Arch. Microbiol. 106, 195-200 (1975) - 9 by Springer-Verlag 1975
Origin and Ultrastructure of Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum J. F. FARLEY, R. A. JERSILD, and D. J. NIEDERPRUEM Departments of Microbiologyand Anatomy, Indiana University Medical Center, Indianapolis, Indiana Received June 30, 1975 Abstract. A cell observationchamber was designedto perform continuous photomicroscopicobservations of hyphal anastomosis and the origin of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum. These data were correlated with ultrastructural features of intra-hyphal hyphae. Hyphal fusions occurred commonly in either species of Trichophyton when incubated alone. In T. terrestre,empty hyphal segments adjoined by live units were invaded at the septa from both
directions by new hyphal ingrowth. Continuous observations revealed that the intra-hyphal hyphae subsequently anastomosed via a lateral fusion peg. Similar intra-hyphal hyphae were shown in T. rubrum. Electron microscopic studies revealed ascomycetoussepta in both conventionalhyphae and intra-hyphal hyphae. For the latter, the cytoplasm and wall of the inner hypha were bounded by cytoplasmic organelles and another cell wall of the outer hypha.
Key words. Trichophyton terrestre - Trichophyton rubrum - Hyphal fusions - Origin of intra-hyphal hyphae - Electron microscopy.
Hyphal invasion by intra-hyphal hyphae (endohyphae, "proliferations internes") occurs in most major classes of fungi including some fungi pathogenic for man. The latter representatives are either dermatophytes such as Trichophyton mentagrophytes (Urabe and Izu, 1969) or pathogenic fungi which elicit systemic disease, as exemplified by Blastomyces dermatitidis and Paracoccidioides brasiliensis (Carbonell and Rodriguez, 1968). In addition to these pathogens, Ascomycetes which show intra-hyphal hyphae include Neurospora crassa (Lowry and Sussman, 1966), Ceratocystis dryocoetidus (Kendrick and Molnar, J965), Ascobolus magn~'cus (Dodge, 1920), Sclerotinia fructigena (Calonge, 1968) and Podospora anserina (Esser, 1972; Esser and Minuth, 1972) among others. In contrast, only a few Basidiomycetes contain intra-hyphal hyphae and members showing this behavior include Rhizoctonia solani (Butler and Bracker, 1970), Schizophyllum commune (Mayfield, 1974) and Gloeophyllum (Lenzites) saepiarum (States, 1975). Even fewer examples of intra-hyphal hyphae exist for Phycomycetes and among these are Saprolegnia (Dodge, 1920) and Linderina (Chan and Stephen, 1967). Interestingly, intra-hyphal hyphae not only grow within the recipient but they may also sporulate therein, as is the case of Aspergillus niger (Miller and Anderson, 1961), Ceratocystis ulmi (Ouellette and Gagnon, 1960) and Ceratocystis dryoeoetidis (Kendrick and Molnar, 1965). Our present attention was focused on the origin and ultrastructure of intra-hyphal hyphae in species
of Trichophyton whose pathogenic representatives elicit ringworm lesions on keratin-rich tissues of man. These circinate, annular lesions spread peripherally and bear a superficial resemblance to the growth bands of a "clock" mutant of Neurospora crassa cultivated on laboratory medium. Examination of the latter near the end of a growth band revealed the frequent occurrence of intra-hyphal hyphae (Lowry and Sussman, 1966). This suggested to us that the emergence of intra-hyphal hyphae during infection might serve as a virulence determinant for fungi which colonize human skin, possibly as a localized fungal growth manifestation to the immune response of the host. To begin to explore this particular issue, we studied the origin and ultrastructure of intra-hyphal hyphae in the non-pathogenic soil inhabitant T. terrestre (Durie and Frey, 1957) and compared it to the behavior of the human pathogen T. rubrum. Materials and Methods Organisms and Culture Conditions. Trichophyton terrestre str. 459, mating type (-), supplied as Arthroderma quadrifidum (-, no. 459) was generouslyprovided by Dr. L. Ajello, Mycology Division (Bureau of Laboratories), Center for Disease Control, Atlanta, Georgia30333. Trichophytonrubrum (str. S-R-24) was kindly supplied by Dr. W.W. Epinette (Department of Dermatology, Indiana University Medical Center, Indianapolis, Indiana 46202). All cultures were grown on complete plus yeast extract agar (C + Y) of the following composition (g/l): glucose, 20; peptone, 2; KHzPO4, 0.5; K2HPO4, 1; MgSO4.7H20, 0.5; yeast extract, 1; agar, 20. The growth incubation temperature for cultures was 23~
196
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Fig. 1. Fungal growth observation chamber
(+ 1) maintained in a Precision Scientific Incubator (model 806) with constant illumination (GE cool white lamp). Growth Chamber for Photomicroscopy. Microscopic observations of live cellular behavior and photomicroscopy were performed using bright-field optics and a Zeiss Photoscope II. Film was Kodak Ektachrome-X. A fungal growth chamber for direct microscopic observations was designed as Shown in Fig. 1. The advantages of this particular design are the superior optics provided by a sandwich of nutrient agar between two cover slips and the property of the chamber to withstand autoclave-sterilization after being cemented together by the epoxy resin mixture (Nu. 0151 Clear EpoxiPatch Kit, Hysol Division, The Dexter Corporation, Olean, New York 14760). Sterile C + Y agar was added to the chamber and inoculated with an aqueous suspension of conidia from a 20- 30 day culture, the chamber plugged with sterile cotton, and maintained in a Petri dish containing moist cotton to preserve high humidity. Incubation prior to microscopic observations was as above. All direct microscopic studies were performed at room temperature (i.e., 25~ + 3). Electron Microscopy. Cultures were incubated as above on C + Y agar medium. Mycelium from various portions of the colony were prepared for electron microscopy by fixation in 3~o phosphate-buffered glutaraldehyde (pH 7.1; 0.1 M) at room temperature for 1 hr, followed by a phosphate buffer rinse, and post-fixation in 1 ~o phosphate-buffered O~O4 (pH 7.3) (Millonig, 1961) at room temperature for 1 hr. Specimens were dehydrated through a graded series of ethyl alcohol and embedded in Epon (Luft, 1961). Sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Results
Initial studies were concerned with the capability of the growth chamber to sustain germination of microconidia of T. terrestre. In most instances the emergence of one germ tube preceeded the other by several hours. In additio n, septation of the initial germ tube and primary branch emergence ensued before these events occurred in the second germ tube of the same microconidium. Apical growth occurred in both germ tubes when the distal septa were employed as reference points. In addition, primary branch emergence always occurred in a region adjacent to a particular septum. Finally, intercalary cell division also occurred frequently in the young hyphae derived from both germ tubes to further partition the established proximal units into yet smaller compartments.
After 60 hrs of incubation in the growth chamber, a high frequency of hyphal anastomoses was evident among adjacent hyphae of T. terrestre, and eventually a complicated lattice of interconnecting hyphae resulted. Following branch outgrowth each apex exhibited a directed turning towards the other and subsequent fusion of the apices established cytoplasmic continuity between the adjacent hyphae. It is conceivable that a chemotactic substance may be operative here to govern directed growth of the apices towards one another but direct proof remains to be provided. More detailed study of the hyphae of T. terrestre incubated in the growth chamber over 60 hrs revealed intra-hyphal hyphae. Continuous microscopic examination of the behavior of live intra-hyphal hyphae are demonstrated in Figs. 2 and 3, in which a 85-hr culture of T. terrestre was studied microscopically for about 7 hrs. Note the presence of one intra-hyphal hypha from the left (Fig.2A and B) and the emergence of another from the right (Fig.2C). By 85 min the two intra-hyphal hyphae nearly made contact (Fig. 2H). Continued observations revealed contact at 90 min (Fig. 3 B) followed by the presence of a lateral fusion peg at 170 min (Fig. 3 D - H). Note also that the lower intra-hyphal hyphae continued to grow towards the right while the upper intra-hyphal hypha ceased to grow. Finally, observe the expansion of the intrahyphal hypha which originally arose from the left, to now nearly fill the previously empty segment through which it grew (Fig. 3G, 410rain). These results clearly demonstrate the importance of continuous microscopic observations of live cellular behavior before assessing the occurrence, mode of origin and fate of intra-hyphal hyphae in fungi, as has been the case so often in past investigations. The ultrastructural features of conventional vegetative hyphae of T. terrestre consisted of a typical ascomycetous septum with adjoining granules (Woro-. nin bodies?) which delimited the two compartments of the hypha. An intra-hyphal hypha is shown in longitudinal section in Fig, 4. The inner hypha contains typical cytoplasmic organelles and is bounded by a plasma membrane and cell wall, which in turn is surrounded by normal cytoplasm and cell wall of the recipient in which it appears housed. The apparent out-growth of an intra-hyphal hypha i s shown in Fig. 5A. Note that the endohypha also contains an ascomycetous septum with an adjacent granule. The cross-section shown in Fig. 5 B suggests the occurrence of two intra-hyphal hyphae within the same compartment, a finding not surprising in view of the live behavior- of intra-hyphal hyphae shown in Figs.2 and 3. While the occurrence of intra-hyphal hyphae was readily demonstrated in the saprophytic fungus T.
J. F. Farley et al. : Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum
197
2 3 Fig. 2 A - H. Origin and growth of intra-hyphal hyphae towards one another in T. terrestre. (A) 84-hr culture; (B) 20 min after (A); (C) 40 rain; (D) 50 rain; (E) 55 rain; (F) 70 rain; (G) 80 rain; (H) 84 rain. Note intrahyphal growth from left (A) while intra-hyphal ingrowth from right initiated at (C). x 800
Fig. 3 A - G. Continued microscopic observations of intra-hyphal hyphae seen in Fig. 2. (A) 85 rain; (B) 90 rain; (C) 115 rain; (D) 170 rain; (E) 230 rnin; (F) 290 rain; (G) 410 rain. Note meeting of intra-hyphal hyphae in (B), emergence of fusion peg (D and E), continued growth of lower intrahyphal hypha to right (E-G) and lateral expansion of intra-hyphal hyphae to essentially fill recipient hypha (G). x 1200
terrestre, these structures were not immediately apparent when the human pathogen T. rubrum was cultivated on C + Y agar at 23 ~ Since stress of any general nature may induce intra-hyphal hyphae in fungi (cf Bullet, 1933), we purposely stressed T. rubrum by temperature extremes (e.g., 23~ 7 days; 37~ 2 days; 4~ 12 hrs; 37~ 12 hrs). Under this regimen, intra-hyphal hyphae similar to thai: described in T. terrestre were evident in T. rubrum. Discussion
hyphal hyphae in live cells of the saprophyte T. terrestre and the human pathogen T. rubrum. The common occurrence of conventional hyphal fusions in either fungus when cultivated alone was not unexpected since hyphal anastomosis occurs in various other dermatophytes including Microsporum audouinii, M. lanosum and T. gypseum (Davidson et'al., 1932) nor do hyphal fusions, as studied here, need bear any relation to sexuality. Hyphae of the mycelial phase of the human pathogen Histoplasma capsulatum likewise undergo anastomosis (Berliner, 1968).
The present study provides direct microscopic evidence for the occurrence and subsequent fusion of intra-
The oriented growth of vegetative hyphal branches towards one another in T. terrestre may be controlled by
198
Arch. Microbiol., Vol. 106, No. 3 (1975)
4
5 Fig. 4. Ultrastructure of intra-hyphal hypha in T. terrestre, x 9000
Fig. 5A and B. Ultrastructure of intra-hyphal hypha duriiag apparent outgrowth (A) and two intra-hyphal hyphae in cross section (B) in T. terrestre. Note ascomycetous septum and associated Woronin Body in intra-hyphal hypha (A). Arrows indicate recipient hyphal cytoplasm, x 13 000
chemotactic substances of the type found in Ascobolus stereorarius (Bistis, 1956, 1957) and species o f Achlya (Raper, 1951) among others. In the latter cases, however, hormonal mechanisms play a vital role in sexuality. Moreover, in mating of Schizophyllum commune, direct evidence has been provided for the A incompatibility factor to genetically govern hyphal
fusion in this Basidiomycete (Ahmad and Miles, 1970a). Further studies showed that the nutritional milieu also exerted an influence on the degree of hyphal fusions in S. commune in that little difference occurred in fusion frequency when the glucose concentration was above 0.5 ~o but in the absence of carbohydrate the frequency was greatly increased (Ahmad and
J. F. Farley et al. : Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum Miles, 1970b). In the case of T. terrestre, hyphal fusions only occurred in older cultures and this, again, may reflect the exhaustion of exogenous nutrients although direct data are lacking on this issue. While conventional hyphal fusions were expected here, quite unexpected was the subsequent fusion of the two intra-hyphal hyphae as they grew towards one a n o t h e r t h r o u g h an apparently e m p t y cell o f the septate hypha. Previous w o r k employing electron microscopic examination o f sectioned material o f Sclerotinia fructigena alluded to ~Ihe possibility of anastomosis between intra-hyphal hyphae but this could not be distinguished f r o m the alternate possibility t h a t the section merely represented a site of branch initiation (Calonge, 1968). The present data gathered on the live behavior of intra-hyphal hyphae of T. terrestre clearly show fusion of the two endoh y p h a e via a lateral fusion peg. In all probability, this also occurs in T. rubrum. It should be noted that other phenomena may also be operative for T. rubrum. Observations of live hyphae of T. rubrum showed a "sudden transfer within a few minutes of the entire contents of one cell into the adjacent cell"; thereafter, the cytoplasm from the neighboring proximal cell gradually moved forward to refill the empty segment (Taplin and Blank, 1961). Whether the latter phenomenon actually represented ingrowth by an intra-hyphal hypha remains unknown because ultrastructural examination of T. rubrum by these workers did not reveal their presence. The current work does provide electron microscopic evidence describing the double structure of outer and inner hyphae in T. terrestre, which we interpret as representing intra-hyphal hyphae. Similar evidence has been offered in the case of T. mentagrophytes although no mention was made of intra-hyphat hyphae in fine structural studies of other dermatophytes including M. canis (Werner et al., 1966), Epidermophytonfloccosum ([,aden and Erickson, 1958; Werner et al., 1964). M. audouinii (Werner et al., 1968), M. gypseum (Werner et aL, 1967), and T. violaceum (Ito et al., 1967). Finally, the question arises as to the role(s) of intra-hyphal hyphae in fungi including those pathogenic for man or plants. From data gained in the present work, we feel that one function of the endohyphae is to reestablish cytoplasmic continuity in septate hyphae which bear senescent or "empty" segments. This could clearly be imagined where two endohyphae meet and fuse after ingrowth through an empty segment from opposite directions. Intra-hyphal hyphae occur in the fungus Ceratocystis dryocoetidis during experimental infection of the alpine fir (Kendrick and Molnar, 1965) and this could conceivably be a fungal defense reaction to some as yet unidentified toxic product elaborated by the invaded plant. Regarding human infection and dermatophytosis elicited by Triehophyton, we know of no cytological evidence for the occurrence of intra-hyphal hyphae in skin lesions of man. Moreover, present evidence shows a high frequency of intra-hyphal hyphae in the saprophyte T. terrestre and only a low frequency in the human pathogen T. rubrum. We are examining Tinea in man in the hope that this matter can be clarified. Acknowledgements. We accord our thanks to Mrs. Vera McAdoo for technical assistance. We also wish to thank Mrs. Marylyn Bartlett for helpful consultation.
199 References
Ahmad, S. S., Miles, P. G. : Hyphal fusions in the woodrotting fungus Schizophyllum commune. I. The effects of incompatibility factors. Genet. Res. Camb. 15, 19-28 (1970a) Ahmad, S. S., Miles, P. G. : Hyphal fusions in Schizophyllum commune. II. Effects of environmental and chemical factors. Mycologia (N.Y.) 62, 1008-1017 (1970b) Berliner, M. D. : Primary subcultures of Histoplasma capsulatum. I. Macro- and micro-morphology of the mycelial phase. Sabouraudia 6, 111 - 118 (~968) Bistis, G. N. : Sexuality in Ascobolus stercorarius. I. Morphology of the ascogonium; Plasmogamy; evidence for a sexual hormonal mechanism. Amer. J. Bot. 43, 389-394 (1956) Bistis, G. N. : Sexuality in Ascobolus stercorarius. II. Preliminary experiments on various aspects of the sexual process. Amer. J. Bot. 44, 436-443 (1957) Buller, A. H . R . : Researches on fungi, Vol. V. Toronto: Longmans, Green and Co. 1933 Butler, E. E., Bracker, C. E. : Morphology and cytology of Rhizoctonia solani. Biology and pathology, J. R. Parmeter, ed., pp. 32-51. Berkeley-Los Angeles-London: University of California Press 1970 Calonge, F. D.: Origin and development of intra-hyphal hyphae in Sclerotinia fi'uct!gena. Mycologia (N.Y.) 60, 932- 942 (1968) Carbonell, L. M., Rodriguez, J. : Mycelial phase of Paracoccidioides brasiliensis and BIastomyces" dermatitidis: An electron microscope study. J. Bact. 96, 533-543 (1968) Chan, C., Stephen, R. C. : Intrahyphal hyphae in the genus Linderina. Canad. J. Bot. 45, 1995-1998 (1967) Davidson, A. M., Dowding, E. S., Butler, A. H. R. : Hyphal fusions in dermatophytes. Canad. J. Res. 6, 1 - 2 0 (1932) Dodge, B. O. : The life history ofAscobolus magnificus. Mycologia (N.Y.) 12, 115- I34 (1920) Durie, E. B., Frey, D. : A new species of Trichophyton from New South Wales. Mycologia (N.Y.) 49, 401- 411 (1957) Esser, K. : Genetic and biochemical analysis of rhythmically growing mycelia in the Ascomycete Podospora anserina. J. Interdiscipi. Cycle Res. 3, 123-128 (1972) Esser, K., Minuth, W. : Abnormal cell wall structure in rhythmically growing mycelia of Podospora anserina. Cytobiologie 5, 319-323 (1972) Ito, Y., Setaguti, T., Nozawa, Y., Sakurai, S. : An electron microscope observation of Trichophyton violaceum. J. invest. Derm. 48, 124-127 (1967) Kendrick, W. B., Molnar, A, C. : A new Ceratocystis and its Verticicladiella imperfect state associated with the bark beetle Dryocoetes confusus on Abies lasiocarpa. Canad. J. Bot. 43, 39-43 (1965) Laden, L. E., Erickson, J. O. : Electron microscopic study of Epidermophyton floccosum. J. invest. Derm. 31, 51-58 (1958) Lowry, R. J., Sussman, A. S. : Iutra-hyphal hyphae in "clock" mutants of Neurospora. Mycologia (N.Y.) 58, 541-548 (1966) Luft, J. H. : Improvements in epoxy resin embedding materials. J. biophys, biochem. Cytol. 9, 409-414 (1961) Mayfield, J. E. : Septal involvement in nuclear migration in Schizophyllum commune. Arch. Microbiol. 95, 115-124 (1974) Miller, C. W., Anderson, N.A.: Proliferation of conidiophores and intrahyphal hyphae in Aspergillus niger. Mycologia (N.Y.) 53, 433-436 (1961)
200 Millonig, G. : The advantages of a phosphate buffer for OsO4 solutions in fixation. J. appl. Phys. 32, 1637 (1961) Ouellette, G. B., Gagnon, C. : Formation ofmicroendospores in Ceratocystis ulmi (Buism.) C. Moreau. Canad. J. Bot. 38, 235-241 (1960) Raper, J. R. : Sexual hormones in Achlya. Amer. Sci. 39, 1 1 0 - 1 2 0 (1951) Reynolds, E. S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. cell Biol. 17, 208-212 (1963) States, J. S. : Intrahyphal hyphae in the Basidiomycete, Gloeophyllum (Lenzites) saepiarum. Mycologia (N.Y.)67, 417-420 (1975) Taplin, D., Blank, H. : Microscopic morphology o f Trichophyton rubrum. J, invest. Derm. 37, 523-528 (1961)
Prof. D. J. Niederpruem Department of Microbiology,. Indiana University Medical Center 1100 West Michigan Street, Indianapolis, Indiana 46202, u.S.A.
Arch. Microbiol., Vol. 106, No. 3 (1975) Urabe, H., Izu, T.: The ultrastructure of Trichophyton and a double cell wall in the hypha. J. invest. Derm. 52, 508-513 (1969) Werner, H. J., Catsulis, C., Jolly, H. W., Jr., Carpenter, C. L. : Electron microscope observations of the fine structure of Microsporum gypseum. J. invest. Derm. 48, 481-484 (1967) Werner, H.J., Jolly, H.W., Jr., Carpenter, C.L.: Observation on the fine structure of Microsporurn audouinii. J. invest. Derm. 50, 276-279 (1968) Werner, H. J., Jolly, H. W., Jr., Lee, J. H. : Electron microscopic observations of Epidermophyton floccosum. J. invest. Derm. 43, 139-143 (1964) Werner, H. J., Jolly, H. W., Jr., Spurlock, B. O.: Electron microscopic observations of the fine structure of Microsporum canis. J. invest. Derm. 46, 130-134 (1966)
Origin and Ultrastructure of Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum J. F. FARLEY, R. A. JERSILD, and D. J. NIEDERPRUEM Departments of Microbiologyand Anatomy, Indiana University Medical Center, Indianapolis, Indiana Received June 30, 1975 Abstract. A cell observationchamber was designedto perform continuous photomicroscopicobservations of hyphal anastomosis and the origin of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum. These data were correlated with ultrastructural features of intra-hyphal hyphae. Hyphal fusions occurred commonly in either species of Trichophyton when incubated alone. In T. terrestre,empty hyphal segments adjoined by live units were invaded at the septa from both
directions by new hyphal ingrowth. Continuous observations revealed that the intra-hyphal hyphae subsequently anastomosed via a lateral fusion peg. Similar intra-hyphal hyphae were shown in T. rubrum. Electron microscopic studies revealed ascomycetoussepta in both conventionalhyphae and intra-hyphal hyphae. For the latter, the cytoplasm and wall of the inner hypha were bounded by cytoplasmic organelles and another cell wall of the outer hypha.
Key words. Trichophyton terrestre - Trichophyton rubrum - Hyphal fusions - Origin of intra-hyphal hyphae - Electron microscopy.
Hyphal invasion by intra-hyphal hyphae (endohyphae, "proliferations internes") occurs in most major classes of fungi including some fungi pathogenic for man. The latter representatives are either dermatophytes such as Trichophyton mentagrophytes (Urabe and Izu, 1969) or pathogenic fungi which elicit systemic disease, as exemplified by Blastomyces dermatitidis and Paracoccidioides brasiliensis (Carbonell and Rodriguez, 1968). In addition to these pathogens, Ascomycetes which show intra-hyphal hyphae include Neurospora crassa (Lowry and Sussman, 1966), Ceratocystis dryocoetidus (Kendrick and Molnar, J965), Ascobolus magn~'cus (Dodge, 1920), Sclerotinia fructigena (Calonge, 1968) and Podospora anserina (Esser, 1972; Esser and Minuth, 1972) among others. In contrast, only a few Basidiomycetes contain intra-hyphal hyphae and members showing this behavior include Rhizoctonia solani (Butler and Bracker, 1970), Schizophyllum commune (Mayfield, 1974) and Gloeophyllum (Lenzites) saepiarum (States, 1975). Even fewer examples of intra-hyphal hyphae exist for Phycomycetes and among these are Saprolegnia (Dodge, 1920) and Linderina (Chan and Stephen, 1967). Interestingly, intra-hyphal hyphae not only grow within the recipient but they may also sporulate therein, as is the case of Aspergillus niger (Miller and Anderson, 1961), Ceratocystis ulmi (Ouellette and Gagnon, 1960) and Ceratocystis dryoeoetidis (Kendrick and Molnar, 1965). Our present attention was focused on the origin and ultrastructure of intra-hyphal hyphae in species
of Trichophyton whose pathogenic representatives elicit ringworm lesions on keratin-rich tissues of man. These circinate, annular lesions spread peripherally and bear a superficial resemblance to the growth bands of a "clock" mutant of Neurospora crassa cultivated on laboratory medium. Examination of the latter near the end of a growth band revealed the frequent occurrence of intra-hyphal hyphae (Lowry and Sussman, 1966). This suggested to us that the emergence of intra-hyphal hyphae during infection might serve as a virulence determinant for fungi which colonize human skin, possibly as a localized fungal growth manifestation to the immune response of the host. To begin to explore this particular issue, we studied the origin and ultrastructure of intra-hyphal hyphae in the non-pathogenic soil inhabitant T. terrestre (Durie and Frey, 1957) and compared it to the behavior of the human pathogen T. rubrum. Materials and Methods Organisms and Culture Conditions. Trichophyton terrestre str. 459, mating type (-), supplied as Arthroderma quadrifidum (-, no. 459) was generouslyprovided by Dr. L. Ajello, Mycology Division (Bureau of Laboratories), Center for Disease Control, Atlanta, Georgia30333. Trichophytonrubrum (str. S-R-24) was kindly supplied by Dr. W.W. Epinette (Department of Dermatology, Indiana University Medical Center, Indianapolis, Indiana 46202). All cultures were grown on complete plus yeast extract agar (C + Y) of the following composition (g/l): glucose, 20; peptone, 2; KHzPO4, 0.5; K2HPO4, 1; MgSO4.7H20, 0.5; yeast extract, 1; agar, 20. The growth incubation temperature for cultures was 23~
196
Arch. Microbiol., Vol. 106, No. 3 (1975) NUTRIENT
AGAR
Fig. 1. Fungal growth observation chamber
(+ 1) maintained in a Precision Scientific Incubator (model 806) with constant illumination (GE cool white lamp). Growth Chamber for Photomicroscopy. Microscopic observations of live cellular behavior and photomicroscopy were performed using bright-field optics and a Zeiss Photoscope II. Film was Kodak Ektachrome-X. A fungal growth chamber for direct microscopic observations was designed as Shown in Fig. 1. The advantages of this particular design are the superior optics provided by a sandwich of nutrient agar between two cover slips and the property of the chamber to withstand autoclave-sterilization after being cemented together by the epoxy resin mixture (Nu. 0151 Clear EpoxiPatch Kit, Hysol Division, The Dexter Corporation, Olean, New York 14760). Sterile C + Y agar was added to the chamber and inoculated with an aqueous suspension of conidia from a 20- 30 day culture, the chamber plugged with sterile cotton, and maintained in a Petri dish containing moist cotton to preserve high humidity. Incubation prior to microscopic observations was as above. All direct microscopic studies were performed at room temperature (i.e., 25~ + 3). Electron Microscopy. Cultures were incubated as above on C + Y agar medium. Mycelium from various portions of the colony were prepared for electron microscopy by fixation in 3~o phosphate-buffered glutaraldehyde (pH 7.1; 0.1 M) at room temperature for 1 hr, followed by a phosphate buffer rinse, and post-fixation in 1 ~o phosphate-buffered O~O4 (pH 7.3) (Millonig, 1961) at room temperature for 1 hr. Specimens were dehydrated through a graded series of ethyl alcohol and embedded in Epon (Luft, 1961). Sections were stained with uranyl acetate and lead citrate (Reynolds, 1963). Results
Initial studies were concerned with the capability of the growth chamber to sustain germination of microconidia of T. terrestre. In most instances the emergence of one germ tube preceeded the other by several hours. In additio n, septation of the initial germ tube and primary branch emergence ensued before these events occurred in the second germ tube of the same microconidium. Apical growth occurred in both germ tubes when the distal septa were employed as reference points. In addition, primary branch emergence always occurred in a region adjacent to a particular septum. Finally, intercalary cell division also occurred frequently in the young hyphae derived from both germ tubes to further partition the established proximal units into yet smaller compartments.
After 60 hrs of incubation in the growth chamber, a high frequency of hyphal anastomoses was evident among adjacent hyphae of T. terrestre, and eventually a complicated lattice of interconnecting hyphae resulted. Following branch outgrowth each apex exhibited a directed turning towards the other and subsequent fusion of the apices established cytoplasmic continuity between the adjacent hyphae. It is conceivable that a chemotactic substance may be operative here to govern directed growth of the apices towards one another but direct proof remains to be provided. More detailed study of the hyphae of T. terrestre incubated in the growth chamber over 60 hrs revealed intra-hyphal hyphae. Continuous microscopic examination of the behavior of live intra-hyphal hyphae are demonstrated in Figs. 2 and 3, in which a 85-hr culture of T. terrestre was studied microscopically for about 7 hrs. Note the presence of one intra-hyphal hypha from the left (Fig.2A and B) and the emergence of another from the right (Fig.2C). By 85 min the two intra-hyphal hyphae nearly made contact (Fig. 2H). Continued observations revealed contact at 90 min (Fig. 3 B) followed by the presence of a lateral fusion peg at 170 min (Fig. 3 D - H). Note also that the lower intra-hyphal hyphae continued to grow towards the right while the upper intra-hyphal hypha ceased to grow. Finally, observe the expansion of the intrahyphal hypha which originally arose from the left, to now nearly fill the previously empty segment through which it grew (Fig. 3G, 410rain). These results clearly demonstrate the importance of continuous microscopic observations of live cellular behavior before assessing the occurrence, mode of origin and fate of intra-hyphal hyphae in fungi, as has been the case so often in past investigations. The ultrastructural features of conventional vegetative hyphae of T. terrestre consisted of a typical ascomycetous septum with adjoining granules (Woro-. nin bodies?) which delimited the two compartments of the hypha. An intra-hyphal hypha is shown in longitudinal section in Fig, 4. The inner hypha contains typical cytoplasmic organelles and is bounded by a plasma membrane and cell wall, which in turn is surrounded by normal cytoplasm and cell wall of the recipient in which it appears housed. The apparent out-growth of an intra-hyphal hypha i s shown in Fig. 5A. Note that the endohypha also contains an ascomycetous septum with an adjacent granule. The cross-section shown in Fig. 5 B suggests the occurrence of two intra-hyphal hyphae within the same compartment, a finding not surprising in view of the live behavior- of intra-hyphal hyphae shown in Figs.2 and 3. While the occurrence of intra-hyphal hyphae was readily demonstrated in the saprophytic fungus T.
J. F. Farley et al. : Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum
197
2 3 Fig. 2 A - H. Origin and growth of intra-hyphal hyphae towards one another in T. terrestre. (A) 84-hr culture; (B) 20 min after (A); (C) 40 rain; (D) 50 rain; (E) 55 rain; (F) 70 rain; (G) 80 rain; (H) 84 rain. Note intrahyphal growth from left (A) while intra-hyphal ingrowth from right initiated at (C). x 800
Fig. 3 A - G. Continued microscopic observations of intra-hyphal hyphae seen in Fig. 2. (A) 85 rain; (B) 90 rain; (C) 115 rain; (D) 170 rain; (E) 230 rnin; (F) 290 rain; (G) 410 rain. Note meeting of intra-hyphal hyphae in (B), emergence of fusion peg (D and E), continued growth of lower intrahyphal hypha to right (E-G) and lateral expansion of intra-hyphal hyphae to essentially fill recipient hypha (G). x 1200
terrestre, these structures were not immediately apparent when the human pathogen T. rubrum was cultivated on C + Y agar at 23 ~ Since stress of any general nature may induce intra-hyphal hyphae in fungi (cf Bullet, 1933), we purposely stressed T. rubrum by temperature extremes (e.g., 23~ 7 days; 37~ 2 days; 4~ 12 hrs; 37~ 12 hrs). Under this regimen, intra-hyphal hyphae similar to thai: described in T. terrestre were evident in T. rubrum. Discussion
hyphal hyphae in live cells of the saprophyte T. terrestre and the human pathogen T. rubrum. The common occurrence of conventional hyphal fusions in either fungus when cultivated alone was not unexpected since hyphal anastomosis occurs in various other dermatophytes including Microsporum audouinii, M. lanosum and T. gypseum (Davidson et'al., 1932) nor do hyphal fusions, as studied here, need bear any relation to sexuality. Hyphae of the mycelial phase of the human pathogen Histoplasma capsulatum likewise undergo anastomosis (Berliner, 1968).
The present study provides direct microscopic evidence for the occurrence and subsequent fusion of intra-
The oriented growth of vegetative hyphal branches towards one another in T. terrestre may be controlled by
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4
5 Fig. 4. Ultrastructure of intra-hyphal hypha in T. terrestre, x 9000
Fig. 5A and B. Ultrastructure of intra-hyphal hypha duriiag apparent outgrowth (A) and two intra-hyphal hyphae in cross section (B) in T. terrestre. Note ascomycetous septum and associated Woronin Body in intra-hyphal hypha (A). Arrows indicate recipient hyphal cytoplasm, x 13 000
chemotactic substances of the type found in Ascobolus stereorarius (Bistis, 1956, 1957) and species o f Achlya (Raper, 1951) among others. In the latter cases, however, hormonal mechanisms play a vital role in sexuality. Moreover, in mating of Schizophyllum commune, direct evidence has been provided for the A incompatibility factor to genetically govern hyphal
fusion in this Basidiomycete (Ahmad and Miles, 1970a). Further studies showed that the nutritional milieu also exerted an influence on the degree of hyphal fusions in S. commune in that little difference occurred in fusion frequency when the glucose concentration was above 0.5 ~o but in the absence of carbohydrate the frequency was greatly increased (Ahmad and
J. F. Farley et al. : Intra-Hyphal Hyphae in Trichophyton terrestre and T. rubrum Miles, 1970b). In the case of T. terrestre, hyphal fusions only occurred in older cultures and this, again, may reflect the exhaustion of exogenous nutrients although direct data are lacking on this issue. While conventional hyphal fusions were expected here, quite unexpected was the subsequent fusion of the two intra-hyphal hyphae as they grew towards one a n o t h e r t h r o u g h an apparently e m p t y cell o f the septate hypha. Previous w o r k employing electron microscopic examination o f sectioned material o f Sclerotinia fructigena alluded to ~Ihe possibility of anastomosis between intra-hyphal hyphae but this could not be distinguished f r o m the alternate possibility t h a t the section merely represented a site of branch initiation (Calonge, 1968). The present data gathered on the live behavior of intra-hyphal hyphae of T. terrestre clearly show fusion of the two endoh y p h a e via a lateral fusion peg. In all probability, this also occurs in T. rubrum. It should be noted that other phenomena may also be operative for T. rubrum. Observations of live hyphae of T. rubrum showed a "sudden transfer within a few minutes of the entire contents of one cell into the adjacent cell"; thereafter, the cytoplasm from the neighboring proximal cell gradually moved forward to refill the empty segment (Taplin and Blank, 1961). Whether the latter phenomenon actually represented ingrowth by an intra-hyphal hypha remains unknown because ultrastructural examination of T. rubrum by these workers did not reveal their presence. The current work does provide electron microscopic evidence describing the double structure of outer and inner hyphae in T. terrestre, which we interpret as representing intra-hyphal hyphae. Similar evidence has been offered in the case of T. mentagrophytes although no mention was made of intra-hyphat hyphae in fine structural studies of other dermatophytes including M. canis (Werner et al., 1966), Epidermophytonfloccosum ([,aden and Erickson, 1958; Werner et al., 1964). M. audouinii (Werner et al., 1968), M. gypseum (Werner et aL, 1967), and T. violaceum (Ito et al., 1967). Finally, the question arises as to the role(s) of intra-hyphal hyphae in fungi including those pathogenic for man or plants. From data gained in the present work, we feel that one function of the endohyphae is to reestablish cytoplasmic continuity in septate hyphae which bear senescent or "empty" segments. This could clearly be imagined where two endohyphae meet and fuse after ingrowth through an empty segment from opposite directions. Intra-hyphal hyphae occur in the fungus Ceratocystis dryocoetidis during experimental infection of the alpine fir (Kendrick and Molnar, 1965) and this could conceivably be a fungal defense reaction to some as yet unidentified toxic product elaborated by the invaded plant. Regarding human infection and dermatophytosis elicited by Triehophyton, we know of no cytological evidence for the occurrence of intra-hyphal hyphae in skin lesions of man. Moreover, present evidence shows a high frequency of intra-hyphal hyphae in the saprophyte T. terrestre and only a low frequency in the human pathogen T. rubrum. We are examining Tinea in man in the hope that this matter can be clarified. Acknowledgements. We accord our thanks to Mrs. Vera McAdoo for technical assistance. We also wish to thank Mrs. Marylyn Bartlett for helpful consultation.
199 References
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Prof. D. J. Niederpruem Department of Microbiology,. Indiana University Medical Center 1100 West Michigan Street, Indianapolis, Indiana 46202, u.S.A.
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