STUDIES OF STREPTOCOCCAL CELL WALLS

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1 STUDIES OF STREPTOCOCCAL CELL WALLS V. AMINO ACID COMPOSITION OF CELL WALLS OF VIRULENT AND AVIRULENT GROUP A HEMOLYTIC STREPTOCOCCI' B. S. TEPPER, J. A. HAYASHI, AND S. S. BARKULIS Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois Received for publication June 22, 1959 Investigations of the cellular components concerned with the antigenicity and virulence of group A streptococci indicate that the typespecific M protein is the antigen most consistently associated with virulence (Lancefield, 1940). The ability of these organisms to resist phagocytosis is, perhaps, the most important factor in conferring virulence. Wiley and Wilson (1956) have made an extensive study of the phagocytosis of group A streptococci, confirming and extending earlier findings (Lancefield and Todd, 1928; Rothbard, 1945, 1948) that organisms which have M protein resist ingestion by leucocytes, and that little or no M protein is found in strains which are readily phagocytized and are avirulent. However, not all M containing organisms are virulent (Lancefield, 1940), indicating that other factors are important in determining whether a culture can establish infection when inoculated in small numbers. The investigations of Lancefield (1943), which showed that digestion of the streptococci with trypsin removed the M protein without killing the cells, indicated that it is located at or near the cell surface. A serologically active, but poorly antigenic M preparation can be obtained from the streptococcal cell by acid-heat extraction (Lancefield, 1928). More recent investigations have shown that the M protein is located on the cell wall (Salton, 1953) and can be extracted from the cell walls by acid-heat extraction (Barkulis and Jones, 1957). Approximately 40 per cent of the cell wall of group A streptococci is rendered soluble by treatment with trypsin (Barkulis and Jones, 1957), leaving a trypsinresistant residue composed, for the most part, of L-rhamnose, glucosamine, and muramic acid (the C polysaccharide) and the amino acids, glutamic acid, alanine, and lysine (Salton, 1953; Cummings and Harris, 1956; Hayashi and Barkulis, 1959). It appears that the C polysaccharide and these amino acids comprise the basic framework of the cell walls of group A streptococci and that the type-specific proteins are attached to this structure (Hayashi and Barkulis, 1959). The protein constituents of the streptococcal cell wall thus play an important part as determinants of the antigenicity and virulence of group A streptococci. The present paper deals with quantitative analyses of the amino acids of the cell walls of two group A streptococci which differ in their virulence for mice. Analyses have been made of cell walls, trypsin-treated cell walls, and a purified acid-heat extract of the cell walls of both strains in an attempt to correlate the amino acid composition of the cell walls of these organisms with their virulence. MATERIALS AND METHODS Growth of the organism and preparation of cell walls. Two strains of group A hemolytic streptococci, differing in virulence for mice, were used in these experiments. Strain Q496 is a nontypable strain having an LD50 for mice of greater than 500,000 streptococcal chains. The second strain, Q43x, was derived from strain Q496 by 43 serial mouse passages. This strain, which is highly virulent for mice, has an LD50 of 25 streptococcal chains and is identified serologically as a type 14 streptococcus. Details on the biological properties of these two streptococcal strains have been reported (Leedom and Barkulis, 1959). The organisms were grown in 1-L volumes of I Supported by a grant from the Life Insurance Medical Research Fund. A preliminary report of Todd-Hewitt broth supplemented with glucosesalt solution (Barkulis and Jones, 1957). Several this work was presented at the 59th annual meeting of the Society of American Bacteriologists, 16-hr cultures were pooled, harvested in the St. Louis, Missouri, May Sharples centrifuge and washed twice in distilled 33

2 34 TEPPER, HAYASHI, AND BARKULIS [VOL. 79 water. The average yield was 3 g wet weight of cells per L of culture. Cell walls of the two strains of streptococci were prepared as described by Barkulis and Jones (1957) with the modification that the Nossal disintegrator (Nossal, 1953) was used in preference to the AIickel disintegrator for breaking cells. Trypsin-treated cell walls were prepared as described by Hayashi and Barkulis (1959). Washed preparations of cell walls and trypsintreated cell walls were lyophilized and stored in vacuo over P205. The M protein of strain Q43x and a comparable protein fraction from strain Q496 were obtained from cell walls as previously described (Barkulis and Jones, 1957). Hydrolysis and chromatographic analyses. Samples of 25 mg or less of the proteins to be analyzed were dissolved or suspended in 100 ml of N HCI and hydrolyzed by refluxing for 36 hr. The hydrolyzates were concentrated to dryness under reduced pressure, a small amount of water was added to the residue, and the solution was again concentrated to dryness to insure the removal of the greater part of the HCl. The residue was dissolved in 5 or 10 ml of ph 2.2, 0.2 N sodium citrate buffer. The amino acids of the hydrolyzates were separated by ion-exchange chromatography on 150 by 0.9 cm columns of Dowex 50 by the method of Moore and Stein (1954a). The resin used was a mixture of 75 per cent Dowex 50 x 4-25 per cent Dowex 50 x 5, corresponding to a nominal 4.25 per cent crosslinked Dowex 50. The column was initially equilibrated at ph 3.1. The amino acids were eluted from the column by buffers of increasing ph and ionic strength. One-ml fractions were collected throughout the elution. The individual compounds eluted from the resin were identified by paper chromatography. Chemical methods. The nitrogen content of the hydrolyzates was determined by the use of micro- Kjeldahl digestion and the colorimetric Nessler procedure described by Wilson and Knight (1952). The amino acids in the effluent of the Dowex 50 column were analyzed using the ninhydrin method of Moore and Stein (1954b). Hexosamine was determined by the method of Dische and Borenfreund (1950). Muramic acid was identified by the Elsen-Morgan reaction (Strange and Powell, 1954) E E Aspatc ocid E Ehrucne Serlne 0.0 ~~~~~~~Murom,c acid Pro/ine ocid SOEffluent ml. 1. * 300 t so 4 - Influent 0.2N, ph 3.1 buffer - Figure 1. Partial elution diagram of the hydrolyzate of cell walls of the avirulent group A streptococcal strain Q496. Separation on Dowex 50 x 4.25 by the method of AMoore and Stein (1954a). Diagram presented to show the location of muramic acid in this elution scheme. RESULTS Examination of the amino acids eluted from the ion-exchange column revealed an elution pattern identical to that reported by Moore and Stein (1954a). There was, however, one unknown compound which was eluted from the resin among the first few peaks. The location of this new peak (peak 4) in relation to other eluted amino acids is shown in figure 1. Samples of the unknown compound were tested and found to give the reactions of a hexosamine. The distinct orange color in the Elsen-illorgan reaction and the absorption spectrum of this chromogen with a maximum at 510 m,t identified the unknown compound as muramic acid (Strange and Powell, 1954). The results of duplicate analyses of the cell walls of two streptococcal strains Q43x and Q496 are shown in table 1 (columns 1 and 2, 4 and 5). No qualitative differences in the composition of the cell walls were detected. Comparison of the mean values for these determinations (columns 3 and 6) shows no large quantitative differences in amino acid content. The major components of both strains are glutamic acid, lysine, alanine, and glucosamine. The mole ratios for these components relative to moles of glutamic acid are 1:0.7:2.2:0.8 for both strains. These analyses, indicate that the cell walls of strain Q496 contain slightly more aspartic acid, glycine, valine, isoleucine, and leucine than strain Q43x; however, these differences are not remarkable.

3 1960] STREPTOCOCCAL CELL WALLS 35 TABLE 1 Amino acid composition of the cell walls of streptococcal strains Q43x and Q496 Amino Acid (jumoles) of Cell Walls of Strains Amino Acid Q43x CWa,b Q496 CWb Q43x TTCWC Q496 TTCWC TTCW Q43xd Q496e I ILr Mean I II Mean I II I II MIean TLP TLP (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) Glutamic acid Lysine Alanine Glucosamine Muramic acid Aspartic acid Threonine Serine Glycine Valine Isoleucine Leucine Tyrosine Phenylalanine Arginine Methionine Proline Ammonia a Abbreviations used in this table: CW = cell walls, TTCW = trypsin-treated cell walls, TLP = trypsin-labile protein. bmicromoles amino acid per mg cell wall nitrogen. c Values adjusted to represent the trypsin-treated residues of the cell walls analyzed in columns 1, 2, 4, and 5. See text for explanation. d Calculated by subtracting data in column II from data in column 3. e Calculated by subtracting data in column 11 from data in column 6. The data in columns 7 through 10 in table 1 summarize the analyses of the trypsin-treated cell walls of the two streptococcal strains. The values reported in these analyses have been adjusted to the same glucosamine concentration as that found in their respective cell walls. Analyses of the data for the cell walls have shown that glucosamine represents 8.7 per cent of the dry weight of the cell walls of both strains. Although 40 per cent (range 38 to 41 per cent) of the cell wall is solubilized on trypsin treatment (Barkulis and Jones, 1957), no glucosamine or muramic acid is removed from the cell wall by this treatment. The adjusted values for the trypsintreated cell walls represent, therefore, that part of the cell wall remaining after the enzyme treatment. These data show that the trypsintreated cell walls are simpler in composition than the intact cell walls. All of the valine, arginine, methionine, and proline has been removed by the action of the enzyme. The residue is composed primarily of glutamic acid, lysine, and alanine with mole ratios of these compounds of 1:1:3.6. These analyses show good agreement between duplicate analyses for each strain (columns 7 and 8, 9 and 10) and reveal no differences apparent between strains. From these analyses, an average amino acid content was calculated (column 11, table 1) which is considered representative of the composition of the trypsin-resistant structure of the cell walls of both the virulent strain Q43x and the avirulent strain Q496. The data on the composition of the cell walls and the trypsin-treated cell walls were used to

4 36 TEPPER, HAYASHI, AND BARKULIS [VOL. 79 TABLE 2 Amino acid conmposition of the acid-extractable proteins from the cell walls of streptococcal strains Q43x and Q496 Amino Acid (pmoles) per Mg Protein Nitrogen Amino Acid Q43x M protein Q496 cell wall proteina I II Mean I II NMean Glutamic acid Lysine Alanine Glucosamine... Muramic acid _ Aspartic acid Threonine Serine Glycine Valine Isoleucine Leucine Tyrosine Phenylalanine Arginine Methionine Proline Ammonia a Prepared from Q496 cell walls in the same manner as was M protein from Q43x cell walls. calculate the composition of the trypsin-labile proteins of the two streptococci (columns 12 and 13, table 1). The calculated data reflect the same differences in amino acid content that were shown in the cell walls and also indicate that strain Q496 contains increased amounts of most of the amino acids found in the trypsin-labile protein fraction. These increased concentrations, however, represent a difference of only 3 per cent in the total weight of the cell walls, a value within the range of variations in the amount of protein solubilized by trypsin. As stated above, the trypsin-labile protein represents that portion of the cell wall of group A streptococci which contains the type-specific M protein. An M protein preparation has been isolated from cell walls of the virulent strain Q43x, and a comparable protein fraction without M activity from the avirulent strain Q496. The results of amino acid analyses of these proteins are shown in table 2. The M protein of Q43x has more lysine, aspartic acid, and threonine and slightly less glycine and alanine than the corresponding protein of strain Q496. Comparison of the data in table 2 with the amino acid composition of the trypsin-labile protein of strains Q43x and Q496 (columns 12 and 13, table 1) shows that in both strains the protein fraction isolated by acid-heat is not identical with the trypsin-labile protein. DISCUSSION Our quantitative data on the amino acid composition of the cell wall of this type 14 streptococcus (Q43x) agree with the qualitative results presented by Salton (1953) for a type 4 streptococcal cell wall with the exception that cysteine was not detected in walls of the type 14 streptococcus. Trypsin treatment of the Q43x and Q496 strains leaves residual structures which are identical in amino acid composition with mole ratios of glutamic acid:lysine: alanine of 1:1:3.6, values almost identical to those reported earlier (Hayashi and Barkulis, 1959). The analyses indicate that the changes which take place in acquiring antigenicity and virulence do not significantly modify the composition of the cell wall. All the amino acids which were detected

5 19601 STREPTOCOCCAL CELL WALLS 37 in the cell walls of the virulent strain were also found in the cell walls of the avirulent strain. There were also no large differences in the concentration of the amino acids found in the cell walls and trypsin-labile proteins of the cell walls of these strains. Evidence has been reported, however, which indicates that the M protein represents only a part of the trypsin-labile protein of the cell wall. Barkulis and Jones (1957) have shown that two acid-heat extractions remove most of the serologically active M protein from the cell walls and that the total protein in these extracts accounts for less than 15 per cent of the cell wall weight. These results suggest that the extraction procedure solubilizes only a small portion of the protein of the cell wall. The present analyses show marked differences in the amino acid composition of the trypsin-labile protein and the isolated M protein fraction from the virulent strain Q43x. The isolated M protein is therefore not representative of the total trypsin-labile protein of the cell wall. The data indicate that the acid-heat extraction solubilizes specific fragments of the surface protein which contain the configuration necessary for the biological activity of the M protein. A protein apparently having similar physical properties to M protein, but not the same serological activity can be extracted from the cell walls of the avirulent strain Q496. As with strain Q43x, the protein extracted from Q496 does not have the amino acid composition of the total trypsin-labile fraction. Comparison of the two acid-heat extractable proteins showed many similarities in composition, although the M protein was found to contain significantly larger amounts of lysine and aspartic acid. It is not possible, at present, to interpret these data in terms of the biological activity of the acid-heat extractable proteins. The data presented here indicate that the changes in configuration of the cell wall protein which are associated with the acquisition of type-specific antigenicity and virulence are accompanied by relatively small changes in amino acid composition. ACKNOWLEDGMENTS We are indebted to Dr. Eugene Fox of Western Reserve University for furnishing the avirulent culture used in this work. The technical assistance of Mr. Gerald Walsh and of Miss Pearl Hillman is gratefully acknowledged. SUMMARY Quantitative amino acid analyses were made on cell walls and protein fractions of cell walls from a virulent, type 14 group A streptococcus (strain Q43x) and an avirulent, nontypable group A streptococcus (strain Q496) in an attempt to determine any changes in the composition of the streptococcal cell wall which could be correlated with virulence or antigenicity. The amino acid composition and content of the trypsin-resistant protein of the cell walls of the two strains were found to be identical. Small quantitative differences and no qualitative differences in the amino acid composition of the cell walls of these strains were detected. The M protein of the virulent strain Q43x and a comparable protein fraction without M activity isolated from the avirulent strain Q496 showed many similarities in composition; however, the M protein contains significantly larger amounts of lysine and aspartic acid. Neither protein fraction was found to be representative of the trypsin-labile protein of their respective cell walls. The data presented indicate that the changes in the proteins of the cell walls which are associated with the acquisition of antigenicity and virulence of the streptococcal cell are not accompanied by any large changes in amino acid composition. REFERENCES BARKULIS, S. S. AND JONES, M. F Studies of streptococcal cell walls. I. Isolation, chemical composition, and preparation of M protein. J. Bacteriol., 74, DISCHE, Z. AND BORENFREUND, E A spectrophotometric method for the microdetermination of hexosamines. J. Biol. Chem., 184, CUMMINGS, C. S. AND HARRIS, H The chemical composition of the cell wall in some gram-positive bacteria and its possible value as a taxonomic character. J. Gen. Microbiol., 14, HAYASHI, J. A. AND BARKULIS, S. S Studies of streptococcal cell walls. III. The amino acids of the trypsin-treated cell wall. J. Bacteriol., 77, LANCEFIELD, R. C The antigenic complex of Streptococcus haemolyticus. I. Demonstration of a type-specific substance in extracts of Streptococcus haemolyticus. J. Exptl. Med., 47, LANCEFIELD, R. C Specific relationship

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