimmune rabbit antiserum used in this study was prepared as previously described 7. Sugar estimation was carried out after hydrolysis using 2 M HC1 at lOO-lO2 ° for 2 h followed by evaporation of acid i ~ vacuo. Reducing sugar activity was measured by the method of MOMOSE8,9 using 3,6-dinitrophthalic acid, amino sugar by the method of BELCHER'°, organic phosphorus by the method of ALLENn and the free N H 2 group reaction as described by GHUVSEN A~D STROmN~ER 12, but with a slight modification. Culture filtrates were lyophilized after dialysis and concentration. Partial purification was obtained by repeated treatments with cold trichloroacetic acid and by precipitation with ethanol. The partially purified starting material still contains four components, as shown by immunoelectrophoresis at pH 7.2 with a WILEY antiserum. The partially purified starting material was filtered on a Sephadex G-5o column (1.8 cm x 66 cm) using water as eluent. (Flow rate, 18 ml/h; collected fraction, 3.o ml.) The fraction which was eluted in the external volume showed the same peak for the four different chemical assays performed. This fraction, which still contained four components, was chromatographed on a DEAE-cellulose column (1. 5 cm ~ 22 cm) equilibrated with 0.02 M phosphate buffer (pH 7.2). Elution with the same buffer gave a fraction containing only the positively charged components, P1 and P2. This fraction gave reactions for reducing sugar activity, for amino sugar and for the free NHz group, but not for organic phosphorus. After desalting, tile fraction was again filtered on the Sephadex G-5o under the conditions described above. The internal volume contained a sharp, single peak, the P I component, which gave the free N H 2 group reaction. On immunoelectrophoresis, using the Wiley antiserum, a sharp, single precipitation band migrated toward the negative pole. The eleetrophoretic mobility indicates that the P1 component has a considerable positive net charge, and it gives a precipitin reaction. Ultracentrifugation (5264 ° rev./min, 2o °) of an aqueous solution of the PI component failed to form a boundary. The behavior of this component in Sephadex G-5o and in the ultracentrifuge suggested that it has a rather small molecular weight, below 1oooo. The P1 component without hydrolysis was shown to be positive for the free N H 2 group and the ninhydrin reaction. The hydrolysate (2 M HCt, lOO-io2 °, 2 h, and 4 M HC1, IOO-lO2 °, 6 h) failed to show reducing sugar activity and the presence of amino sugar. The result of amino acid analysis using the technique of S P A C K M A N , STEIN AND Biochim. Biophys. dcta, I 2 I
(I960) 2to -212
PRELIMINARY NOTES
211
TABLE I MOLAR RATIOS OF THE
CELL
OF A M I N O ACIDS OF T H E WALLS
OF
Staphylococcus Pz
G l u t a m i c acid Lysine Alanine Glycine NH 3 T o t a l r e c o v e r y ***
I I ,oo 2, I o 4.55 o.99 94.5
PI
COMPONENT
A N D OF T H E "MUCOPEPTIDE
'~ F R A C T I O N
aureus
" Mucopeptide" fraction of, the cell walls of S. aureus 1 0.85 2.14 4 .61 Not determined
* C a l c u l a t e d on t h e basis of I mole of g l u t a m i c acid. ** Q u o t e d fro m t h e r e s u l t s of STROMINGER19. This " m u c o p e p t i d e " f r a c t i o n is t h e i n s o l u b l e m a t e r i a l w h i c h w a s o b t a i n e d a f t e r r e m o v i n g t e i c h o i c a c i d of t h e cell w a l l s w i t h t r i c h l o r o a c e t i c acid. *** The r e c o v e r y was c a l c u l a t e d on t h e basis of t o t a l a m i n o a c i d r e s i d u e p e r p e p t i d e hyd r o l y s e d (%).
MOORE13 is shown in Table I. Only glutamic acid, lysine, alanine and glycine were found in the hydrolysate (6 M HC1, 100-102 °, 24 h). In addition to these amino acids, NH3 was also found. There were also traces of two or three other unknown ninhydrinpositive materials. These results indicate that the P I component is most probably a peptide. The molecular ratios of amino acids and N H 3, within the experimental error, were i : I : 2 : 5 : I for G l u - L y s - A l a - G l y - N H 3. When the general losses of these amino acid residues from the analysis and hydrolysis were considered, the contamination by minor components other than the peptide seemed rather unlikely. The presence of the N H 3 in the hydrolysate indicates the presence of the amide structure in this peptide. Optical rotation of the alanine residue was examined using YOSHIDA'S highly specific, purified L-alanine dehydrogenase (EC 1.4.1.1) 1~, and showed that approx. 2/3 of the alanine residue was D-alanine. The glutamic acid residue was also found to be composed entirely of I)-glutamic acid b y enzymatic assay using L-glutamate dehydrogenase (EC 1.4.1.2)15,16, (Sigma Chemical Co.). The determination of the N-terminal amino acid residue was carried out by the dinitrophenylation methodlL is. DNP-alanine was determined as DNP-N~terminal residue of this peptide in thin-layer chromatography using a silica gel G layer. It has been generally accepted that the cell-wall peptide, composed of the four component amino acids (n-glutamic acid, lysine and I)- and L-alanine), is linked to the N-acetylmuramic acid-N-acetylglucosamine polysaccharide backbone of the mucopeptide. Especially in Staphylococcal cell walls, it has been thought that the cell-wall peptide chains are further cross-linked with each other through pentaglycyl peptide bridges in some unknown way 19. The amino acid sequence of the staphylococcal cell-wall peptide was postulated by STROMINGER to be L-Ala-I)-Glu-LysI)-Ala2~. Our results are compatible with the interpretation that the component is a cell-wall peptide polymer itself which is perhaps cross-linked b y pentaglycyl peptide bridges. The molar ratios of the amino acids also seem to coincide well with those of the mucopeptide fraction obtained from staphylococcal cell walls 19 (Table I). In this case, one ~ of the hypotheses of STROMINGER as to the19, ~° pentaglycyl Biochim. Biophys. Mcta, 121 (1966) 21o-212
212
PRELIMINARY NOTES
peptide cross-linkage in the cell walls seems to be most acceptable. Total recovery from the amino acid analysis, a single peak in chromatography and a single precipitation band on immunoelectrophoresis indicates that this PI component is pure peptide. The component was shown to be immunologically active by the precipitin reaction. This is believed to be the first report of the cell-wall peptide to be obtained in immunologically active form. This is also considered to be the first report of the isolation and purification of the cell-wall peptide polymer. Further chemical characterization and biological studies on the component, especially the determination of the amino acid sequence as regards the pentaglycyl peptide cross-linkage, are under investigation. The authors wish to express their appreciation to Dr. S. UTSUMI a n d Dr. A. YOSHIOA for their aid in the amino acid analysis and the L-alanine determination, and also to Dr. I. SUZ~'KA for the ultracentrifugal determination. This investigation was supported by the U.S. Public Health Service Grant AI-o5473 and by the U.S. Veterans Administration Central Office Research Service.
Department of Public Healih, School of Medicine, University of Pennsylvania, The U.S. Veterans Administration and Philadelphia General Hospitals, Philadelphia, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20
K . HISATSUNE*
S. J.
DECOuRCY,
JR.
S. MUDI)
M. R. J. SALTON, "The Bacterial Cell Wall", Elsevier, Amsterdam, 1964, p. 133. E. M. ABDULLA AND J, H. SCH\VAB, Proc. Soc. Expll. Biol. Med., 118 (1965) 359K. J-IISATSUNE, S. J. DECouRcY, JR. AND S. MUDD, Bact. Proc., (1965) 62. t3. t3. WILEY, Can. J. 3/licrobiol., 7 (1961) 933. 13. t3. WILEY, Can. J. iVIicrobiol., 9 (1963) 27. 13. 13. WILEY AND J. C. ~VONNACOTT, J. Bacteriol., 83 (1962) 1169. S. MEnD AND S. J. DECouRCY, JR., J. Bacteriol., 89 (1965) 874. T. MOMOSE, A. INABA, Y. MUKAI AND M. WATANABE, Talanta, 4 (196°) 33. T. MOMOSE, Y'. MUKAI AND M. WATANABE, Talanta, 5 (196o) 275. R. BELCHER, A. J. NUTTEN AND C. M. SAMBROOK, Analyst, 79 (1954) 2Ol. R. J. L. ALLEN, Biochem. J., 34 (194 ° ) 858J. M. GHUYSEN AND J. L. STROMINOER, Biochemistry, 2 (1963) 111o. D. H. SPACKMAN, XV. I-I. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. A. YOSHIDA, Anal. Biochem., I I (1965) 383S. J. AOELSTEIN AND 13. L. VALLEE, J. Biol. Chem., 233 (1958) 589 . H. J. STRECKER, in S. P. COLOWICK ANn ~N~.O. I~APLAN, Methods in Enzymology, VoI. 2, Academic Press, N e w York, 1955, p. 220. H. FRAENKEL-CONRAT, J. I. HARRIS, ANn A. L. LEVY, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, Interscience, New York, 1955, p. 359F. SAI~GER, E. O. P. THOMPSON, Biochem. J., 53 (1953) 353. M. H. MANDELSTAM ANn J. L. STROMINGER, Biochem. Biophys. Res. Commun., 5 (I96I) 466. J. L. STROMINGER, Ann. N . Y . Acad. Sci., 128 (1965) 58 , 59.
Received January 6th, 1966 * Fellow of the Theresa F. and Joseph Felsen Memorial Fund.
Biochim. Biophys. Acta, i2i (i966) 2io--212
BBA
PRELIMINARY
NOTES
21130
An immunologically active cell-wall peptide polymer obtained from the culture filtrates of Staphylococcus aureus Cell-walt peptide is one of the two elemental structural components of the "mucopeptide" which is considered to exist in the cell walls of all bacterial species as their "basal structure 'u. Immunological activity of the bacterial cell-wall peptide (polymer) has not hitherto been demonstrated, although immunological activity has recently been reported for streptococcal mucopeptide 2. In the present study, an immunologically active cell-wall peptide polymer has been obtained and purified from culture filtrate a of a viscid wound strain of Staphylococcus 4-6. The cultivation of cells was based on WILEY'S original method. Hyperimmune rabbit antiserum used in this study was prepared as previously described 7. Sugar estimation was carried out after hydrolysis using 2 M HC1 at lOO-lO2 ° for 2 h followed by evaporation of acid i ~ vacuo. Reducing sugar activity was measured by the method of MOMOSE8,9 using 3,6-dinitrophthalic acid, amino sugar by the method of BELCHER'°, organic phosphorus by the method of ALLENn and the free N H 2 group reaction as described by GHUVSEN A~D STROmN~ER 12, but with a slight modification. Culture filtrates were lyophilized after dialysis and concentration. Partial purification was obtained by repeated treatments with cold trichloroacetic acid and by precipitation with ethanol. The partially purified starting material still contains four components, as shown by immunoelectrophoresis at pH 7.2 with a WILEY antiserum. The partially purified starting material was filtered on a Sephadex G-5o column (1.8 cm x 66 cm) using water as eluent. (Flow rate, 18 ml/h; collected fraction, 3.o ml.) The fraction which was eluted in the external volume showed the same peak for the four different chemical assays performed. This fraction, which still contained four components, was chromatographed on a DEAE-cellulose column (1. 5 cm ~ 22 cm) equilibrated with 0.02 M phosphate buffer (pH 7.2). Elution with the same buffer gave a fraction containing only the positively charged components, P1 and P2. This fraction gave reactions for reducing sugar activity, for amino sugar and for the free NHz group, but not for organic phosphorus. After desalting, tile fraction was again filtered on the Sephadex G-5o under the conditions described above. The internal volume contained a sharp, single peak, the P I component, which gave the free N H 2 group reaction. On immunoelectrophoresis, using the Wiley antiserum, a sharp, single precipitation band migrated toward the negative pole. The eleetrophoretic mobility indicates that the P1 component has a considerable positive net charge, and it gives a precipitin reaction. Ultracentrifugation (5264 ° rev./min, 2o °) of an aqueous solution of the PI component failed to form a boundary. The behavior of this component in Sephadex G-5o and in the ultracentrifuge suggested that it has a rather small molecular weight, below 1oooo. The P1 component without hydrolysis was shown to be positive for the free N H 2 group and the ninhydrin reaction. The hydrolysate (2 M HCt, lOO-io2 °, 2 h, and 4 M HC1, IOO-lO2 °, 6 h) failed to show reducing sugar activity and the presence of amino sugar. The result of amino acid analysis using the technique of S P A C K M A N , STEIN AND Biochim. Biophys. dcta, I 2 I
(I960) 2to -212
PRELIMINARY NOTES
211
TABLE I MOLAR RATIOS OF THE
CELL
OF A M I N O ACIDS OF T H E WALLS
OF
Staphylococcus Pz
G l u t a m i c acid Lysine Alanine Glycine NH 3 T o t a l r e c o v e r y ***
I I ,oo 2, I o 4.55 o.99 94.5
PI
COMPONENT
A N D OF T H E "MUCOPEPTIDE
'~ F R A C T I O N
aureus
" Mucopeptide" fraction of, the cell walls of S. aureus 1 0.85 2.14 4 .61 Not determined
* C a l c u l a t e d on t h e basis of I mole of g l u t a m i c acid. ** Q u o t e d fro m t h e r e s u l t s of STROMINGER19. This " m u c o p e p t i d e " f r a c t i o n is t h e i n s o l u b l e m a t e r i a l w h i c h w a s o b t a i n e d a f t e r r e m o v i n g t e i c h o i c a c i d of t h e cell w a l l s w i t h t r i c h l o r o a c e t i c acid. *** The r e c o v e r y was c a l c u l a t e d on t h e basis of t o t a l a m i n o a c i d r e s i d u e p e r p e p t i d e hyd r o l y s e d (%).
MOORE13 is shown in Table I. Only glutamic acid, lysine, alanine and glycine were found in the hydrolysate (6 M HC1, 100-102 °, 24 h). In addition to these amino acids, NH3 was also found. There were also traces of two or three other unknown ninhydrinpositive materials. These results indicate that the P I component is most probably a peptide. The molecular ratios of amino acids and N H 3, within the experimental error, were i : I : 2 : 5 : I for G l u - L y s - A l a - G l y - N H 3. When the general losses of these amino acid residues from the analysis and hydrolysis were considered, the contamination by minor components other than the peptide seemed rather unlikely. The presence of the N H 3 in the hydrolysate indicates the presence of the amide structure in this peptide. Optical rotation of the alanine residue was examined using YOSHIDA'S highly specific, purified L-alanine dehydrogenase (EC 1.4.1.1) 1~, and showed that approx. 2/3 of the alanine residue was D-alanine. The glutamic acid residue was also found to be composed entirely of I)-glutamic acid b y enzymatic assay using L-glutamate dehydrogenase (EC 1.4.1.2)15,16, (Sigma Chemical Co.). The determination of the N-terminal amino acid residue was carried out by the dinitrophenylation methodlL is. DNP-alanine was determined as DNP-N~terminal residue of this peptide in thin-layer chromatography using a silica gel G layer. It has been generally accepted that the cell-wall peptide, composed of the four component amino acids (n-glutamic acid, lysine and I)- and L-alanine), is linked to the N-acetylmuramic acid-N-acetylglucosamine polysaccharide backbone of the mucopeptide. Especially in Staphylococcal cell walls, it has been thought that the cell-wall peptide chains are further cross-linked with each other through pentaglycyl peptide bridges in some unknown way 19. The amino acid sequence of the staphylococcal cell-wall peptide was postulated by STROMINGER to be L-Ala-I)-Glu-LysI)-Ala2~. Our results are compatible with the interpretation that the component is a cell-wall peptide polymer itself which is perhaps cross-linked b y pentaglycyl peptide bridges. The molar ratios of the amino acids also seem to coincide well with those of the mucopeptide fraction obtained from staphylococcal cell walls 19 (Table I). In this case, one ~ of the hypotheses of STROMINGER as to the19, ~° pentaglycyl Biochim. Biophys. Mcta, 121 (1966) 21o-212
212
PRELIMINARY NOTES
peptide cross-linkage in the cell walls seems to be most acceptable. Total recovery from the amino acid analysis, a single peak in chromatography and a single precipitation band on immunoelectrophoresis indicates that this PI component is pure peptide. The component was shown to be immunologically active by the precipitin reaction. This is believed to be the first report of the cell-wall peptide to be obtained in immunologically active form. This is also considered to be the first report of the isolation and purification of the cell-wall peptide polymer. Further chemical characterization and biological studies on the component, especially the determination of the amino acid sequence as regards the pentaglycyl peptide cross-linkage, are under investigation. The authors wish to express their appreciation to Dr. S. UTSUMI a n d Dr. A. YOSHIOA for their aid in the amino acid analysis and the L-alanine determination, and also to Dr. I. SUZ~'KA for the ultracentrifugal determination. This investigation was supported by the U.S. Public Health Service Grant AI-o5473 and by the U.S. Veterans Administration Central Office Research Service.
Department of Public Healih, School of Medicine, University of Pennsylvania, The U.S. Veterans Administration and Philadelphia General Hospitals, Philadelphia, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20
K . HISATSUNE*
S. J.
DECOuRCY,
JR.
S. MUDI)
M. R. J. SALTON, "The Bacterial Cell Wall", Elsevier, Amsterdam, 1964, p. 133. E. M. ABDULLA AND J, H. SCH\VAB, Proc. Soc. Expll. Biol. Med., 118 (1965) 359K. J-IISATSUNE, S. J. DECouRcY, JR. AND S. MUDD, Bact. Proc., (1965) 62. t3. t3. WILEY, Can. J. 3/licrobiol., 7 (1961) 933. 13. t3. WILEY, Can. J. iVIicrobiol., 9 (1963) 27. 13. 13. WILEY AND J. C. ~VONNACOTT, J. Bacteriol., 83 (1962) 1169. S. MEnD AND S. J. DECouRCY, JR., J. Bacteriol., 89 (1965) 874. T. MOMOSE, A. INABA, Y. MUKAI AND M. WATANABE, Talanta, 4 (196°) 33. T. MOMOSE, Y'. MUKAI AND M. WATANABE, Talanta, 5 (196o) 275. R. BELCHER, A. J. NUTTEN AND C. M. SAMBROOK, Analyst, 79 (1954) 2Ol. R. J. L. ALLEN, Biochem. J., 34 (194 ° ) 858J. M. GHUYSEN AND J. L. STROMINOER, Biochemistry, 2 (1963) 111o. D. H. SPACKMAN, XV. I-I. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. A. YOSHIDA, Anal. Biochem., I I (1965) 383S. J. AOELSTEIN AND 13. L. VALLEE, J. Biol. Chem., 233 (1958) 589 . H. J. STRECKER, in S. P. COLOWICK ANn ~N~.O. I~APLAN, Methods in Enzymology, VoI. 2, Academic Press, N e w York, 1955, p. 220. H. FRAENKEL-CONRAT, J. I. HARRIS, ANn A. L. LEVY, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, Interscience, New York, 1955, p. 359F. SAI~GER, E. O. P. THOMPSON, Biochem. J., 53 (1953) 353. M. H. MANDELSTAM ANn J. L. STROMINGER, Biochem. Biophys. Res. Commun., 5 (I96I) 466. J. L. STROMINGER, Ann. N . Y . Acad. Sci., 128 (1965) 58 , 59.
Received January 6th, 1966 * Fellow of the Theresa F. and Joseph Felsen Memorial Fund.
Biochim. Biophys. Acta, i2i (i966) 2io--212