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Destruction of hair by Chrysosporium keratinophilum
[ 247 ] Trans. Br. mycol. Soc. 52 (2) , 247-255 (196g)
Printed in Great Britain
DESTRUCTION OF HAIR BY CHRYSOSPORIUM KERATINOPHILUM By MARY P. ENGLISH Pathology Department, General Hospital, Bristol,
1
(With Plates 15-16 and 3 Text-figures) The means by which the keratinolytic fungus Chrysosporium keratinophilum (Frey) Carmichael attacks and destroys human and bovine hair in vitro are described. While fronded mycelium plays a part, especially in the attack on the cuticle, the destruction of the cortex is mainly by boring hyphae which are at first typical of those of non-keratinolytic fungi but later penetrate the hair in all directions giving rise to large individual cells, wide-celled columns, and masses of mycelium of extraordinarily varied morphology. True perforating organs as found in the dermatophytes were never seen, neither was erosion of the keratin around the mycelial elements demonstrable. It is concluded that the fungus digests keratin in a manner chemically distinct from the dermatophytes and that on this difference may depend the difference in the morphology of the digesting mycelium. In certain ways this is intermediate between that of non-keratinolytic fungi and of true dermatophytes, thus providing support for the hypothesis that the latter may have evolved from the former.
Chrysosporium (Aleurisma) keratinophilum, a geophilic fungus, was first described by Frey (1959). She stated that it was keratinolytic but gave no details as to how it attacked hair. Carmichael (1962) remarked that it did this by means of 'penetrating bodies' and Otcenasek & Dvorak (1964) used the term ' penetrating organs'. Evolceanu, Alteras & Stoian (1963), describing a fungus which they called Microsporum nanum, but which was later shown by Ajello , Varsavsky, Ginther & Bubash (1963) to be C. keratinophilum, used the expression 'organes perforateurs, semblables a ceux du M. gypseum' to describe the mycelium attacking hair. Bohme & Ziegler (1967) also thought that the fungus attacked hair by means of perforating organs typical of dermatophytes, and Pinetti & Lostia (1966), though less specific, also suggest this. No illustrations are given by any of these authors. In contrast, Ajello et al. (1963), while not describing the organs, publish a photomicrograph of a hair attacked by C. keratinophilum, which closely resembles one colonized by the boring hyphae of a non-keratinolytic fungus (English, 1965). In view of these contradictory observations, this investigation was designed to discover the actual method of attack by C. keratinophilum on hair in vitro and to compare this with the behaviour of the dermatophytes and of non-keratinolytic fungi.
Transactions British Mycological Society MATERIALS AND METHODS
One Australian (no. RNSH 688), and three Czechoslovak (nos. 39 V, 95 V and 163) isolates of C. keratinophilum were used. Preliminary investigations showed that all behaved in an identical manner on hair and they were used indiscriminately for the investigation. They were maintained on glucose-peptone agar slopes, master cultures being kept under paraffin oil. The fair hair of a rz-year-old boy and the white hair of a cow were used as substrata. The hair was prepared and inoculated as described by English (1968). Hairs were examined at different stages of attack by three techniques. Whole hairs were mounted in lactophenol-cotton blue and examined microscopically. This gave a reasonably clear picture of the method of attack but, as in previous work (English, 1963), details of the structure of the buried mycelium were impossible to distinguish. Other hairs were therefore examined by the KOH-phase contrast technique (English, 1968) which enabled the morphology of the invading mycelium to be observed. Finally, histological sections were prepared and stained by the periodic acid-Schiff (PAS) method with haematoxylin counter-stain. Drawings were made with the aid of a camera lucida. DEFINITIONS
Usages of the words 'keratinolytic', 'keratinophilic' and 'dermatophyte' vary greatly. For this reason 'keratinophilic' will not be used in this article and the other two words will be used only in the following senses: keratinolytic-a fungus able to digest keratinized substrata completely; dermatophyte-s-« fungus belonging to one of the genera Keratinomyces, Microsporum, T richoph:Yton or Epidermophyton, whether pathogenic to man or animals or not. All dermatophytes are keratinolytic. RESULTS
Growth on human hair Attack on cuticle. This is the first part of the hair to be attacked. The behaviour of C. keratinophilum closely resembles that of a dermatophyte (English, 1963, fig. I) except that, while some lateral hyphae give rise to fronded mycelium (PI. 15, fig. I), others produce the terminal or intercalary appressoria typical of many non-keratinolytic fungi (English, 1965, text-fig. 2). As with other fungi, the thrust of the fronds may cause cracks in the cuticle but, if penetration is deep, a curious undulation of the cuticle is seen in side view (PI. 15, fig. 2) which has not been observed with other fungi. The fraying noted by Pinetti & Lostia (1966) in dermatophytes and by English (1965) in non-keratinolytic fungi also occurs. It may take place in cuticle overlying a frond, (Text-fig. I D), or where there is no visible frond formation but where the cortex below has been strongly attacked. Unlike that of a hair attacked by a dermatophyte, the
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cuticle is digested only slowly, the pattern of its scales remaining clear, though ragged, for 3-4 weeks, long after the cortex has been severely ravaged. Each appressorium gives rise to one or two borers penetrating into the C
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Text-Fig. I. A-D. Boring hyphae penetrating hairs from cuticle and showing varying degrees of swelling. A and B produced from appressoria, C and D from subcuticular fronds. C terminates in a normal borer. E, F. Grossly enlarged and proliferating borers, F penetrating right through hair, and both causing splitting of surrounding cortex. Lactophenol-cotton blue mounts of whole hairs. x 1350. c= cuticle, f = frond, cc splits in cortex.
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cortex and, usually, entirely typical of those of non-keratinolytic fungi (English, 1965). Borers may also be produced from fronds. Absolutely no evidence was found of the true perforating organs so typical of dermatophytes. Attack on the medulla. The medulla is usually first colonized from borers that have entered the hair from the cuticle (PI. 15, fig. 5) : this may occur within a few days of the exposure of a hair to the fungus. In the medulla, the tip of a borer gives rise to a rope of mycelium very reminiscent of those of dermatophytes (PI. 15, figs. 3, 4) (cf. English, 1968, pI. I G) but lacking the sharply pointed terminal cells typical of those fungi. As with the dermatophytes, growth in the medulla is much' faster than in the cortex, the keratin of which is less easily digested than the trichohyalin of the former. The medullary mycelium quickly produces more borers which grow back towards the cuticle (PI. 15, fig. 5), and it is impossible, after 5 or 6 days, to tell whether any given piece of cortical mycelium originated from the medulla or the cuticle (PI. 15, fig. 3). Invasion oj cortex. The first attack is by borers, some typical of other non-dermatophytes and others wider, which produce in the hair an appearance identical to that illustrated by Ajello et al. (1964). Wide borers develop when the tip of a normal one, after penetrating some way through the cortex, or occasionally immediately on entering it, swells to produce a cell or column of cells (a 'swollen borer ') of varying shape and width, often quite as large as the perforating organ of a dermatophyte (PI. 15, figs. 6,8; Text-fig. I A-D). Occasionally a normal borer ends in a knob (Text-fig. 2 C) which may be an early stage in the formation of a swollen borer. A swollen borer may penetrate right through the cortex or may stop abruptly while the distal cell gives rise once more to a typical boring hypha (Text-figs. I C, 2 C, D, F). Sometimes a swollen borer proliferates in all directions to form a large, solid mass of mycelium (Text-fig. I E, F). At advanced stages of invasion this mass projects backwards out of the hair but in a manner quite unlike the 'handle' formation of a dermatophyte, the projecting cells being in a bulging mass, not in a single, often collapsed, column. Other forms of proliferation from borers in the matrix of the cortex are very varied. Longitudinal attack is usually by means of a torpedo-shaped row of very wide cells (Pl. 15, figs. 3, 4; PI. 16, figs. 5, 6; Text-fig. 2A). They eventually proliferate laterally as well as penetrating further along the hair, forming elongated masses of cells which occasionally end in a small frond (Pl. 15, figs. 6,7; Text-fig. 2B, G). KOH-phase contrast preparations sometimes showed a sheath of narrow-celled mycelium closely pressed round a hyphae of wide-celled, cortical mycelium (PI. 15, fig. 7), even enclosing its growing tip. Such a sheath may account for the effect shown in PI. 16, fig. 4, where one hypha appears to be growing within a larger one. Occasionally walls of cortical mycelium grow along the hair from swollen borers, resembling dermatophytes (English, 1968, fig. 19). Much more often, however, growth proceeds at any angle in relation to the hair, swollen cells, either singly like balloons, or in columns or masses (PI. 15,
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Text-fig. 2 . A, B. Lactophenol-corton blue mounts of whole hair. A, successive focal planes from cuticle through cortex ofa human hair. Dotted Unes represent out-of-focus cortical mycelium. A" cuticle, stripped of aerial mycelium, showing origin of two borers with cortical myceUum below them ; A2 , 4Jtm below AI , the borers have given rise to two longitudinal cortical hyphae from their tips. Examined a t a lower magnification the hypha which is out of focus has the appearance of an eroded space around that which is in focus . AS, I I p.m below A2, shows the end of a swollen borer from the cortical mycelium above. The cortex has split both at the growing ends of the cortical mycelium and around the swollen borer. B, Cortical mycelium with serrated outer cell walls in situ in the hair cortex. e-G, KOH-phase contrast preparations: C, serrated cortical mycelium with knobbed borer; D, swollen borer with ringed indentations; a normal borer grows from its tip. ; E, F, borers and cortical mycelium of very varied morphology; G, tip of cortical mycelium ending in two branches with rudimentary fronds . All x '350. c cuticle or cu ticular scale, b borer, sb = swollen borer, oh = origin of borer, cc = cortical splitting, mr = rope of medullary mycelium.
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fig. 8; PI. 16, figs. 1,2) being indiscriminately mixed with narrow borers. It is this unorientated branching of elements of very varied shape which distinguishes a hair in an advanced stage of digestion by C. keratinophilum from one attacked by a dermatophyte. Many extraordinary variations in the morphology of cortical mycelium can be demonstrated in KOHphase contrast preparations (T ext-fig. 2 E-G, and illustrations discussed in other contexts).
Mechanism of attack. On exposure to a fungus a hair is usually attacked at a number of points. C. keratinophilum often, though not always, causes a marked swelling of the hair at these sites (PI. 16, fig. 3) indicating that, on such occasions, the invading mycelium is bulkier than the keratin it has replaced, and that therefore the hair must be under some pressure from the mycelium. This has not been noted for dermatophytes. I-
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Text-fig. 3. A. PAS-stained section of cortex of bovine hair showing cor tical mycelium and borer partially surrounded by a clear zone (z ) of unknown constitution. B. KOHphase contrast preparation of cortical mycelium and swollen borer partially surrounded by firmly adherent, phase-dense material. Both x 1350.
Keratin at the ends of penetrating mycelial masses often appears longitudinally split (Text-fig. 2 A) as also does that surrounding swollen borers (PI. 16, fig. 4; Text-fig. 1 D-F). This again suggests that the mycelium exerts pressure in the act of penetration, both at its tip and round its circumference. Pressure is also indicated by the occasional finding of a mass of cortical mycelium, both in situ in the hair (PI. 16, fig. 6) and in KOH-phase contrast mounts (Text-fig. 2 B, C), with serrated outer walls, apparently moulded by pressing against partially digested keratinized cells; stepped rings also occur round smaller, radially penetrating, fungal elements, apparentlyfrom the same cause (PI. 16, fig. 5; Text-fig. 2 D). No amount of searching could establish that keratin was dissolved at a distance from the mycelium, as it is by dermatophytes. Where, in an initial examination of a lactophenol-cotton blue preparation, cavities were apparently present round the mycelium, investigation under a higher power of the microscope always showed that the 'clear' space was, in fact, another wider piece of mycelium over- or underlying the first, at a slightly different focus (PI. 16, fig. 7; Text-fig. 2A). That digestion must take place is clear, as an attacked hair is entirely replaced by mycelium in 6-8 weeks. Certain limited observations gave a
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clue as to the method. In PAS-stained sections (Text-fig. 3A) the mycelium embedded in the cortex was sometimes partially surrounded by what appeared to be either a gap or, more likely in view of the observations just described, a zone of altered keratin. Text-figure 3 B shows a KOHphase contrast preparation of cortical mycelium with a borer, partially surrounded by structureless, phase-dense material with no cell wall, of exactly the same shape relative to the mycelium as the mark on the keratin in the PAS section. That this material must be solid or semisolid can be concluded from its ability to adhere to the mycelium during the drastic process of digestion with KOH. That it is not dissolved by this treatment implies also a marked chemical change from the original keratin. It seems probable that it represents a transitional stage in the digestion of keratin. Growth in bovine hair The differences between the method of attack of C. keratinophilum on bovine and human hair appears, as with other fungi (English, 1963, 1965), to be related to differences in the structure of the hairs. The wide medulla is quickly penetrated by the fungus and the initial attack on the cortex is consequently from there rather than from the cuticle (PI. 15, fig. 6). Again, as with other fungi, no rope of medullary mycelium is formed, the hyphae remaining distinct and separate, some of normal width, some swollen (English, 1965, pl. 18, fig. 4). The cortex is invaded from these hyphae in three ways. It may be penetrated by borers or swollen borers in the same way as in human hair; fronds may be found pressed to, or probably just under, the surface of the medulla; and masses of mycelium may bulge into the cortex, themselves often giving rise to borers. From this stage both the cortex and cuticle are attacked in the same way as in human hair. DISCUSSION
It has been confirmed that C. keratinophilum is keratinolytic, but detailed observations show that both the morphology of the active mycelium, except the fronds, and the chemistry of its action are different from those of the dermatophytes. The examination offronded mycelium during this work has suggested a hypothesis which could explain, for all fungi, the way in which it is formed. When a hypha first makes its way between two closely adherent layers of a substance it not only forces them apart where they are directly in contact with it, but also rips them slightly apart on either side of itself (English, 1965). Lateral hyphae formed behind the leading hypha would, in normal circumstances, diverge from the leader at more or less wide angles. If this were to happen in a layered substratum each new lateral hypha would have to make a fresh penetration of the intact substratum thus using extra energy. Clearly if these hyphae were to follow the line of least resistance they would be channelled into the slits in the keratin on either side of the leading hypha, which would entail their growing in the same plane as that hypha and closely pressed to it. This is exactly what appears to occur in a frond. In growing in this way the lateral hyphae would enlarge the slits further and so give the next generation of
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laterals the same advantage. This process could continue indefinitely, resulting in the building up of the very large fronds produced by many fungi in layered substrata. C. keratinophilum resembles the dermatophytes in that pressure clearly plays an important part in penetration. It differs from them in that the mycelium buried in the hair is always closely pressed to the walls of the cavity in which it lies. The chemistry of keratin digestion by C. keratinophilum must therefore differ from that of the dermatophytes. The demonstration of what is possibly a zone of altered keratin adjacent to the cortical mycelium suggests that digestion by C. keratinophilum occurs in two stages. First the keratin is chemically altered to a structureless but still solid form. Secondly this is digested, the fungus simultaneously growing into the space so formed. Biochemical studies are necessary to elucidate these observations. On its digestive process is probably dependent the distinctive morphology of the keratin-invading mycelium of the fungus. The swollen borers characteristic of it are intermediate between its own narrow borers, which are identical with those of non-keratinolytic fungi, and the perforating organs of the dermatophytes. They indicate how perforating organs could have evolved from borers in species which developed keratinolytic properties, and provide support for this suggestion, put forward by me in 1965 and followed by Kunert (1967) from his observations on Keratinophyton terreum, but disputed by Pinetti & Lostia (1966). I am most grateful to Dr D. Frey and Dr M. Otcenasek for the cultures of C. keratinophilum; to Mr J. Washer for some of the photomicrographs and to Mr J. Hancock for assistance with the remainder. REFERENCES
AJELLO, L., VARSAVSKY, E., GINTHER, O.J. & BUBASH, G. (1964). The natural history of Microsporum nanum. Mycologia 54, 873-884. BOHME, H. & ZmGLER, H. (1967)' Keratinabbau durch Pilze. Arch. Mikrobiol. 67, 93- I 10. CARMICHAEL, J. ( I962). Chrysosporium and some other aleuriosporic hyphomycetes, Can.]. Bot. 40, 1137-1173. ENGLISH, M. P. (1963). The saprophytic growth of keratinophilic fungi on keratin. Sabouraudia 2, I 15- I 30. ENGLISH, M. P. (1965). The saprophytic growth of non-keratinophilic fungi on keratinized substrata, and a comparison with keratinophilic fungi. Trans. Br. mycol. Soc. 4 8, 2 19-325. ENGLISH, M. P. (1968). The developmental morphology of the perforating organs of dermatophytes. Sabouraudia 6, 218-227. EVOLCEANu, R., ALTERAS, I. & STOIAN, M. (1963)' Considerations sur la morphologie, Ie pouvoir pathogene et la geophilie du Microsporum nanum. Mycopath, Mycol. appl. Ig,24-36. FREY, D. (1959). Isolation of a new species of Aleurisma from soil in Australia and New Guinea. Mycologia 51, 641-646. KUNERT, J. (1967). Zur Mikromorphologie des Haarabbaues durch Dermatophyten in vitro. Derm, Wschr. 153, 1141-1150. OTCENASEK, M. & DvoRAK,J. (1964). The isolation ofChrysosporium keratinophilum (Frey) Carmichael 1962 and similar fungi from Czechoslovakian soil. Mycopath. Mycol. appl.23, 121-124. PlNETTI, P. & LOSTIA, A. (1966). L'infezione dermatofitica del pelo in vitro. Monograph 13, Rass, med. Sarda, Cagliari.
Trans. Br. myeol. Soc.
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Chrysosporium. Mary P. English EXPLANATION OF PLATES
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15 AND 16
PLATE 15
Fig. Fig.
Detail of fronded mycelium. x 1250. 2. Undulation of cuticle of human hair following deep penetration by fronded mycelium (f). No lysis of the cuticle is apparent. x 1250. Fig. 3. Rope of medullary mycelium (r) from human hair produced from, and giving rise to polymorphous boring hyphae and cortical mycelium. x 500. Fig. 4. Borer (b) arising from medullary mycelium from human hair and giving rise to lateral torpedo-shaped cortical mycelium (ts). x 1050. Fig. 5. Early stage in colonization of medulla of human hair, the mycelial rope producing borers which are growing back towards the cuticle. x 550. Fig. 6. Cortex of bovine hair colonized from medulla by swollen borer (sb) and torpedo-shaped mass of lateral mycelium (1$). x 1050. Fig. 7. Tip of torpedo-shaped cortical hypha sheathed by flattened cells. Small frond (1) produced from tip. x 1250. Fig. 8. Cortical mycelium of very varied morphology and orientation colonizing human hair I.
x 550. Figs. I, 3, 4, 7: KOH-phase contrast preparations of mycelium. Figs. mounts of whole hairs.
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PLATE 16
Fig. I. Polymorphous cortical mycelium showing swollen borers (sb), borer (b) and torpedoshaped mycelium (ts). x 1250. Fig. 2. As fig. I, but showing balloon-shaped cells and borers (b). x 1250. Fig. 3. Human hair showing swelling at sites of maximal fungal attack. x 138. Fig. 4. End-on view of swollen borer in cortex of human hair, showing marked cracking of surrounding keratin. x 1250. Fig. 5. Swollen borer showing indented rings on cell wall and an early stage in the formation of torpedo-shaped lateral mycelium. x 1250. Fig. 6. Torpedo-shaped mass of cortical mycelium showing end-on view of swollen borer (sb) from which it originated, and serrated growing tip. x 1050. Fig. 7. Polymorphous mycelium in cortex of human hair. The comparatively narrow borer (b) overlies a swollen borer (sb) giving a false impression ofa zone oflysis round the former. x 1250. Figs. 1,2,5: KOH-phase contrast preparations of mycelium. Figs. 3, 4, 6, 7: cotton blue mounts of whole hairs.
(Accepted for publication 6 July 1968)
Printed in Great Britain
DESTRUCTION OF HAIR BY CHRYSOSPORIUM KERATINOPHILUM By MARY P. ENGLISH Pathology Department, General Hospital, Bristol,
1
(With Plates 15-16 and 3 Text-figures) The means by which the keratinolytic fungus Chrysosporium keratinophilum (Frey) Carmichael attacks and destroys human and bovine hair in vitro are described. While fronded mycelium plays a part, especially in the attack on the cuticle, the destruction of the cortex is mainly by boring hyphae which are at first typical of those of non-keratinolytic fungi but later penetrate the hair in all directions giving rise to large individual cells, wide-celled columns, and masses of mycelium of extraordinarily varied morphology. True perforating organs as found in the dermatophytes were never seen, neither was erosion of the keratin around the mycelial elements demonstrable. It is concluded that the fungus digests keratin in a manner chemically distinct from the dermatophytes and that on this difference may depend the difference in the morphology of the digesting mycelium. In certain ways this is intermediate between that of non-keratinolytic fungi and of true dermatophytes, thus providing support for the hypothesis that the latter may have evolved from the former.
Chrysosporium (Aleurisma) keratinophilum, a geophilic fungus, was first described by Frey (1959). She stated that it was keratinolytic but gave no details as to how it attacked hair. Carmichael (1962) remarked that it did this by means of 'penetrating bodies' and Otcenasek & Dvorak (1964) used the term ' penetrating organs'. Evolceanu, Alteras & Stoian (1963), describing a fungus which they called Microsporum nanum, but which was later shown by Ajello , Varsavsky, Ginther & Bubash (1963) to be C. keratinophilum, used the expression 'organes perforateurs, semblables a ceux du M. gypseum' to describe the mycelium attacking hair. Bohme & Ziegler (1967) also thought that the fungus attacked hair by means of perforating organs typical of dermatophytes, and Pinetti & Lostia (1966), though less specific, also suggest this. No illustrations are given by any of these authors. In contrast, Ajello et al. (1963), while not describing the organs, publish a photomicrograph of a hair attacked by C. keratinophilum, which closely resembles one colonized by the boring hyphae of a non-keratinolytic fungus (English, 1965). In view of these contradictory observations, this investigation was designed to discover the actual method of attack by C. keratinophilum on hair in vitro and to compare this with the behaviour of the dermatophytes and of non-keratinolytic fungi.
Transactions British Mycological Society MATERIALS AND METHODS
One Australian (no. RNSH 688), and three Czechoslovak (nos. 39 V, 95 V and 163) isolates of C. keratinophilum were used. Preliminary investigations showed that all behaved in an identical manner on hair and they were used indiscriminately for the investigation. They were maintained on glucose-peptone agar slopes, master cultures being kept under paraffin oil. The fair hair of a rz-year-old boy and the white hair of a cow were used as substrata. The hair was prepared and inoculated as described by English (1968). Hairs were examined at different stages of attack by three techniques. Whole hairs were mounted in lactophenol-cotton blue and examined microscopically. This gave a reasonably clear picture of the method of attack but, as in previous work (English, 1963), details of the structure of the buried mycelium were impossible to distinguish. Other hairs were therefore examined by the KOH-phase contrast technique (English, 1968) which enabled the morphology of the invading mycelium to be observed. Finally, histological sections were prepared and stained by the periodic acid-Schiff (PAS) method with haematoxylin counter-stain. Drawings were made with the aid of a camera lucida. DEFINITIONS
Usages of the words 'keratinolytic', 'keratinophilic' and 'dermatophyte' vary greatly. For this reason 'keratinophilic' will not be used in this article and the other two words will be used only in the following senses: keratinolytic-a fungus able to digest keratinized substrata completely; dermatophyte-s-« fungus belonging to one of the genera Keratinomyces, Microsporum, T richoph:Yton or Epidermophyton, whether pathogenic to man or animals or not. All dermatophytes are keratinolytic. RESULTS
Growth on human hair Attack on cuticle. This is the first part of the hair to be attacked. The behaviour of C. keratinophilum closely resembles that of a dermatophyte (English, 1963, fig. I) except that, while some lateral hyphae give rise to fronded mycelium (PI. 15, fig. I), others produce the terminal or intercalary appressoria typical of many non-keratinolytic fungi (English, 1965, text-fig. 2). As with other fungi, the thrust of the fronds may cause cracks in the cuticle but, if penetration is deep, a curious undulation of the cuticle is seen in side view (PI. 15, fig. 2) which has not been observed with other fungi. The fraying noted by Pinetti & Lostia (1966) in dermatophytes and by English (1965) in non-keratinolytic fungi also occurs. It may take place in cuticle overlying a frond, (Text-fig. I D), or where there is no visible frond formation but where the cortex below has been strongly attacked. Unlike that of a hair attacked by a dermatophyte, the
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cuticle is digested only slowly, the pattern of its scales remaining clear, though ragged, for 3-4 weeks, long after the cortex has been severely ravaged. Each appressorium gives rise to one or two borers penetrating into the C
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cortex and, usually, entirely typical of those of non-keratinolytic fungi (English, 1965). Borers may also be produced from fronds. Absolutely no evidence was found of the true perforating organs so typical of dermatophytes. Attack on the medulla. The medulla is usually first colonized from borers that have entered the hair from the cuticle (PI. 15, fig. 5) : this may occur within a few days of the exposure of a hair to the fungus. In the medulla, the tip of a borer gives rise to a rope of mycelium very reminiscent of those of dermatophytes (PI. 15, figs. 3, 4) (cf. English, 1968, pI. I G) but lacking the sharply pointed terminal cells typical of those fungi. As with the dermatophytes, growth in the medulla is much' faster than in the cortex, the keratin of which is less easily digested than the trichohyalin of the former. The medullary mycelium quickly produces more borers which grow back towards the cuticle (PI. 15, fig. 5), and it is impossible, after 5 or 6 days, to tell whether any given piece of cortical mycelium originated from the medulla or the cuticle (PI. 15, fig. 3). Invasion oj cortex. The first attack is by borers, some typical of other non-dermatophytes and others wider, which produce in the hair an appearance identical to that illustrated by Ajello et al. (1964). Wide borers develop when the tip of a normal one, after penetrating some way through the cortex, or occasionally immediately on entering it, swells to produce a cell or column of cells (a 'swollen borer ') of varying shape and width, often quite as large as the perforating organ of a dermatophyte (PI. 15, figs. 6,8; Text-fig. I A-D). Occasionally a normal borer ends in a knob (Text-fig. 2 C) which may be an early stage in the formation of a swollen borer. A swollen borer may penetrate right through the cortex or may stop abruptly while the distal cell gives rise once more to a typical boring hypha (Text-figs. I C, 2 C, D, F). Sometimes a swollen borer proliferates in all directions to form a large, solid mass of mycelium (Text-fig. I E, F). At advanced stages of invasion this mass projects backwards out of the hair but in a manner quite unlike the 'handle' formation of a dermatophyte, the projecting cells being in a bulging mass, not in a single, often collapsed, column. Other forms of proliferation from borers in the matrix of the cortex are very varied. Longitudinal attack is usually by means of a torpedo-shaped row of very wide cells (Pl. 15, figs. 3, 4; PI. 16, figs. 5, 6; Text-fig. 2A). They eventually proliferate laterally as well as penetrating further along the hair, forming elongated masses of cells which occasionally end in a small frond (Pl. 15, figs. 6,7; Text-fig. 2B, G). KOH-phase contrast preparations sometimes showed a sheath of narrow-celled mycelium closely pressed round a hyphae of wide-celled, cortical mycelium (PI. 15, fig. 7), even enclosing its growing tip. Such a sheath may account for the effect shown in PI. 16, fig. 4, where one hypha appears to be growing within a larger one. Occasionally walls of cortical mycelium grow along the hair from swollen borers, resembling dermatophytes (English, 1968, fig. 19). Much more often, however, growth proceeds at any angle in relation to the hair, swollen cells, either singly like balloons, or in columns or masses (PI. 15,
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Text-fig. 2 . A, B. Lactophenol-corton blue mounts of whole hair. A, successive focal planes from cuticle through cortex ofa human hair. Dotted Unes represent out-of-focus cortical mycelium. A" cuticle, stripped of aerial mycelium, showing origin of two borers with cortical myceUum below them ; A2 , 4Jtm below AI , the borers have given rise to two longitudinal cortical hyphae from their tips. Examined a t a lower magnification the hypha which is out of focus has the appearance of an eroded space around that which is in focus . AS, I I p.m below A2, shows the end of a swollen borer from the cortical mycelium above. The cortex has split both at the growing ends of the cortical mycelium and around the swollen borer. B, Cortical mycelium with serrated outer cell walls in situ in the hair cortex. e-G, KOH-phase contrast preparations: C, serrated cortical mycelium with knobbed borer; D, swollen borer with ringed indentations; a normal borer grows from its tip. ; E, F, borers and cortical mycelium of very varied morphology; G, tip of cortical mycelium ending in two branches with rudimentary fronds . All x '350. c cuticle or cu ticular scale, b borer, sb = swollen borer, oh = origin of borer, cc = cortical splitting, mr = rope of medullary mycelium.
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fig. 8; PI. 16, figs. 1,2) being indiscriminately mixed with narrow borers. It is this unorientated branching of elements of very varied shape which distinguishes a hair in an advanced stage of digestion by C. keratinophilum from one attacked by a dermatophyte. Many extraordinary variations in the morphology of cortical mycelium can be demonstrated in KOHphase contrast preparations (T ext-fig. 2 E-G, and illustrations discussed in other contexts).
Mechanism of attack. On exposure to a fungus a hair is usually attacked at a number of points. C. keratinophilum often, though not always, causes a marked swelling of the hair at these sites (PI. 16, fig. 3) indicating that, on such occasions, the invading mycelium is bulkier than the keratin it has replaced, and that therefore the hair must be under some pressure from the mycelium. This has not been noted for dermatophytes. I-
I
Text-fig. 3. A. PAS-stained section of cortex of bovine hair showing cor tical mycelium and borer partially surrounded by a clear zone (z ) of unknown constitution. B. KOHphase contrast preparation of cortical mycelium and swollen borer partially surrounded by firmly adherent, phase-dense material. Both x 1350.
Keratin at the ends of penetrating mycelial masses often appears longitudinally split (Text-fig. 2 A) as also does that surrounding swollen borers (PI. 16, fig. 4; Text-fig. 1 D-F). This again suggests that the mycelium exerts pressure in the act of penetration, both at its tip and round its circumference. Pressure is also indicated by the occasional finding of a mass of cortical mycelium, both in situ in the hair (PI. 16, fig. 6) and in KOH-phase contrast mounts (Text-fig. 2 B, C), with serrated outer walls, apparently moulded by pressing against partially digested keratinized cells; stepped rings also occur round smaller, radially penetrating, fungal elements, apparentlyfrom the same cause (PI. 16, fig. 5; Text-fig. 2 D). No amount of searching could establish that keratin was dissolved at a distance from the mycelium, as it is by dermatophytes. Where, in an initial examination of a lactophenol-cotton blue preparation, cavities were apparently present round the mycelium, investigation under a higher power of the microscope always showed that the 'clear' space was, in fact, another wider piece of mycelium over- or underlying the first, at a slightly different focus (PI. 16, fig. 7; Text-fig. 2A). That digestion must take place is clear, as an attacked hair is entirely replaced by mycelium in 6-8 weeks. Certain limited observations gave a
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clue as to the method. In PAS-stained sections (Text-fig. 3A) the mycelium embedded in the cortex was sometimes partially surrounded by what appeared to be either a gap or, more likely in view of the observations just described, a zone of altered keratin. Text-figure 3 B shows a KOHphase contrast preparation of cortical mycelium with a borer, partially surrounded by structureless, phase-dense material with no cell wall, of exactly the same shape relative to the mycelium as the mark on the keratin in the PAS section. That this material must be solid or semisolid can be concluded from its ability to adhere to the mycelium during the drastic process of digestion with KOH. That it is not dissolved by this treatment implies also a marked chemical change from the original keratin. It seems probable that it represents a transitional stage in the digestion of keratin. Growth in bovine hair The differences between the method of attack of C. keratinophilum on bovine and human hair appears, as with other fungi (English, 1963, 1965), to be related to differences in the structure of the hairs. The wide medulla is quickly penetrated by the fungus and the initial attack on the cortex is consequently from there rather than from the cuticle (PI. 15, fig. 6). Again, as with other fungi, no rope of medullary mycelium is formed, the hyphae remaining distinct and separate, some of normal width, some swollen (English, 1965, pl. 18, fig. 4). The cortex is invaded from these hyphae in three ways. It may be penetrated by borers or swollen borers in the same way as in human hair; fronds may be found pressed to, or probably just under, the surface of the medulla; and masses of mycelium may bulge into the cortex, themselves often giving rise to borers. From this stage both the cortex and cuticle are attacked in the same way as in human hair. DISCUSSION
It has been confirmed that C. keratinophilum is keratinolytic, but detailed observations show that both the morphology of the active mycelium, except the fronds, and the chemistry of its action are different from those of the dermatophytes. The examination offronded mycelium during this work has suggested a hypothesis which could explain, for all fungi, the way in which it is formed. When a hypha first makes its way between two closely adherent layers of a substance it not only forces them apart where they are directly in contact with it, but also rips them slightly apart on either side of itself (English, 1965). Lateral hyphae formed behind the leading hypha would, in normal circumstances, diverge from the leader at more or less wide angles. If this were to happen in a layered substratum each new lateral hypha would have to make a fresh penetration of the intact substratum thus using extra energy. Clearly if these hyphae were to follow the line of least resistance they would be channelled into the slits in the keratin on either side of the leading hypha, which would entail their growing in the same plane as that hypha and closely pressed to it. This is exactly what appears to occur in a frond. In growing in this way the lateral hyphae would enlarge the slits further and so give the next generation of
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laterals the same advantage. This process could continue indefinitely, resulting in the building up of the very large fronds produced by many fungi in layered substrata. C. keratinophilum resembles the dermatophytes in that pressure clearly plays an important part in penetration. It differs from them in that the mycelium buried in the hair is always closely pressed to the walls of the cavity in which it lies. The chemistry of keratin digestion by C. keratinophilum must therefore differ from that of the dermatophytes. The demonstration of what is possibly a zone of altered keratin adjacent to the cortical mycelium suggests that digestion by C. keratinophilum occurs in two stages. First the keratin is chemically altered to a structureless but still solid form. Secondly this is digested, the fungus simultaneously growing into the space so formed. Biochemical studies are necessary to elucidate these observations. On its digestive process is probably dependent the distinctive morphology of the keratin-invading mycelium of the fungus. The swollen borers characteristic of it are intermediate between its own narrow borers, which are identical with those of non-keratinolytic fungi, and the perforating organs of the dermatophytes. They indicate how perforating organs could have evolved from borers in species which developed keratinolytic properties, and provide support for this suggestion, put forward by me in 1965 and followed by Kunert (1967) from his observations on Keratinophyton terreum, but disputed by Pinetti & Lostia (1966). I am most grateful to Dr D. Frey and Dr M. Otcenasek for the cultures of C. keratinophilum; to Mr J. Washer for some of the photomicrographs and to Mr J. Hancock for assistance with the remainder. REFERENCES
AJELLO, L., VARSAVSKY, E., GINTHER, O.J. & BUBASH, G. (1964). The natural history of Microsporum nanum. Mycologia 54, 873-884. BOHME, H. & ZmGLER, H. (1967)' Keratinabbau durch Pilze. Arch. Mikrobiol. 67, 93- I 10. CARMICHAEL, J. ( I962). Chrysosporium and some other aleuriosporic hyphomycetes, Can.]. Bot. 40, 1137-1173. ENGLISH, M. P. (1963). The saprophytic growth of keratinophilic fungi on keratin. Sabouraudia 2, I 15- I 30. ENGLISH, M. P. (1965). The saprophytic growth of non-keratinophilic fungi on keratinized substrata, and a comparison with keratinophilic fungi. Trans. Br. mycol. Soc. 4 8, 2 19-325. ENGLISH, M. P. (1968). The developmental morphology of the perforating organs of dermatophytes. Sabouraudia 6, 218-227. EVOLCEANu, R., ALTERAS, I. & STOIAN, M. (1963)' Considerations sur la morphologie, Ie pouvoir pathogene et la geophilie du Microsporum nanum. Mycopath, Mycol. appl. Ig,24-36. FREY, D. (1959). Isolation of a new species of Aleurisma from soil in Australia and New Guinea. Mycologia 51, 641-646. KUNERT, J. (1967). Zur Mikromorphologie des Haarabbaues durch Dermatophyten in vitro. Derm, Wschr. 153, 1141-1150. OTCENASEK, M. & DvoRAK,J. (1964). The isolation ofChrysosporium keratinophilum (Frey) Carmichael 1962 and similar fungi from Czechoslovakian soil. Mycopath. Mycol. appl.23, 121-124. PlNETTI, P. & LOSTIA, A. (1966). L'infezione dermatofitica del pelo in vitro. Monograph 13, Rass, med. Sarda, Cagliari.
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Chrysosporium. Mary P. English EXPLANATION OF PLATES
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15 AND 16
PLATE 15
Fig. Fig.
Detail of fronded mycelium. x 1250. 2. Undulation of cuticle of human hair following deep penetration by fronded mycelium (f). No lysis of the cuticle is apparent. x 1250. Fig. 3. Rope of medullary mycelium (r) from human hair produced from, and giving rise to polymorphous boring hyphae and cortical mycelium. x 500. Fig. 4. Borer (b) arising from medullary mycelium from human hair and giving rise to lateral torpedo-shaped cortical mycelium (ts). x 1050. Fig. 5. Early stage in colonization of medulla of human hair, the mycelial rope producing borers which are growing back towards the cuticle. x 550. Fig. 6. Cortex of bovine hair colonized from medulla by swollen borer (sb) and torpedo-shaped mass of lateral mycelium (1$). x 1050. Fig. 7. Tip of torpedo-shaped cortical hypha sheathed by flattened cells. Small frond (1) produced from tip. x 1250. Fig. 8. Cortical mycelium of very varied morphology and orientation colonizing human hair I.
x 550. Figs. I, 3, 4, 7: KOH-phase contrast preparations of mycelium. Figs. mounts of whole hairs.
2,
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PLATE 16
Fig. I. Polymorphous cortical mycelium showing swollen borers (sb), borer (b) and torpedoshaped mycelium (ts). x 1250. Fig. 2. As fig. I, but showing balloon-shaped cells and borers (b). x 1250. Fig. 3. Human hair showing swelling at sites of maximal fungal attack. x 138. Fig. 4. End-on view of swollen borer in cortex of human hair, showing marked cracking of surrounding keratin. x 1250. Fig. 5. Swollen borer showing indented rings on cell wall and an early stage in the formation of torpedo-shaped lateral mycelium. x 1250. Fig. 6. Torpedo-shaped mass of cortical mycelium showing end-on view of swollen borer (sb) from which it originated, and serrated growing tip. x 1050. Fig. 7. Polymorphous mycelium in cortex of human hair. The comparatively narrow borer (b) overlies a swollen borer (sb) giving a false impression ofa zone oflysis round the former. x 1250. Figs. 1,2,5: KOH-phase contrast preparations of mycelium. Figs. 3, 4, 6, 7: cotton blue mounts of whole hairs.
(Accepted for publication 6 July 1968)