Pray Ch,
For.\ orkrr L,p,r,\ Vol. 16. pp 171-177 Pcry;,mon Press. IY7X. PrInted ,n Greal Bnh,n
DERMATOPHYTE JAN
LIPIDS
VINCENT
CONTENTS 171
1. INTKOULICTION II.
171
TOTAL
LIPIDS
IN DEKMATOPHYTES
III.
FATTY
Arm
Iv.
STEROLS
175
173
PHOSPHOLIPIDS
176
ACKNOWLEI)GEMENTS
176
RFFERENCES
176
V.
I. INTRODUCTION
This review concerns the distribution of dermatophyte lipids, their possible physiological role and their importance in pathogenesis of dermatophytosis. The text is divided into two parts: the first part covers the increasing amount of information regarding the total lipid composition of dermatophytes with special reference to the various factors influencing the lipid content; the second part covers the three major lipid classes occurring in dermatophytes, i.e. fatty acids, sterols and phospholipids. Although lipids were among the first natural products investigated,‘O they were neglected until the last decades when sophisticated analytical techniques became available. This has, of course, also stimulated more extensive investigations on dermatophyte lipids. Dermatophytes are a highly specialized group of filamentous fungi, causing lesions of the skin and its appendages in man and animals. Dermatophytes, according to their imperfect forms. include species of three genera: Epidermophyton, Microsporum and Trichoph~ton (Fungi imperfecti). All known perfect forms belong to genera Arthroderma and Nannizicr (Ascomycetes).” Beside the chemotaxonomical implications, this review may provide useful information for further investigations concerning the role that dermatophyte lipids play in the inflammatory skin reactions in dermatophytosis. II.
TOTAL
LIPIDS
IN
DERMATOPHYTES
Even before dermatophyte lipids were known, their lipolytic activity was described.sS2* The use of classical staining methods first demonstrated the presence of lipids in dermatophytes. ’ *9 Later, mycelial extractions were carried out for evaluation of total lipids in order to elucidate their relationship in the pathogenesis of dermatophytosis 2~3,17~21.22~3h~41~44.51 The total lipid abundances in dermatophytes are summarized in Table I. Regarding the relatively large range of abundances, it is apparent from the table that consideration must be given to growth conditions, age and the analytical techniques. Dermatophytes demonstrate a relatively low growth activity when compared with the majority of saprophytic molds. 4o For example, the mycelia of E. JIoccosum contain total lipids as a function of age, in the range 7&19.8% of the dry weight, which is also true for most dermatophytes studied. The total lipids of E. Joccosum significantly increase with age, except during the early growth period when there is a decrease, mainly at the expense of the triglycerides.41 Thus, the energy requirements of the developing mycelia would appear to be met, at least in part, by fatty acid oxidation. The 171
Jan Vincent
172 TABLE
Genus
.
Epidermophyton Microsporum
Trichophyton
Reported
1. Total Lipids of Dermatophytes Species
j?occosum jloccosum audouinii canis canis gypseum ajel/oi* ajelloi (mutant) gallinae nGgninii** mentagrophytes mentagrophytes mentagrophytes mentagrophytes*** rybrum rubrum rubrum schb;nleinii schiinleinii tonsurans uerrucosum**** violaceum violaceum
Total lipid (“, dry wt.)
Reference
7.0-19.8” 14.77b 14.63b 10.81b 26.77” 13.57h
41 3 3 2 3 3
15.5’
22
20.5’ 12.98” 8.7’ 15.3J 18.13h 8.2’ 18.8’ 18.49h 11.2’ 7.7’ I 9.48h 8.3’ 10.85” 43.7’ 10.lOh 16.5’
22 3 51 36 3 17 51 3 17 44 3 17 3 51 3 51
uncinatum (stat. ascosp.); **T. rosaceum; ***T. asteroides; ****T. fauiforme. ochraceum. Growth conditions: “Sabouraud dextrose broth. 30°C; kparagine liquid medium. 20°C; ‘Sabouraud dextrose broth. 20°C; ‘dextrose (1%) and peptone (l:,,,) broth, 20°C; ‘Griitz-III-medium, 20°C. as: ‘Arthroderma
gypseum,
lipid accumulation in older mycelia as a store of energy is confirmed also by cytological studies.47 The decrease of total lipids in E. ~OCCOSU~during germination and in the early phase of growth may be a consequence of their utilization as a prime carbon source and substrate for polysaccharide synthesis.” Environmental conditions such as substrate, temperature, agitation, etc. have been recognized to have a particular influence on the total lipid content of dermatophytes. Lipid production of dermatophytes, similar to the majority of filamentous fungi, depends on the nature of the carbon source. In this connection, the richest carbohydrates are, in descending order, glucose, sucrose and fructose. Also amino acids, present in certain concentrations, such as alanine or phenylalanine, can affect the total lipid content3’ It is generally agreed that a relatively high carbon-nitrogen ratio in the media is required in order to attain maximum lipid production. In common with most living organisms, dermatophytes show a tendency to enhance lipid content with increasing environmental temperature (unpublished data). Thus, investigations should be carried out on the species grown under optimal temperatures. Moreover, some microorganisms subcultured at low temperatures show a relatively high degree of unsaturation. l3 This alters some physical properties of the lipids, which can be of importance for the survival of the dermatophytes in unfavorable environments. Aeration of culture has also a pronounced effect on the general metabolism of fungi and their lipid composition. The degree of aeration required depends on the technique applied, e.g. semisolid media, shake or still cultures. Thus, significant differences in the amount of total lipids in still and shake cultures have been reported.3 Early stages of germination are characterized by a dependency on the endogenous substrate. It has been suggested l2 that the macroconidia of M. gypseum are capable of immediate response to a favorable environment and therefore constitute an ideal means for the transmittance of infection. The longevity of the macroconidia may depend more on a high concentration of essential materials in the spore rather than any well
Dermatophyte TABLE 2. Dermatophyte
Genus Epidermophyton
173
lipids Cell Wall
Cell wall lipid (% dry wt.1
Species jloccosum
j7occowm Microsporum Trichophyton
Lipids
canis gypseum mentagrophyres mentagrophytes
Reference
3.3 4.6
37 33
4.0 3.6
37 37 37 32
3.1 6.6
developed dormancy mechanisms. This confirms the changes of dermatophyte M. quinceanum during starvation which indicates that the carbohydrates do not form the principal endogenous reserve. In this case, lipids may represent a primary reserve material because they decrease rapidly from 23% in the spores to 13% in a 24 hr old mycelium. The same picture was true during starvation.40 Cell walls from filamentous fungi, particularly dermatophytes, have been extensively studied.32*33*37 However, relatively little is known about their lipid composition. Lipid abundancies of dermatophyte cell walls are given in Table 2. Shah and Knight”’ showed that these lipids may be of importance in conferring rigidity or protection against drying, since morphological distortion was observed after solvent extraction. The function of dermatophyte lipids is as yet not well understood. Historically, lipids have been considered as reserve material which may be converted to energy and carbon skeletons during growth and reproduction. This is true for those lipids which accumulate in cytoplasma that are composed primarily of triglycerides. However, as we know more about the structure and biosynthesis of less abundant lipids, it is apparent that they have more specific roles to play. For example, lipids are essential components in the membrane structures when they may serve as protective coatings or may be involved in the immunological reactions in dermatophytosis. According to Peck,35 lipids of pathogenic fungi have a significant role in the mechanism of infectious disease. Moreover, interest in dermatophyte metabolites leads to the detection of specific antigenic components extracted from their mycelia. Lipid fractions, especially fatty acids, have been reported to possess antigenic activity.4*‘8 . III.
FATTY
ACIDS
Fungal fatty acids and their biosynthesis have been extensively reviewed.20*38 Generally, dermatophyte fatty acids consist of a homologous series of saturated and unsaturated aliphatic acids ranging from C6 to Cz2 with a predominance of Cl6 and Cis acids. These acids can account for over 80% of the total fatty acid content. The ethylenic bonds in the unsaturated acids are in the cis configuration and, where more than one ethylenic bond is present, they occur normally in methylene-interrupted sequence. Fatty acid distribution in some species of dermatophytes from the genus Trichophyand Microsporum to11 5923.29--31.42,4*-51 43 has been determined mainly by paper chromatography. The fatty acid composition of the remaining monotype genus Epidermophyton was established by combined gas chromatography-mass spectrometry in this laboratory.41 The distribution of fatty acids encountered in dermatophytes is given in Table 3. For instance, fatty acids of E. Jlocc~~um decrease significantly with age, which may be due partly to their conversion to glycerides and partly to their complete degradation. However, the composition of the predominating fatty acids was unaffected by age.41 The narrow range of dermatophyte fatty acids confirms that they compose a homogenous group of filamentous fungi. Recently, the enzymatic system responsible for fatty acid synthesis in dermatophyte was investigated.24 It has been postulated that an equilibrium between malonyl CoA (the intermediate in de nouo synthesis) and acetyl CoA
(41)
‘T.
reported as: ‘T. gypseum;
WIRTH (43)
schiinleinii MERKEL (30)3 tonsurans ZAMIECH. (48)4 ZAMIECH. (49)s quincheanum MEXKEL (31)6 migninii ZAMIECH. (SO)’ ZAMIECH. (51)8 verrucosum ZAMIECH. (51)9 violaceum ZAhSECH. (51) rubrum WIRTH (42) KOSTIW (23) gypseum
j7occosum VINCENT
Epidermophyton
Microsporum
mentagrophytes
Trichophyton
Analyzed with
GC
’ PC
gypseum,
GC-MS
i
j
’
MERKEL (29)’ PC ZAMIECH (5 1)’ 1 AIJDETTE(5) GC
Species
Genus
9
10
0.1
tr 0.3
1.1 0.5 0.7
0.1
8
asteroides; 3Achorion;
tr
6
“T.
0.2 plicatile;
1.7 0.3 5T. sulphureum;
1.4
6Achorion;
2.7 0.2
‘T.
18.9
17.2
23.8 17.6
X
x
0.8 0.7
0.7 rosaceum;
2.3
9.6
7.7
7.4 16.1
X
X
*T. rosaceum;
tr
X
x
42.8
64.7
52.4 35.6
‘T. faviforme,
16.2
10.4
13.1 19.2
X
X
X
x
X
X
X
x
X X
18:2
1710 45.2
X X
1;12
x X
2.6
1.0
X
18:l
X
X
tr.
0.2
X
18
x
218
x
Fatty acids 16 16:l 17 17:l
X
0.8 2.1 0.8 0.1 0.5
0.9
15
X X
1.0
0.3
14 14:l
X
OYl
13:l
3. Fatty Acids of Dermatophytes4’
12 13
0.2 0.1 tr
11
TABLE
1.1
1.1
20
0.5
0.3
2O:l
ochraceum.
0.9
19
0.1
0.2
20:2
tr.
21
tr. 0.8
22
Dermatophyte lipids
175
(the intermediate in chain elongation) may provide the site for the mechanism controlling these two processes. The taxonomical value of fungal fatty acids is limited.3* However, as Phycomycetes are characterized by a predominant occurrence of y-linolenic acid, its absence in dermatophytes is of taxonomical significance, which thus confirms the relevance of dermatophytes to Ascomycetes. Moreover, the fatty acid composition may be of importance in membrane biological activity6 and may be involved in the allergic inflammatory delayed skin reactions in dermatophytosis.4 The Landstainer-Draise test of these compounds confirms that the middle chain (C,a-CiJ fatty acids are able to sensitize the mammalian skin and elicit the delayed inflammatory skin reactions.18 IV. STEROLS
The presence of ergosterol in dermatophytes, such as 27 mentagrophytes,’ T. mentagrophytes (asteroides)34 and T. rubrum, was primarily reported. Brassicasterol and a trace of squalene have been isolated from T. rubrum. 44 The occurrence of squalene in trace quantities in the mycelia of T. rubrum is consistent with its known position in the biosynthetic pathway between acetate and sterols, and with the fact that squalene does not normally accumulate, to a significant extent, under aerobic conditions. Subsequently, Blank ef al.’ identified two sterols, brassicasterol and ergosterol, from different species of dermatophytes. Ergosterol was found to be more widely distributed than brassicasterol. These sterols occurred together in some species but the second was only present in very small amounts. In a number of species, ergosterol was present to the exclusion of the other. The distribution of these two sterols among the dermatophytes is rather interesting from the taxonomic point of view (Table 4). Although there are several reports on the subject, the exact function of sterols in fungi is uncertain.” The sterol content of E. fIoccosum reaches a maximum during the early growth period, 41 but its relationship with an eventual germination control or cell permeability remains open for investigation.16 Summarizing, sterols are required for growth and reproduction as well as being implicated in membrane structures and permeability. Relationships have been established between the sterols of fungal membranes and the action of polyene antifungal antibiotics. 15*46 For instance, the effect of nystatin is probably due to its insertion among the sterols of the lipid bilayer, leading to molecular rearrangements, loss in rigidity and mechanical failure of the membrane.26 TABLE 4. Dermatophyte
Genus
Sterols (% dry 4 Brassicasterol Ergosterol
Species
Epidermophyton Microsporum
Sterols’
-
jloccowm audouinii canis
Reported discoides.
*T.
granulosum;
**T.
0.026 0.270 0.250 -
-
quinckeanum ferrugineum mbgninii mentagrophytes* mentagrophytes** mentagrophytes*** rubrum tonsurans verrucosum**** violaceum as:
0.150 0.016
-
gweum Trichophyton
0.083
0.039 -
0.130 1.50 0.280 0.056
0.126 -
0.010 0.015 persicolor;
***T.
interdigitale,
****T.
17b
Jan Vincent TABLE 5. Phospholipids Genus
Trichophyton
Reported
Phospholipids (% dry wt.1
Species
l
Epidermophyton Microsporum
in Dermatophytes
jloccosum audouinii
6.96 5.02
conis
0.048
canis
4.48 4.04 2.6 5 4.97
gweum ajelloi* ajelloi (mutant) gallinae mentagrophytes mentagrophytes rubrum schijnleinii tonsurans violaceum as: *Arthroderma
uncinatum
3.13 0.3 4.87 3.59
1.33 2.58
Reference 3 3 2 3 3 22 22 3 3 36 3 3 3 3
(stat. ascosp.)
V. PHOSPHOLIPIDS
Phospholipids are considered to be involved mainly in membrane functions. Their composition in dermatophytes (Table 5) have been, until recently, rather poorly investigated.2*3v36 Ghoshal l4 found 41% phosphatidylcholine and some sphingomyelin in T. rubrum, but the presence of ‘other phospholipids could not be confirmed. However, in M. cookei, five major components as phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylinositol were identified.25 The phospholipid composition of a mutant strain of the dermatophyte Arthrodermo uncinatum (stat. ascosp. of T. ajelloi) was compared with that of the wild type.22 Both strains contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diphosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, but important differences in the amounts of phosphatidylcholine, phosphatidylserine, and phosphatidic acid were observed. In both strains, the predominant fatty acid was 18:2 with 54.0% in the wild type and 46.7% in the mutant. Qualitatively, the same fatty acids, with the exception of the CzO acids, were found in all phospholipid classes. T. rubrum contains phosphatidylinositol, polyphosphatidylinocitol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid.” The relative proportions of these components in total phospholipid fraction remain unchanged with age. Acknowledgements-1 would like to express my gratitude to Professor Sixten Abrahamsson, Associate Professor Ng. Dinh-Nguyen and Assistant Professor Lars Hellgren for their valuable advice, and to Mr Weston Pimlott for reviewing the text. Special thanks go also to Mrs Greta Sonnhagen for her contributions in the preparation of the entire manuscript. The study was supported by the Swedish Medical Research Council.
REFERENCES 1. AKASAKA, T. Jpn. Dermatol. 63. 471 (1953). 2. AL-DCORY, Y. J. Bacterial. 80. 565 (1960). 3. AL-DOORY, Y. and LARSH. H. W. Appl. Microbial. 10. 492 (1962). 4. ANDERSSON,9. A., HELLGREN, L. and- VINCENT, J. Sabouraudia 14, 237 (1976). 5. AUDETIX, R. C. S.. BAXTER. R. M. and WALKER. G. C. Can. J. Microbial. 7. 282 (1961). 6. BERTOLI,.E., CHAPMAN, D.,~GIUFFITHS,D. E. and STARCH, S. J. Biochem. Sot. Trans. 2,‘964 (1974). 7. BLANK, F., SHORTUIND, F. E. and JUST, G. J. Invest. Dermatol. 39, 91 (1962). 8. B~~HKE,H. Mycopathologia 46. 221 (1972). 9. BURDON. K. L. J. Bacterial. 52, 665 (1946). 10. CHEVREUL,M. E. Recherches chimiques sur /es corps gras d’origine animale. Levrault. Paris, 1823. 11. DAS, S. K. and BANFXJEE.A. 9. Sabouraudia 12, 281 (1974). 12. DILL, 9. C., LEIGHTON,T. J. and SKXX. J. J. Appl. Microbial. 24. 977 (1972). 13. GAUGHIUN, E. R. L. Bacterial. Rev. II. 189 (1947). 14. GHOSHAL, J. Sci. Cult. 35. 694 (1969).
Dermatophyte 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
47. 48. 49. 50. 51.
lipids
177
GOTTLIEB, D., CARTER, H. E., SU)NEKER, J. H. and AM?+NNN. A. Science 128, 361 (1958). GOTTLIEB, D. and VAN ELM, J. Z. J. Bacterial. 91, 161 (1966). GOTZ, H. and PASCHER, G. Dermatologica 124. 31 (1962). HELLGREN, L. and VINCENT, J. Sabouraudia 14, 243 (1976). HENDRIX, J. W. A~I. Rev. Phytopathol. 8. 111 (1970). HILDITCH, T. P. and WILLIAMS, P. N. The Chemical Constitution of Natural Fats, 4th Ed., Chapman Hall, London (1964). Iro, Y. and FUJII. T. Inst. Lombard0 (Rend. SC.) B 92, 313 (1958). KISH, Z. and JACK, R. C. Lipids 9, 264 (1974). Kosrrw, L. L., VICHER, E. E. and LYON, I. Mycopathologia 29, 145 (1966). Kosnw, L. L., VICHER, E. E. and LYON, I. Mycopathologia 49, 67 (1973). KUDRYK, B. Ph.D. Thesis, St. John’s University, New York, 1970. LAMPEN, J. 0.. ARNOW, P. M., BOROWSKA, Z. and LASKIN, A. I. J. Bacterial. 84, 1152 (1962). LOEFFLER, W. Zbl. Bakt. Hyg. I. Abt. Orig. A 220, 247 (1972). MALLINCKRODT-HAUPT. A. V. Zbl. Bakt. Hyg. I. Abt. Orig. A 103, 73 (1927). MERKEL, M. Bull. Acad. Polon. Sci. Cl. 2 5, 341 (1957). MERKEL, M. Bull. Acad. Polon. Sci. Cl. 2 6, 417 (1958). MERKEL, M. Bull. Acad. Polon. Sci. CL 2 7, 501 (1959). N~GUCHI, T’.. KITAZIMA, Y., NOZAWA, Y. and Ire, Y. Arch. Biochem. Biophys. 146. 506 (1971). NOZAWA, Y., KITAJIMA, Y. and ITO. Y. Biochim. biophys. Acta 307. 92 (1973). OKAZAKI, K. and TAMEMASA, 0. J. Pharm. Sot. Japan 75. 1087 (1955). PECK, R. L. In Biology of Pathogenic Fungi, Chronica Botanica, Waltham, Mass. (1947). PRINCE, H. N. J. Bacreriol. 79. 154 (1960). SHAH, V. K. and KNIGHT, S. G. Arch. Biochem. Biophys. 127, 229 (1968). SHAW, R. Adv. Lipid Res. 4, 107 (1966). SINGH, B., GUPTA, K. G. and BHATNAGAR, L. Indian J. Exp. Biol. 11. 555 (1973). SWANSON, R. and STANK, J. J. Appl. Microbial. 14. 438 (1966). VINCENT, J. Zbl. Bakt. Hyg. I. Abt. Orig. A. 233. 410 (1975). WIRTH, J. C. and ANAND, S. R. Can. J. Microbial. 10, 23 (1964). WIRTH, J. C., ANAND, S. R. and KISH. Z. L. Can. J. Microbial. 10, 811 (1964). WIRTH, J. C., BEESLEY, T. and MILLER, W. J. Invest. Dermatol. 37. 153 (196lj. WIRTH. J. C.. O’BRIEN. P. J.. SCHMITT. F. L. and SOHLER. A. J. Invest. Dermatol. 29. 47 (1957). . I WOOD;, R. A. .I. Bactdriol. iO8. 69 (1671). ZALOKAR, M. In The Fungi, Ed. G. C. AINSWORTH and A. F. SUSSMAN, Vol. 1, Academic Press, New York and London (1965). ZAMIECHOWSKA-MIAZG~WA, J. Bull. Acad. Polon. Sci. CL 2 6, 423 (1958). ZAMIECHOWSKA-MIAZG~WA, J. Bull. Acad. Polon. Sci. Cl. 2 7, 445 (1959). ZAMIECHOWSKA-MIAZG~WA, J. Bull. Acad. Polon. Sci. Cl. 2 10, 3 (1962). ZAMIECHOWSKA-MIAZC~WA, J. Bull. Acad. Polon. Sci. Cl. 2 12, 67 (1964).
For.\ orkrr L,p,r,\ Vol. 16. pp 171-177 Pcry;,mon Press. IY7X. PrInted ,n Greal Bnh,n
DERMATOPHYTE JAN
LIPIDS
VINCENT
CONTENTS 171
1. INTKOULICTION II.
171
TOTAL
LIPIDS
IN DEKMATOPHYTES
III.
FATTY
Arm
Iv.
STEROLS
175
173
PHOSPHOLIPIDS
176
ACKNOWLEI)GEMENTS
176
RFFERENCES
176
V.
I. INTRODUCTION
This review concerns the distribution of dermatophyte lipids, their possible physiological role and their importance in pathogenesis of dermatophytosis. The text is divided into two parts: the first part covers the increasing amount of information regarding the total lipid composition of dermatophytes with special reference to the various factors influencing the lipid content; the second part covers the three major lipid classes occurring in dermatophytes, i.e. fatty acids, sterols and phospholipids. Although lipids were among the first natural products investigated,‘O they were neglected until the last decades when sophisticated analytical techniques became available. This has, of course, also stimulated more extensive investigations on dermatophyte lipids. Dermatophytes are a highly specialized group of filamentous fungi, causing lesions of the skin and its appendages in man and animals. Dermatophytes, according to their imperfect forms. include species of three genera: Epidermophyton, Microsporum and Trichoph~ton (Fungi imperfecti). All known perfect forms belong to genera Arthroderma and Nannizicr (Ascomycetes).” Beside the chemotaxonomical implications, this review may provide useful information for further investigations concerning the role that dermatophyte lipids play in the inflammatory skin reactions in dermatophytosis. II.
TOTAL
LIPIDS
IN
DERMATOPHYTES
Even before dermatophyte lipids were known, their lipolytic activity was described.sS2* The use of classical staining methods first demonstrated the presence of lipids in dermatophytes. ’ *9 Later, mycelial extractions were carried out for evaluation of total lipids in order to elucidate their relationship in the pathogenesis of dermatophytosis 2~3,17~21.22~3h~41~44.51 The total lipid abundances in dermatophytes are summarized in Table I. Regarding the relatively large range of abundances, it is apparent from the table that consideration must be given to growth conditions, age and the analytical techniques. Dermatophytes demonstrate a relatively low growth activity when compared with the majority of saprophytic molds. 4o For example, the mycelia of E. JIoccosum contain total lipids as a function of age, in the range 7&19.8% of the dry weight, which is also true for most dermatophytes studied. The total lipids of E. Joccosum significantly increase with age, except during the early growth period when there is a decrease, mainly at the expense of the triglycerides.41 Thus, the energy requirements of the developing mycelia would appear to be met, at least in part, by fatty acid oxidation. The 171
Jan Vincent
172 TABLE
Genus
.
Epidermophyton Microsporum
Trichophyton
Reported
1. Total Lipids of Dermatophytes Species
j?occosum jloccosum audouinii canis canis gypseum ajel/oi* ajelloi (mutant) gallinae nGgninii** mentagrophytes mentagrophytes mentagrophytes mentagrophytes*** rybrum rubrum rubrum schb;nleinii schiinleinii tonsurans uerrucosum**** violaceum violaceum
Total lipid (“, dry wt.)
Reference
7.0-19.8” 14.77b 14.63b 10.81b 26.77” 13.57h
41 3 3 2 3 3
15.5’
22
20.5’ 12.98” 8.7’ 15.3J 18.13h 8.2’ 18.8’ 18.49h 11.2’ 7.7’ I 9.48h 8.3’ 10.85” 43.7’ 10.lOh 16.5’
22 3 51 36 3 17 51 3 17 44 3 17 3 51 3 51
uncinatum (stat. ascosp.); **T. rosaceum; ***T. asteroides; ****T. fauiforme. ochraceum. Growth conditions: “Sabouraud dextrose broth. 30°C; kparagine liquid medium. 20°C; ‘Sabouraud dextrose broth. 20°C; ‘dextrose (1%) and peptone (l:,,,) broth, 20°C; ‘Griitz-III-medium, 20°C. as: ‘Arthroderma
gypseum,
lipid accumulation in older mycelia as a store of energy is confirmed also by cytological studies.47 The decrease of total lipids in E. ~OCCOSU~during germination and in the early phase of growth may be a consequence of their utilization as a prime carbon source and substrate for polysaccharide synthesis.” Environmental conditions such as substrate, temperature, agitation, etc. have been recognized to have a particular influence on the total lipid content of dermatophytes. Lipid production of dermatophytes, similar to the majority of filamentous fungi, depends on the nature of the carbon source. In this connection, the richest carbohydrates are, in descending order, glucose, sucrose and fructose. Also amino acids, present in certain concentrations, such as alanine or phenylalanine, can affect the total lipid content3’ It is generally agreed that a relatively high carbon-nitrogen ratio in the media is required in order to attain maximum lipid production. In common with most living organisms, dermatophytes show a tendency to enhance lipid content with increasing environmental temperature (unpublished data). Thus, investigations should be carried out on the species grown under optimal temperatures. Moreover, some microorganisms subcultured at low temperatures show a relatively high degree of unsaturation. l3 This alters some physical properties of the lipids, which can be of importance for the survival of the dermatophytes in unfavorable environments. Aeration of culture has also a pronounced effect on the general metabolism of fungi and their lipid composition. The degree of aeration required depends on the technique applied, e.g. semisolid media, shake or still cultures. Thus, significant differences in the amount of total lipids in still and shake cultures have been reported.3 Early stages of germination are characterized by a dependency on the endogenous substrate. It has been suggested l2 that the macroconidia of M. gypseum are capable of immediate response to a favorable environment and therefore constitute an ideal means for the transmittance of infection. The longevity of the macroconidia may depend more on a high concentration of essential materials in the spore rather than any well
Dermatophyte TABLE 2. Dermatophyte
Genus Epidermophyton
173
lipids Cell Wall
Cell wall lipid (% dry wt.1
Species jloccosum
j7occowm Microsporum Trichophyton
Lipids
canis gypseum mentagrophyres mentagrophytes
Reference
3.3 4.6
37 33
4.0 3.6
37 37 37 32
3.1 6.6
developed dormancy mechanisms. This confirms the changes of dermatophyte M. quinceanum during starvation which indicates that the carbohydrates do not form the principal endogenous reserve. In this case, lipids may represent a primary reserve material because they decrease rapidly from 23% in the spores to 13% in a 24 hr old mycelium. The same picture was true during starvation.40 Cell walls from filamentous fungi, particularly dermatophytes, have been extensively studied.32*33*37 However, relatively little is known about their lipid composition. Lipid abundancies of dermatophyte cell walls are given in Table 2. Shah and Knight”’ showed that these lipids may be of importance in conferring rigidity or protection against drying, since morphological distortion was observed after solvent extraction. The function of dermatophyte lipids is as yet not well understood. Historically, lipids have been considered as reserve material which may be converted to energy and carbon skeletons during growth and reproduction. This is true for those lipids which accumulate in cytoplasma that are composed primarily of triglycerides. However, as we know more about the structure and biosynthesis of less abundant lipids, it is apparent that they have more specific roles to play. For example, lipids are essential components in the membrane structures when they may serve as protective coatings or may be involved in the immunological reactions in dermatophytosis. According to Peck,35 lipids of pathogenic fungi have a significant role in the mechanism of infectious disease. Moreover, interest in dermatophyte metabolites leads to the detection of specific antigenic components extracted from their mycelia. Lipid fractions, especially fatty acids, have been reported to possess antigenic activity.4*‘8 . III.
FATTY
ACIDS
Fungal fatty acids and their biosynthesis have been extensively reviewed.20*38 Generally, dermatophyte fatty acids consist of a homologous series of saturated and unsaturated aliphatic acids ranging from C6 to Cz2 with a predominance of Cl6 and Cis acids. These acids can account for over 80% of the total fatty acid content. The ethylenic bonds in the unsaturated acids are in the cis configuration and, where more than one ethylenic bond is present, they occur normally in methylene-interrupted sequence. Fatty acid distribution in some species of dermatophytes from the genus Trichophyand Microsporum to11 5923.29--31.42,4*-51 43 has been determined mainly by paper chromatography. The fatty acid composition of the remaining monotype genus Epidermophyton was established by combined gas chromatography-mass spectrometry in this laboratory.41 The distribution of fatty acids encountered in dermatophytes is given in Table 3. For instance, fatty acids of E. Jlocc~~um decrease significantly with age, which may be due partly to their conversion to glycerides and partly to their complete degradation. However, the composition of the predominating fatty acids was unaffected by age.41 The narrow range of dermatophyte fatty acids confirms that they compose a homogenous group of filamentous fungi. Recently, the enzymatic system responsible for fatty acid synthesis in dermatophyte was investigated.24 It has been postulated that an equilibrium between malonyl CoA (the intermediate in de nouo synthesis) and acetyl CoA
(41)
‘T.
reported as: ‘T. gypseum;
WIRTH (43)
schiinleinii MERKEL (30)3 tonsurans ZAMIECH. (48)4 ZAMIECH. (49)s quincheanum MEXKEL (31)6 migninii ZAMIECH. (SO)’ ZAMIECH. (51)8 verrucosum ZAMIECH. (51)9 violaceum ZAhSECH. (51) rubrum WIRTH (42) KOSTIW (23) gypseum
j7occosum VINCENT
Epidermophyton
Microsporum
mentagrophytes
Trichophyton
Analyzed with
GC
’ PC
gypseum,
GC-MS
i
j
’
MERKEL (29)’ PC ZAMIECH (5 1)’ 1 AIJDETTE(5) GC
Species
Genus
9
10
0.1
tr 0.3
1.1 0.5 0.7
0.1
8
asteroides; 3Achorion;
tr
6
“T.
0.2 plicatile;
1.7 0.3 5T. sulphureum;
1.4
6Achorion;
2.7 0.2
‘T.
18.9
17.2
23.8 17.6
X
x
0.8 0.7
0.7 rosaceum;
2.3
9.6
7.7
7.4 16.1
X
X
*T. rosaceum;
tr
X
x
42.8
64.7
52.4 35.6
‘T. faviforme,
16.2
10.4
13.1 19.2
X
X
X
x
X
X
X
x
X X
18:2
1710 45.2
X X
1;12
x X
2.6
1.0
X
18:l
X
X
tr.
0.2
X
18
x
218
x
Fatty acids 16 16:l 17 17:l
X
0.8 2.1 0.8 0.1 0.5
0.9
15
X X
1.0
0.3
14 14:l
X
OYl
13:l
3. Fatty Acids of Dermatophytes4’
12 13
0.2 0.1 tr
11
TABLE
1.1
1.1
20
0.5
0.3
2O:l
ochraceum.
0.9
19
0.1
0.2
20:2
tr.
21
tr. 0.8
22
Dermatophyte lipids
175
(the intermediate in chain elongation) may provide the site for the mechanism controlling these two processes. The taxonomical value of fungal fatty acids is limited.3* However, as Phycomycetes are characterized by a predominant occurrence of y-linolenic acid, its absence in dermatophytes is of taxonomical significance, which thus confirms the relevance of dermatophytes to Ascomycetes. Moreover, the fatty acid composition may be of importance in membrane biological activity6 and may be involved in the allergic inflammatory delayed skin reactions in dermatophytosis.4 The Landstainer-Draise test of these compounds confirms that the middle chain (C,a-CiJ fatty acids are able to sensitize the mammalian skin and elicit the delayed inflammatory skin reactions.18 IV. STEROLS
The presence of ergosterol in dermatophytes, such as 27 mentagrophytes,’ T. mentagrophytes (asteroides)34 and T. rubrum, was primarily reported. Brassicasterol and a trace of squalene have been isolated from T. rubrum. 44 The occurrence of squalene in trace quantities in the mycelia of T. rubrum is consistent with its known position in the biosynthetic pathway between acetate and sterols, and with the fact that squalene does not normally accumulate, to a significant extent, under aerobic conditions. Subsequently, Blank ef al.’ identified two sterols, brassicasterol and ergosterol, from different species of dermatophytes. Ergosterol was found to be more widely distributed than brassicasterol. These sterols occurred together in some species but the second was only present in very small amounts. In a number of species, ergosterol was present to the exclusion of the other. The distribution of these two sterols among the dermatophytes is rather interesting from the taxonomic point of view (Table 4). Although there are several reports on the subject, the exact function of sterols in fungi is uncertain.” The sterol content of E. fIoccosum reaches a maximum during the early growth period, 41 but its relationship with an eventual germination control or cell permeability remains open for investigation.16 Summarizing, sterols are required for growth and reproduction as well as being implicated in membrane structures and permeability. Relationships have been established between the sterols of fungal membranes and the action of polyene antifungal antibiotics. 15*46 For instance, the effect of nystatin is probably due to its insertion among the sterols of the lipid bilayer, leading to molecular rearrangements, loss in rigidity and mechanical failure of the membrane.26 TABLE 4. Dermatophyte
Genus
Sterols (% dry 4 Brassicasterol Ergosterol
Species
Epidermophyton Microsporum
Sterols’
-
jloccowm audouinii canis
Reported discoides.
*T.
granulosum;
**T.
0.026 0.270 0.250 -
-
quinckeanum ferrugineum mbgninii mentagrophytes* mentagrophytes** mentagrophytes*** rubrum tonsurans verrucosum**** violaceum as:
0.150 0.016
-
gweum Trichophyton
0.083
0.039 -
0.130 1.50 0.280 0.056
0.126 -
0.010 0.015 persicolor;
***T.
interdigitale,
****T.
17b
Jan Vincent TABLE 5. Phospholipids Genus
Trichophyton
Reported
Phospholipids (% dry wt.1
Species
l
Epidermophyton Microsporum
in Dermatophytes
jloccosum audouinii
6.96 5.02
conis
0.048
canis
4.48 4.04 2.6 5 4.97
gweum ajelloi* ajelloi (mutant) gallinae mentagrophytes mentagrophytes rubrum schijnleinii tonsurans violaceum as: *Arthroderma
uncinatum
3.13 0.3 4.87 3.59
1.33 2.58
Reference 3 3 2 3 3 22 22 3 3 36 3 3 3 3
(stat. ascosp.)
V. PHOSPHOLIPIDS
Phospholipids are considered to be involved mainly in membrane functions. Their composition in dermatophytes (Table 5) have been, until recently, rather poorly investigated.2*3v36 Ghoshal l4 found 41% phosphatidylcholine and some sphingomyelin in T. rubrum, but the presence of ‘other phospholipids could not be confirmed. However, in M. cookei, five major components as phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylinositol were identified.25 The phospholipid composition of a mutant strain of the dermatophyte Arthrodermo uncinatum (stat. ascosp. of T. ajelloi) was compared with that of the wild type.22 Both strains contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diphosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, but important differences in the amounts of phosphatidylcholine, phosphatidylserine, and phosphatidic acid were observed. In both strains, the predominant fatty acid was 18:2 with 54.0% in the wild type and 46.7% in the mutant. Qualitatively, the same fatty acids, with the exception of the CzO acids, were found in all phospholipid classes. T. rubrum contains phosphatidylinositol, polyphosphatidylinocitol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid.” The relative proportions of these components in total phospholipid fraction remain unchanged with age. Acknowledgements-1 would like to express my gratitude to Professor Sixten Abrahamsson, Associate Professor Ng. Dinh-Nguyen and Assistant Professor Lars Hellgren for their valuable advice, and to Mr Weston Pimlott for reviewing the text. Special thanks go also to Mrs Greta Sonnhagen for her contributions in the preparation of the entire manuscript. The study was supported by the Swedish Medical Research Council.
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