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Chemical composition of hyphal walls of dermatophytes
ARCHIVES
OF BIOCHEMISTRY
Chemical
AND
BIOPHYSICS
Composition
127,
229-234
of Hyphal
V. K. SHAH’ Department
of Bacteriology,
Received
(1968)
March
of Dermatophytes’
S. G. KNIGHT’
AND
University
Walls
of
Wisconsin,
9, 1968; accepted
Madison, April
Wisconsin
53706
8, 1968
Quantitative analyses of the purified cell walls of Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum are described. The cell walls consist mainly of a glucose-containing polysaccharide, an N-acetyl glucosamine polymer which is probably chitin, mannan, and protein. In addition, small quantities of galactosamine and lipid have been found. Only minor diflerences could be observed among the walls of these four species. Perhaps the higher glucose and lower mannose contents in T. mentagrophytes and E. fEoccosum cell walls as compared to M. canis and M. gypseum cell walls are significant differences. These studies revealed that the dermatophyte cell walls contain more complex chemical structures than chitin alone.
Studies on the cell wall composition of the filamentous fungi are still rare in contrast to the large amount of work done on the cell walls of bacteria. Most of the earlier work on the fungal cell wall has been directed to the demonstration of chitin or cellulose, and use of these constituents as criteria in establishing taxonomical relationships (1, 2). Many investigators (3-7) have employed chemically prepared walls using procedures that generally involved alkaline digestion. Studies by different investigators (8-10) clearly showed that chemical methods of cell wall preparation remove certain wall constituents. A marked degree of complexity and diversity in fungal wall structure has become evident from the studies on the cell walls isolated by mechanical methods (6, 8, 10, 11). Substantially, however, relatively few fungal species have been examined (9-16) by modern isolation and fractionation procedures. This investigation was initiated to determine and compare the components and ’ This investigation was supported by grant AI01201.10 from the U. S. Public Health Service. ’ Present address: Department of Microbiology Faculty of Science, M. S. University of Baroda, Baroda, India. ’ Deceased November 14, 1966.
structure of the hyphal cell walls of Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum with a view that this information may lead eventually to a better understanding of the physiological significance of cell walls and the action of some antifungal drugs. EXPERIMENTAL
PROCEDURE
Organisms and growth conditions. Stock cultures of T. mentagrophytes, M. canis, M. gypseum and E. floccosum were maintained on slants of Sabouraud’s glucose agar, composed of 4’( glucose, l’( Neopeptone (Difco), and 1.5’~ agar. Conidial suspensions for the inoculation of all cultures were prepared from Roux bottle cultures on Sabouraud’s glucose agar. Growth on this medium in Roux bottles was harvested in 50 ml of sterile distilled water by scraping the mycelial surface. The conidial suspension was filtered through sterile glass wool to remove hyphae, debris and clumps. One-liter Erlenmeyer flasks containing 250 ml of Sabouraud’s liquid medium, composed of 4’, glucose and l’, Neopeptone, were inoculated with 10 ml of the conidial suspensions and incubated at 30° on a New Brunswick rotary shaker. The mycelia were harvested after SO-72 hours incubation by suction filtration through a layer of cheese cloth. The mycelial mass was washed with distilled water until the filtrate was colorless. Preparation of cell walls. Cell walls were prepared by the ballistic disruption method essentially
230
SHAH
AND
that of Carlson (17). Glass-distilled water was used for subsequent work as ordinary distilled water was found to affect the color of the preparation during breaking cycles. The mycelial mass was mixed with an equal volume of acid-washed Superbrite glass beads, about 100 p in diameter (Minnesota Mining and Manufacturing Co., St. Paul, Minn.), and two volumes of cold 0.5 M NaCl in a 400 ml stainless steel container of the Sorvall Omni-Mixer. The container was lowered into an ice water bath and the contents homogenized at the maximum attainable speed for 10 minutes. The homogenate was decanted to separate the bulk of the glass beads, and then centrifuged briefly at low speed to sediment the cells, walls and residual beads. The solubilized cytoplasmic debris was discarded with the supernatant liquid. The pellet was washed with 0.1 M NaCl at room temperature and sedimented again by centrifugation. The resulting pellet was resuspended in the same volume of cold 0.5 M NaCl and glass beads, and the breaking and washing cycles repeated thrice. Breakage efficiency was judged by phase contrast and light microscopy. A cytoplasmic stain, la&phenol cotton blue, was used to enhance differentiation of empty cell walls and intact cells. The wall material was separated from the glass beads by repeatedly resuspending and carefully withdrawing the upper half of the sediment. The final preparations were virtually free of glass beads and unbroken cells. Purification of cell walls. Cell wall material was then washed five times with ten volumes of 1 M NaCl, and five times with distilled water. The crude cell wall preparations were digested with crystalline trypsin (Worthington Biochemical Corp., Freehold, N. J .) , and ribonuclease (Calbiochem, Los Angeles, Calif.). The walls were suspended in 0.05 M phosphate buffer, pH 8.0, containing 0.5 mg/ml trypsin and O.OlY; merthiolate. After 8 hours incubation at 37O, the walls were washed thrice with distilled water, suspended in 0.05 M tris(hydroxymethyl)aminomethane and 0.005 M MgCln buffer, pH 7.4, containing 0.5 mg/ml ribonuclease. After digestion for 3 hours at 37”, the cell wall residue was washed by centrifugation twice with 0.1 M NaCl and five times with distilled water. The cell wall preparations were extracted with cold ethanol, twice with ethanol-ethyl ether (1: 3 v/v) mixture at room temperature, and ethyl ether at room temperature. The combined solvent fraction was concentrated in uacuo, the residual material extracted twice with ethyl ether, and the ether extract dried and weighed. The cell wall material, after enzymatic digestion and solvent extraction, was dried, weighed, and stored in a desiccator for subsequent analyses. Dry weight loss upon heating at 105” for 24 hours was
KNIGHT determined, and all data have been corrected for this loss. The final product was a fluffy, white to gray material, and the results reported are based on analyses of this material. Methods of cell wall analysis. For the analysis of amino acids and amino sugars, cell walls were routinely hydrolyzed in sealed tubes with five volumes of 6 N hydrochloric acid at 100-105” for 12 hours. Undissolved material was separated by centrifugation, washed twice, and the washings added to the hydrolysate. HCl was removed by repeated evapomtion in uacuo at 40” over NaOH pellets. For the analysis of sugars, cell walls were hydrolyzed with formic, sulfuric, or hydrochloric acid. Hydrolysis with formic acid involved heating at 105’ in sealed tubes with 5 ml of 76? (v/v) formic acid for 12-18 hours. The soluble fraction was evaporated almost to dryness under reduced pressure and the formyl esters removed by heating the dried supematant liquid fraction with 2 ml of I N H2SOa for one hour at 105”. The sulfuric acid was neutralized with Ba(OH),, and the BaS04 removed by filtration. The hydrolysate was deionized by passing it through a small column of Amerlite IR-120 (hydrogen form); it was then evaporated to dryness to 40” under reduced pressure. For sulfuric acid hydrolysis, cell wall material was sealed in a tube with 5 ml of 1 N HSSO,, and placed in an oven at 105” for 24 hours. The supernatant liquid fractions containing the soluble wall components were neutralized with Ba(OH), and dried as described above. To minimize destruction of the components, sequential hydrolysis and fractionation was frequently used. The fraction liberated by mild hydrolysis, usually with 2 N HCl for 1-8 hours at 80-105”, was divided and half of it was further hydrolyzed before analysis. The wall material resistant to solution in 2 N HCl was resuspended in 2 or 6 N HCl and hydrolysis continued for 3 hours at 100”. HCI was removed as reported above. Ash analysis. Dried cell walls were weighted into dried, tared crucibles of silica, heated at 500” for 8 hours, and cooled in a desiccator over calcium chloride before weighing. These ash samples were used for the mineral estimations made with a Jarrell-Ash direct reading spectrometer. Nucleic acid determination. Extraction of nucleic acid with hot 5’, trichlomacetic acid was based on the procedure described by Schneider (18). Total nucleic acids were determined by measuring the optical density of the trichloroacetic acid extract at 268.5 rnr in a Beckman Model DU spectrophotometer (19). Total nitrogen was determined with Nessler’s reagent after digesting the cell walls by the method of Wilson and Knight (20). Protein was measured
HYPHAL
WALLS
OF
by the method of Lowry et al. (21), performed on the extracts of the walls prepared by treatment with 2 N NaOh at 100” for 30 minutes. Individual amino acids were separated quantitatively and measured on a Beckman-Spinco automatic amino acid analyzer with ninhydrin (22). Reducing sugars in the cell wall fractions were measured by the method of Somogyi (23) using the arsenomolybdate color reagent of Nelson (24) and a glucose standard. Pentose, methyl pentoses, and uranic acids were sought calorimetrically in the hydrolysates by the methods of Tracey (25), Winzler (26), and Dische (27), respectively. Hexosamine determinations were made on the acid and enzymatic hydrolysates of the cell wall material by the modification (28) of the Elson-Morgan reaction, and Nacetyl hexosamines were measured with the acetylating step omitted. Glucosamine and N-acetyl glucosamine were used as standards. Glucose was assayed with the glucose oxidase reagent of Worthington Biochemical Corp., Freehold, N. J. Snail enzyme digestion of cell walls. Snail enzyme made from the gut juice of Helix pomatiu (Endo Laboratories Inc., Garden City, New York) was a gift from Professor H. 0. Halvorson, University of Wisconsin, Madison, Wisconsin. A 20-mg sample of cell walls was treated with 5 ml of 0.005 M cysteine containing 0.1 ml of the snail juice. The mixture was incubated at 37” for 10 hours and the enzymes then inactivated by immersion of the tube of mixture in boiling water for 3 minutes. Resistant wall material was sedimented and washed twice. The enzymatically digested supernatant liquid and washings were pooled together and dialyzed for 24 hours in the cold against a known amount of distilled water, and the diffusible material was then analyzed. The glucose, N-acetyl hexosamine, and hexosamine values reported are from the determinations carried out on the enzymatically digested walls in which no destruction of the liberated sugars oc curred. Paper chromatogmphic analysis. Cell wall hydrolysates were subjected to descending chromatography on Whatman No. 1 filter paper in a variety of solvent systems. The solvent systems used in separation of amino acids were n-butanol :acetic acid: water (12:3:5 v/v), n-butanol : pyridine : water (1:l:l v/v), and phenol : ethanol : water : ammonia (12:4:4:0.1 v/v). Solvent systems (29) used in the separation of monosaccharides were n-butanol : acetic acid: water (12:3:5 v/v), isoamyl acetate:pyridine : water (3 : 3 : 0.9 v/v), and isopropanol : water (4:l v/v). Amino acids were located by using 0.2Tc isatin in acetone and 0.2% ninhydrin in acetone. Sugars were located with the alkaline silver nitrate reagent (30).
231
DERMATOPHYTES RESULTS
AND
DISCUSSION
The cell walls of the four dermatophyte species examined appear to be composed mainly of a glucose-containing polysaccharide, an N-acetyl glucosamine polymer which is probably chitin, and protein. The major fraction of hexosamine in acid hydrolysates was confirmed as glucosamine, and the N-acetyl hexosamine in the enzymatic digest as N-acetyl glucosamine, by chromatography in different solvent systems. Hexosamine and N-acetyl hexosamine determinations on enzymatically digested cell walls showed that practically all of the hexosamine existed in acetylated form (Table 1) ; this provides strong evidence for the presence of chitin. Blank (4) showed by X-ray diffraction and chemical analyses the presence of chitin alone, with no other polymeric substances in cell walls of fifteen species of dermatophytes. The problem, then, was not one of establishing chitin identity, but to determine its quantity and, if possible, how it was involved with other components in making up the wall structure. Our results reveal that chitin constitutes only a part of the total cell wall of these TABLE PRINCIPAL
I
COMPONENTS
I
OF CELL
WALLS
OF
DERMATOPHYTES Expressed
Constituent
M&5*0-
i
canis
J
-
45.9 7.8 28.1
Glucose Mannose N-acetyl hexosamine Hexosamine Protein Ash Lipid Nitrogen Total nucleic acid Total carbohydrates (anthrone direct
I ~po*u?n
1
.
as % of dry cell walls
-
0.6 7.1 4.7 3.1 3.8 0.35 55.5
37.5 11.4 26.6 1.0 6.8 8.2 4.0 3.4 0.38 48.3
36.6 10.3 31.2
45.8 6.7 29.7
0.8 8.0 7.6 3.6 3.9 0.32
0.3 7.4 5.9 3.3 3.5 0.31
45.9
56.6
lY) -
-
232
SHAH TABLE
AMINO
ACID
AND
II
COMPOSIION
OF
CELL
WALLS
OF
DERMATOPHYTES
-
Micromoles Amino
acid
amin;arsid/lOO
mg dry
cell
-
ITricho$hyton ,nenlagrophytes
Micro‘porum y$WWTZ
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine and Cysteic acid Valine Isoleucine Leucine Tyrosine Phenylalanine Methionine Ornithine Lysine Histidine Arginine Glucosamine Galactosamine a tr
= Trace
2.3 4.2 3.8 3.0 2.6 4.4 3.0 1.6
1.7 2.6 2.2 2.2 1.2 2.5 1.3 tra
2.1 4.1 3.6 2.4 3.2 3.6 2.3 ND”
2.2 4.2 3.9 2.8 4.1 4.2 2.8 0.9
3.3 1.0 7.0 3.1 3.4 tr 2.5 1.9 0.8 tra 80.9 2.2
1.7 0.6 5.7 1.4 2.3 0.7 1.8 1.1 0.4 trQ 75.0 1.7
1.2 0.7 1.5 2.0 2.7 0.8 1.2 0.8 1.0 tra 104.0 1.4
2.0 0.9 2.2 2.3 2.4 tra 3.5 1.7 0.9 tra 73.1 2.4
amounts,
ND
= Not
detectable.
dermatophytes. The preparation procedure used by Blank was so rigorous that little else than chitin could have survived. The results in Table 1 show that the variation in the N-acetyl hexosamine content of these walls is relatively small. Because of large amounts of glucosamine in the cell walls, it was not possible to separate quantitatively the galactosamine in different solvent systems. As evident from the ion-exchange chromatographic data (Table 2), galactosamine constitutes only 3% or less of the total These peaks accounted amino sugars. for the calculated value of less than 0.5:; of the dry weight of the cell walls. Whether the galactosamine is acetylated or not was not established. Aside from the amino sugars, the only monosaccharides detected by paper chro-
KNIGHT
matography in various solvent systems in different hydrolysates of the cell walls of the four species were glucose and mannose. The quantity of mannose was estimated from the chromatographic analysis of the formic acid hydrolysates. Earlier, various investigators reported the detection of glucose and mannose in the walls of other species of dermatophytes (17, 31, 32). However, Cummins and Harris (33) could not detect mannose in the cell walls of T. mentagrophytes and E. floecosum and found only a trace in the walls of M. gypseum. Also, they found only traces of glucose in the wall preparations of M. gypseum and T. mentagrophytes, and a relatively small amount of glucose in the walls of E. floccosum. Although our observations show that glucose is the predominant constituent of the cell walls of all the four species of dermatophytes examined, T. mentagrophytes and E. floecosum walls contain higher amounts of glucose as compared to M. canis and M. gypseum walls; the reverse is true with respect to mannose. When wall material is analyzed for total carbohydrate by the anthrone method, without prior hydrolysis of the walls, values are higher than those of any acid hydrolytic procedure. These data compare reasonably well, however, with those of determinations made after enzymatic digestion. A faint spot corresponding to galactose was observed in the chromatographic analysis of some of the hydrolysates of M. canis and M. gypseum. The amount present was so small that it could not be measured quantitatively. Pentoses or methyl pentoses could not be detected by paper chromatographic analysis of the hydrolysates. Since mannose was found in the mild acid hydrolysates (formic acid, 2 N HCl or 1 N HzS04), and repeating the mild hydrolysis did not release more mannose, it is evident that this sugar is easily liberated and completely removed from the polymer by mild acid hydrolysis. Extraction of hyphal walls by boiling water, and hydrolysis of this water-soluble material revealed the presence of mannose and _ ._glucase in the extracted polysaccharides.
HYPHAL
WALLS
OF
Since this mannose has not been isolated in polymerized form, and water-soluble mannose makes up a small proportion of the total mannose, the occurrence of mannose polymers is uncertain. The structure of the polysaccharide components of the walls is under further investigation. The amino acid composition of the cell walls is presented in Table 2. No effort has been made to correct for losses during hydrolysis. These results show that arginine is present only in trace amounts in the walls of these dermatophytes. Methionine content is high in the cell walls of M. canis and M. gypseum as compared to T. mentagrophytes and E. floccosum; while reverse is true for cysteine. The leutine content is very high in T. mentagrophytes and M. canis walls as compared to M. gypseum and E. floccosum walls. There are no major differences with respect to other amino acids. The total nitrogen and protein content of these cell walls reveal only minor differences. A relatively small proportion of the cell walls is readily extractable with fat solvents. The lipid in hyphae walls somehow may have been instrumental in conferring rigidity or protection against drying, since morphological distortion was observed after solvent extractions. Very little is known regarding the wall lipids of filamentous fungi. Small amounts of nucleic acid were found in the cell wall preparations which survived the treatment with ribonuclease. Subsequent treatment with ribonuclease was ineffective. Presence of nucleic acid was reported in the walls of Mucor rouxii (9) and Allomyces macrogynus (6). Whether the nucleic acid or nucleic acid derivatives extractable from the walls are associated with walls for some biological purposes is not known. Although relatively high values for the ash content of fungal walls are often reported, very little is known about the inorganic wall constituents. We found high levels of phosphorus, potassium and sodium in the walls of these dermatophyte species (Table 3). The T. mentagrophytes cell wall is low in calcium, as compared
233
DERMATOPHYTES TABLE MINERAL
III
COMPOSITION OF CELL DE~MATOPHYTES /
WALLS
OF
R,inera,
Values Calcium Phosphorus Potassium Sodium Magnesium
expressed
1
<0.05 0.22 0.1-l 0.20 0.026
values
expressed
Aluminum Barium Iron Strontium Boron Copper Zinc Manganese Chromium
39 82 1085
as $;, of drjj 0.23 0.17 0.22 0.32 0.012 as ppm
69 107 845
cell walls 0.15 1 O.lG 1 0.19 ’ 0.23 0.011
of di-y~ cell
37 73 670
0.14 0.19 0.12 0.29 0.010
wa/h -18 86 1200
to the remaining three species examined; while the reverse is true with respect to magnesium. Other minerals found in these dermatophyte cell walls are also reported. Bartnicki-Garcia and Nickerson found phosphorus, magnesium and calcium as the major elements in Mucor cell walls (9). How these inorganic constituents are linked to other wall structures is UIlkIlOWIl. ACKNOWLEDGMENTS One of the authors (V.K.S.) thanks Professor P. W. Wilson, Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, for his interest in this work in the later phase of this project, and also Professor H. 0. Halvorson, University of Wisconsin, Madison, Wisconsin for the gift of snail enzyme. REFERENCES 1. WETTSTEIN, F., Sitzber. Akad. Wiss. Wien. Math. Natuno. Kl., Abt. I, 130, 3 (1921). 10, 515 (1939). 2. NABEL, K., Arch. Mikrobiol. 3. NORMAN, A. G., AND PETERSON, W. H., Biochem. J. 26, 1946 (1932). 4. BLANK, F., Biochim. Biophys. Acta 10, 110 (1953). 5. BLANK, F., Can. J. Microbial. 1, l(l954).
234
SHAH
AND
6. ARONSON, J. M., AND MACHLIS, L., Am. J. Botany 46, 292 (1959). 7. PRINCE, H. N., J. Inuest. Dermatol. 35, 1 (1960). 8. KREGER, D. R., Biochim. Biophys. Acta 13, 1 (1954). 9. BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta 58,102 (1962). 10. HAMILTON, P. B., AND KNIGHT, S. G., Arch. Biothem. Biophys. 99,282 (1962). 11. CROOK, E. M., AND JOHNSTON, I. R., Biochem. J. 83, 325 (1962). 12. HORIKOSHI, K., AND IIDA, S., Biochim. biophys. Acta 83, 197 (1964). 13. JOHNSTON, I. R., Biochem. J. 96, 651 (1965). 14. BARTNICKI-GARCIA, S., AND REYES, E., Arch. Biothem. Biophys. 108,125 (1964). 15. APPLEGARTH, D. A., Arch. Biochem. Biophys. 120, 471 (1967). 16. NOVAES-LEDIEU, M., JIMENEZ-MARTINEZ, A., AND VILLANUEVA, J. R., J. Gen. Microbial. 47, 237 (1967). 17. CARLSON, D. G., PH.D. Thesis, University of Wisconsin (1966). 18. SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1945). 19. LOGAN, J. E., MANNELL, W. A., AND ROSSITER, R. J., Biochem. J. 51,480 (1952). 20. WILSON, P. W., AND KNIGHT, S. G., Experiments in bacterial physiology, Burgess Publishing Co., Minneapolis, Minn. (1952).
KNIGHT 21. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 22. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anulyt. Chem. 30, 1185 (1958). 23. SOMOGYI,M., J. Biol. Chem. 195, 19 (1952). 24. NELSON, N., J. Biol. Chem. 153,375 (1944). 25. TRACEY, M. V., Biochem. J. 47,433 (1950). 26. WINZLER, R. J., Methods Biochem. Analy. 2, 294 (1955). 27. DISCHE, Z., Methods Biochem. An&. 2, 342 (1955). 28. REISSIG, J. L., STROMINGER, J. L., AND LELOIR, L. F., J. Biol. Chem. 217, 959 (1955). 29. SMITH, I., Chromatographic and Electrophoretic Techniques, Vol. I: Chromatography, William Heinemann Medical Books, Ltd., London (1960). 30. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S., Nature 166,444 (1950). 31. MCNALL, E. G., in G. Dalldorf (ed.), “Fungi and Charles C Thomas, Fungous Diseases,” Springfield, IIlinois (1962). 32. BISHOP, C. T., BLANK, F., AND HRANISAVIJEVICJAKOVIJEVIC, M., Can. J. Chem. 40, 1816 (1962). 33. CUMMINS,
C. S., AND HARRIS, H., J. Gen. Microbiol. 18, 173 (1958).
OF BIOCHEMISTRY
Chemical
AND
BIOPHYSICS
Composition
127,
229-234
of Hyphal
V. K. SHAH’ Department
of Bacteriology,
Received
(1968)
March
of Dermatophytes’
S. G. KNIGHT’
AND
University
Walls
of
Wisconsin,
9, 1968; accepted
Madison, April
Wisconsin
53706
8, 1968
Quantitative analyses of the purified cell walls of Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum are described. The cell walls consist mainly of a glucose-containing polysaccharide, an N-acetyl glucosamine polymer which is probably chitin, mannan, and protein. In addition, small quantities of galactosamine and lipid have been found. Only minor diflerences could be observed among the walls of these four species. Perhaps the higher glucose and lower mannose contents in T. mentagrophytes and E. fEoccosum cell walls as compared to M. canis and M. gypseum cell walls are significant differences. These studies revealed that the dermatophyte cell walls contain more complex chemical structures than chitin alone.
Studies on the cell wall composition of the filamentous fungi are still rare in contrast to the large amount of work done on the cell walls of bacteria. Most of the earlier work on the fungal cell wall has been directed to the demonstration of chitin or cellulose, and use of these constituents as criteria in establishing taxonomical relationships (1, 2). Many investigators (3-7) have employed chemically prepared walls using procedures that generally involved alkaline digestion. Studies by different investigators (8-10) clearly showed that chemical methods of cell wall preparation remove certain wall constituents. A marked degree of complexity and diversity in fungal wall structure has become evident from the studies on the cell walls isolated by mechanical methods (6, 8, 10, 11). Substantially, however, relatively few fungal species have been examined (9-16) by modern isolation and fractionation procedures. This investigation was initiated to determine and compare the components and ’ This investigation was supported by grant AI01201.10 from the U. S. Public Health Service. ’ Present address: Department of Microbiology Faculty of Science, M. S. University of Baroda, Baroda, India. ’ Deceased November 14, 1966.
structure of the hyphal cell walls of Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum with a view that this information may lead eventually to a better understanding of the physiological significance of cell walls and the action of some antifungal drugs. EXPERIMENTAL
PROCEDURE
Organisms and growth conditions. Stock cultures of T. mentagrophytes, M. canis, M. gypseum and E. floccosum were maintained on slants of Sabouraud’s glucose agar, composed of 4’( glucose, l’( Neopeptone (Difco), and 1.5’~ agar. Conidial suspensions for the inoculation of all cultures were prepared from Roux bottle cultures on Sabouraud’s glucose agar. Growth on this medium in Roux bottles was harvested in 50 ml of sterile distilled water by scraping the mycelial surface. The conidial suspension was filtered through sterile glass wool to remove hyphae, debris and clumps. One-liter Erlenmeyer flasks containing 250 ml of Sabouraud’s liquid medium, composed of 4’, glucose and l’, Neopeptone, were inoculated with 10 ml of the conidial suspensions and incubated at 30° on a New Brunswick rotary shaker. The mycelia were harvested after SO-72 hours incubation by suction filtration through a layer of cheese cloth. The mycelial mass was washed with distilled water until the filtrate was colorless. Preparation of cell walls. Cell walls were prepared by the ballistic disruption method essentially
230
SHAH
AND
that of Carlson (17). Glass-distilled water was used for subsequent work as ordinary distilled water was found to affect the color of the preparation during breaking cycles. The mycelial mass was mixed with an equal volume of acid-washed Superbrite glass beads, about 100 p in diameter (Minnesota Mining and Manufacturing Co., St. Paul, Minn.), and two volumes of cold 0.5 M NaCl in a 400 ml stainless steel container of the Sorvall Omni-Mixer. The container was lowered into an ice water bath and the contents homogenized at the maximum attainable speed for 10 minutes. The homogenate was decanted to separate the bulk of the glass beads, and then centrifuged briefly at low speed to sediment the cells, walls and residual beads. The solubilized cytoplasmic debris was discarded with the supernatant liquid. The pellet was washed with 0.1 M NaCl at room temperature and sedimented again by centrifugation. The resulting pellet was resuspended in the same volume of cold 0.5 M NaCl and glass beads, and the breaking and washing cycles repeated thrice. Breakage efficiency was judged by phase contrast and light microscopy. A cytoplasmic stain, la&phenol cotton blue, was used to enhance differentiation of empty cell walls and intact cells. The wall material was separated from the glass beads by repeatedly resuspending and carefully withdrawing the upper half of the sediment. The final preparations were virtually free of glass beads and unbroken cells. Purification of cell walls. Cell wall material was then washed five times with ten volumes of 1 M NaCl, and five times with distilled water. The crude cell wall preparations were digested with crystalline trypsin (Worthington Biochemical Corp., Freehold, N. J .) , and ribonuclease (Calbiochem, Los Angeles, Calif.). The walls were suspended in 0.05 M phosphate buffer, pH 8.0, containing 0.5 mg/ml trypsin and O.OlY; merthiolate. After 8 hours incubation at 37O, the walls were washed thrice with distilled water, suspended in 0.05 M tris(hydroxymethyl)aminomethane and 0.005 M MgCln buffer, pH 7.4, containing 0.5 mg/ml ribonuclease. After digestion for 3 hours at 37”, the cell wall residue was washed by centrifugation twice with 0.1 M NaCl and five times with distilled water. The cell wall preparations were extracted with cold ethanol, twice with ethanol-ethyl ether (1: 3 v/v) mixture at room temperature, and ethyl ether at room temperature. The combined solvent fraction was concentrated in uacuo, the residual material extracted twice with ethyl ether, and the ether extract dried and weighed. The cell wall material, after enzymatic digestion and solvent extraction, was dried, weighed, and stored in a desiccator for subsequent analyses. Dry weight loss upon heating at 105” for 24 hours was
KNIGHT determined, and all data have been corrected for this loss. The final product was a fluffy, white to gray material, and the results reported are based on analyses of this material. Methods of cell wall analysis. For the analysis of amino acids and amino sugars, cell walls were routinely hydrolyzed in sealed tubes with five volumes of 6 N hydrochloric acid at 100-105” for 12 hours. Undissolved material was separated by centrifugation, washed twice, and the washings added to the hydrolysate. HCl was removed by repeated evapomtion in uacuo at 40” over NaOH pellets. For the analysis of sugars, cell walls were hydrolyzed with formic, sulfuric, or hydrochloric acid. Hydrolysis with formic acid involved heating at 105’ in sealed tubes with 5 ml of 76? (v/v) formic acid for 12-18 hours. The soluble fraction was evaporated almost to dryness under reduced pressure and the formyl esters removed by heating the dried supematant liquid fraction with 2 ml of I N H2SOa for one hour at 105”. The sulfuric acid was neutralized with Ba(OH),, and the BaS04 removed by filtration. The hydrolysate was deionized by passing it through a small column of Amerlite IR-120 (hydrogen form); it was then evaporated to dryness to 40” under reduced pressure. For sulfuric acid hydrolysis, cell wall material was sealed in a tube with 5 ml of 1 N HSSO,, and placed in an oven at 105” for 24 hours. The supernatant liquid fractions containing the soluble wall components were neutralized with Ba(OH), and dried as described above. To minimize destruction of the components, sequential hydrolysis and fractionation was frequently used. The fraction liberated by mild hydrolysis, usually with 2 N HCl for 1-8 hours at 80-105”, was divided and half of it was further hydrolyzed before analysis. The wall material resistant to solution in 2 N HCl was resuspended in 2 or 6 N HCl and hydrolysis continued for 3 hours at 100”. HCI was removed as reported above. Ash analysis. Dried cell walls were weighted into dried, tared crucibles of silica, heated at 500” for 8 hours, and cooled in a desiccator over calcium chloride before weighing. These ash samples were used for the mineral estimations made with a Jarrell-Ash direct reading spectrometer. Nucleic acid determination. Extraction of nucleic acid with hot 5’, trichlomacetic acid was based on the procedure described by Schneider (18). Total nucleic acids were determined by measuring the optical density of the trichloroacetic acid extract at 268.5 rnr in a Beckman Model DU spectrophotometer (19). Total nitrogen was determined with Nessler’s reagent after digesting the cell walls by the method of Wilson and Knight (20). Protein was measured
HYPHAL
WALLS
OF
by the method of Lowry et al. (21), performed on the extracts of the walls prepared by treatment with 2 N NaOh at 100” for 30 minutes. Individual amino acids were separated quantitatively and measured on a Beckman-Spinco automatic amino acid analyzer with ninhydrin (22). Reducing sugars in the cell wall fractions were measured by the method of Somogyi (23) using the arsenomolybdate color reagent of Nelson (24) and a glucose standard. Pentose, methyl pentoses, and uranic acids were sought calorimetrically in the hydrolysates by the methods of Tracey (25), Winzler (26), and Dische (27), respectively. Hexosamine determinations were made on the acid and enzymatic hydrolysates of the cell wall material by the modification (28) of the Elson-Morgan reaction, and Nacetyl hexosamines were measured with the acetylating step omitted. Glucosamine and N-acetyl glucosamine were used as standards. Glucose was assayed with the glucose oxidase reagent of Worthington Biochemical Corp., Freehold, N. J. Snail enzyme digestion of cell walls. Snail enzyme made from the gut juice of Helix pomatiu (Endo Laboratories Inc., Garden City, New York) was a gift from Professor H. 0. Halvorson, University of Wisconsin, Madison, Wisconsin. A 20-mg sample of cell walls was treated with 5 ml of 0.005 M cysteine containing 0.1 ml of the snail juice. The mixture was incubated at 37” for 10 hours and the enzymes then inactivated by immersion of the tube of mixture in boiling water for 3 minutes. Resistant wall material was sedimented and washed twice. The enzymatically digested supernatant liquid and washings were pooled together and dialyzed for 24 hours in the cold against a known amount of distilled water, and the diffusible material was then analyzed. The glucose, N-acetyl hexosamine, and hexosamine values reported are from the determinations carried out on the enzymatically digested walls in which no destruction of the liberated sugars oc curred. Paper chromatogmphic analysis. Cell wall hydrolysates were subjected to descending chromatography on Whatman No. 1 filter paper in a variety of solvent systems. The solvent systems used in separation of amino acids were n-butanol :acetic acid: water (12:3:5 v/v), n-butanol : pyridine : water (1:l:l v/v), and phenol : ethanol : water : ammonia (12:4:4:0.1 v/v). Solvent systems (29) used in the separation of monosaccharides were n-butanol : acetic acid: water (12:3:5 v/v), isoamyl acetate:pyridine : water (3 : 3 : 0.9 v/v), and isopropanol : water (4:l v/v). Amino acids were located by using 0.2Tc isatin in acetone and 0.2% ninhydrin in acetone. Sugars were located with the alkaline silver nitrate reagent (30).
231
DERMATOPHYTES RESULTS
AND
DISCUSSION
The cell walls of the four dermatophyte species examined appear to be composed mainly of a glucose-containing polysaccharide, an N-acetyl glucosamine polymer which is probably chitin, and protein. The major fraction of hexosamine in acid hydrolysates was confirmed as glucosamine, and the N-acetyl hexosamine in the enzymatic digest as N-acetyl glucosamine, by chromatography in different solvent systems. Hexosamine and N-acetyl hexosamine determinations on enzymatically digested cell walls showed that practically all of the hexosamine existed in acetylated form (Table 1) ; this provides strong evidence for the presence of chitin. Blank (4) showed by X-ray diffraction and chemical analyses the presence of chitin alone, with no other polymeric substances in cell walls of fifteen species of dermatophytes. The problem, then, was not one of establishing chitin identity, but to determine its quantity and, if possible, how it was involved with other components in making up the wall structure. Our results reveal that chitin constitutes only a part of the total cell wall of these TABLE PRINCIPAL
I
COMPONENTS
I
OF CELL
WALLS
OF
DERMATOPHYTES Expressed
Constituent
M&5*0-
i
canis
J
-
45.9 7.8 28.1
Glucose Mannose N-acetyl hexosamine Hexosamine Protein Ash Lipid Nitrogen Total nucleic acid Total carbohydrates (anthrone direct
I ~po*u?n
1
.
as % of dry cell walls
-
0.6 7.1 4.7 3.1 3.8 0.35 55.5
37.5 11.4 26.6 1.0 6.8 8.2 4.0 3.4 0.38 48.3
36.6 10.3 31.2
45.8 6.7 29.7
0.8 8.0 7.6 3.6 3.9 0.32
0.3 7.4 5.9 3.3 3.5 0.31
45.9
56.6
lY) -
-
232
SHAH TABLE
AMINO
ACID
AND
II
COMPOSIION
OF
CELL
WALLS
OF
DERMATOPHYTES
-
Micromoles Amino
acid
amin;arsid/lOO
mg dry
cell
-
ITricho$hyton ,nenlagrophytes
Micro‘porum y$WWTZ
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine and Cysteic acid Valine Isoleucine Leucine Tyrosine Phenylalanine Methionine Ornithine Lysine Histidine Arginine Glucosamine Galactosamine a tr
= Trace
2.3 4.2 3.8 3.0 2.6 4.4 3.0 1.6
1.7 2.6 2.2 2.2 1.2 2.5 1.3 tra
2.1 4.1 3.6 2.4 3.2 3.6 2.3 ND”
2.2 4.2 3.9 2.8 4.1 4.2 2.8 0.9
3.3 1.0 7.0 3.1 3.4 tr 2.5 1.9 0.8 tra 80.9 2.2
1.7 0.6 5.7 1.4 2.3 0.7 1.8 1.1 0.4 trQ 75.0 1.7
1.2 0.7 1.5 2.0 2.7 0.8 1.2 0.8 1.0 tra 104.0 1.4
2.0 0.9 2.2 2.3 2.4 tra 3.5 1.7 0.9 tra 73.1 2.4
amounts,
ND
= Not
detectable.
dermatophytes. The preparation procedure used by Blank was so rigorous that little else than chitin could have survived. The results in Table 1 show that the variation in the N-acetyl hexosamine content of these walls is relatively small. Because of large amounts of glucosamine in the cell walls, it was not possible to separate quantitatively the galactosamine in different solvent systems. As evident from the ion-exchange chromatographic data (Table 2), galactosamine constitutes only 3% or less of the total These peaks accounted amino sugars. for the calculated value of less than 0.5:; of the dry weight of the cell walls. Whether the galactosamine is acetylated or not was not established. Aside from the amino sugars, the only monosaccharides detected by paper chro-
KNIGHT
matography in various solvent systems in different hydrolysates of the cell walls of the four species were glucose and mannose. The quantity of mannose was estimated from the chromatographic analysis of the formic acid hydrolysates. Earlier, various investigators reported the detection of glucose and mannose in the walls of other species of dermatophytes (17, 31, 32). However, Cummins and Harris (33) could not detect mannose in the cell walls of T. mentagrophytes and E. floecosum and found only a trace in the walls of M. gypseum. Also, they found only traces of glucose in the wall preparations of M. gypseum and T. mentagrophytes, and a relatively small amount of glucose in the walls of E. floccosum. Although our observations show that glucose is the predominant constituent of the cell walls of all the four species of dermatophytes examined, T. mentagrophytes and E. floecosum walls contain higher amounts of glucose as compared to M. canis and M. gypseum walls; the reverse is true with respect to mannose. When wall material is analyzed for total carbohydrate by the anthrone method, without prior hydrolysis of the walls, values are higher than those of any acid hydrolytic procedure. These data compare reasonably well, however, with those of determinations made after enzymatic digestion. A faint spot corresponding to galactose was observed in the chromatographic analysis of some of the hydrolysates of M. canis and M. gypseum. The amount present was so small that it could not be measured quantitatively. Pentoses or methyl pentoses could not be detected by paper chromatographic analysis of the hydrolysates. Since mannose was found in the mild acid hydrolysates (formic acid, 2 N HCl or 1 N HzS04), and repeating the mild hydrolysis did not release more mannose, it is evident that this sugar is easily liberated and completely removed from the polymer by mild acid hydrolysis. Extraction of hyphal walls by boiling water, and hydrolysis of this water-soluble material revealed the presence of mannose and _ ._glucase in the extracted polysaccharides.
HYPHAL
WALLS
OF
Since this mannose has not been isolated in polymerized form, and water-soluble mannose makes up a small proportion of the total mannose, the occurrence of mannose polymers is uncertain. The structure of the polysaccharide components of the walls is under further investigation. The amino acid composition of the cell walls is presented in Table 2. No effort has been made to correct for losses during hydrolysis. These results show that arginine is present only in trace amounts in the walls of these dermatophytes. Methionine content is high in the cell walls of M. canis and M. gypseum as compared to T. mentagrophytes and E. floccosum; while reverse is true for cysteine. The leutine content is very high in T. mentagrophytes and M. canis walls as compared to M. gypseum and E. floccosum walls. There are no major differences with respect to other amino acids. The total nitrogen and protein content of these cell walls reveal only minor differences. A relatively small proportion of the cell walls is readily extractable with fat solvents. The lipid in hyphae walls somehow may have been instrumental in conferring rigidity or protection against drying, since morphological distortion was observed after solvent extractions. Very little is known regarding the wall lipids of filamentous fungi. Small amounts of nucleic acid were found in the cell wall preparations which survived the treatment with ribonuclease. Subsequent treatment with ribonuclease was ineffective. Presence of nucleic acid was reported in the walls of Mucor rouxii (9) and Allomyces macrogynus (6). Whether the nucleic acid or nucleic acid derivatives extractable from the walls are associated with walls for some biological purposes is not known. Although relatively high values for the ash content of fungal walls are often reported, very little is known about the inorganic wall constituents. We found high levels of phosphorus, potassium and sodium in the walls of these dermatophyte species (Table 3). The T. mentagrophytes cell wall is low in calcium, as compared
233
DERMATOPHYTES TABLE MINERAL
III
COMPOSITION OF CELL DE~MATOPHYTES /
WALLS
OF
R,inera,
Values Calcium Phosphorus Potassium Sodium Magnesium
expressed
1
<0.05 0.22 0.1-l 0.20 0.026
values
expressed
Aluminum Barium Iron Strontium Boron Copper Zinc Manganese Chromium
39 82 1085
as $;, of drjj 0.23 0.17 0.22 0.32 0.012 as ppm
69 107 845
cell walls 0.15 1 O.lG 1 0.19 ’ 0.23 0.011
of di-y~ cell
37 73 670
0.14 0.19 0.12 0.29 0.010
wa/h -18 86 1200
to the remaining three species examined; while the reverse is true with respect to magnesium. Other minerals found in these dermatophyte cell walls are also reported. Bartnicki-Garcia and Nickerson found phosphorus, magnesium and calcium as the major elements in Mucor cell walls (9). How these inorganic constituents are linked to other wall structures is UIlkIlOWIl. ACKNOWLEDGMENTS One of the authors (V.K.S.) thanks Professor P. W. Wilson, Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, for his interest in this work in the later phase of this project, and also Professor H. 0. Halvorson, University of Wisconsin, Madison, Wisconsin for the gift of snail enzyme. REFERENCES 1. WETTSTEIN, F., Sitzber. Akad. Wiss. Wien. Math. Natuno. Kl., Abt. I, 130, 3 (1921). 10, 515 (1939). 2. NABEL, K., Arch. Mikrobiol. 3. NORMAN, A. G., AND PETERSON, W. H., Biochem. J. 26, 1946 (1932). 4. BLANK, F., Biochim. Biophys. Acta 10, 110 (1953). 5. BLANK, F., Can. J. Microbial. 1, l(l954).
234
SHAH
AND
6. ARONSON, J. M., AND MACHLIS, L., Am. J. Botany 46, 292 (1959). 7. PRINCE, H. N., J. Inuest. Dermatol. 35, 1 (1960). 8. KREGER, D. R., Biochim. Biophys. Acta 13, 1 (1954). 9. BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta 58,102 (1962). 10. HAMILTON, P. B., AND KNIGHT, S. G., Arch. Biothem. Biophys. 99,282 (1962). 11. CROOK, E. M., AND JOHNSTON, I. R., Biochem. J. 83, 325 (1962). 12. HORIKOSHI, K., AND IIDA, S., Biochim. biophys. Acta 83, 197 (1964). 13. JOHNSTON, I. R., Biochem. J. 96, 651 (1965). 14. BARTNICKI-GARCIA, S., AND REYES, E., Arch. Biothem. Biophys. 108,125 (1964). 15. APPLEGARTH, D. A., Arch. Biochem. Biophys. 120, 471 (1967). 16. NOVAES-LEDIEU, M., JIMENEZ-MARTINEZ, A., AND VILLANUEVA, J. R., J. Gen. Microbial. 47, 237 (1967). 17. CARLSON, D. G., PH.D. Thesis, University of Wisconsin (1966). 18. SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1945). 19. LOGAN, J. E., MANNELL, W. A., AND ROSSITER, R. J., Biochem. J. 51,480 (1952). 20. WILSON, P. W., AND KNIGHT, S. G., Experiments in bacterial physiology, Burgess Publishing Co., Minneapolis, Minn. (1952).
KNIGHT 21. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 22. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anulyt. Chem. 30, 1185 (1958). 23. SOMOGYI,M., J. Biol. Chem. 195, 19 (1952). 24. NELSON, N., J. Biol. Chem. 153,375 (1944). 25. TRACEY, M. V., Biochem. J. 47,433 (1950). 26. WINZLER, R. J., Methods Biochem. Analy. 2, 294 (1955). 27. DISCHE, Z., Methods Biochem. An&. 2, 342 (1955). 28. REISSIG, J. L., STROMINGER, J. L., AND LELOIR, L. F., J. Biol. Chem. 217, 959 (1955). 29. SMITH, I., Chromatographic and Electrophoretic Techniques, Vol. I: Chromatography, William Heinemann Medical Books, Ltd., London (1960). 30. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S., Nature 166,444 (1950). 31. MCNALL, E. G., in G. Dalldorf (ed.), “Fungi and Charles C Thomas, Fungous Diseases,” Springfield, IIlinois (1962). 32. BISHOP, C. T., BLANK, F., AND HRANISAVIJEVICJAKOVIJEVIC, M., Can. J. Chem. 40, 1816 (1962). 33. CUMMINS,
C. S., AND HARRIS, H., J. Gen. Microbiol. 18, 173 (1958).