Journalof Medical& VeterinaryMycology1997,35, 289-293
Accepted 28 April 1997
Short communication
Contact-sensing by hyphae of dermatophytic and
saprophytic fungi
T. H. S. PERERA, D. W. G R E G O R Y , * D. M A R S H A L L * & N. A. R. G O W
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen
AB25 2ZD, UK and *Department of Medical Microbiology, Medical School Buildings, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK
Contact-sensing or thigmotropism is the directional growth response of cells in
relation to topographical guidance cues. Thigmotropism is thought to play a major
role in the location of infectable sites on plants by phytopathogenic fungi and has
recently been shown to be a property of hyphae in the human pathogenic fungus
Candida albicans. Here we show that hyphae of the dermatophytes Epidermophyton
floccosum, Microsporum canis and Trichophyton mentagrophytes reorientate their
direction of growth in response to grooves and pores of membrane substrata as did
hyphae of the saprophytes Mucor mucedo and Neurospora crassa. This suggests that
the thigmotropic behaviour of hyphae is not a specific property of pathogens, but
rather a general feature of the growth of fungal hyphae that must forage for nutrients
on surfaces and within solid materials.
Keywords contact-sensing, dermatophyte, hyphal growth, thigmotropism
Introduction
A broad range of eukaryotic cells are able to reorientate
their direction of growth in relation to physical or topographical features of the growth substratum [1-3]. This
tropic behaviour is known variously as contact-sensing,
contour guidance or thigmotropism. Examples include
plant roots that can reversibly rotate the root apex or
achieve the circumnavigation of obstacles that they
encounter in soil [4], and the growth of developing
embryo axonal or dendritic processes that must organize
their growth within solid embryonic or regenerating tissues to achieve the innervation of specific targets [2,5]. In
addition, contact-sensing is an important aspect of pathogenesis in the pre-penetration stages of some plant pathogenic fungi such as the rice blast fungus Magnaporthe
grisea [6] and rust fungi such as Uromyces, for which
detailed studies on thigmotropism have been conducted
[3,7 11]. In these fungi contact-sensing is normally
employed to locate the guard cells of stomata through
which penetration of the appressorium infection peg
Correspondence: Professor N.A.R. Gow, Department of Molecular
and Cell Biology, Institute of Medical Sciences, University of
Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
,e~1997 ISHAM
takes place, Appressorium formation is induced by
thigmo-differentiation in responses to the precise heights
of the stomata l guard cell lips [8,11,12]. Similar hyphaI
behaviour has also been observed in the entomopathogenic fungus Metarhizium anisopliae [13,14]. Therefore
the ability to sense and respond to features of the
growth substratum is an adaptation of a large number
of tip growing cells that live on and within solid
materials.
We have already described thigmotropic responses of
hyphae of Candida albicans [15,16]. Hyphae of this dimorphic, human pathogenic fungus were shown to be guided
by pore~, scratches, grooves and ridges of a variety of
artificial membranes. This suggested that clinically
important fungi may employ thigmotropism to potentiate
the invasion of epithelia through wounds, surface invaginations or other points of weakened surface integrity
[17,18]. Further evidence of the clinical significance of
thigmotropism was found when ultrastructural investigations were performed on biopsy specimens from
patients with pseudomembranous oral candidasis [19]. In
this study Candida hyphae were seen to penetrate the
entire epithelium and cross the basal membrane invading
connective tissues through intercellular spaces.
Perera et al.
Fig. 1 Scanning electron micrographs of
hyphae responding to grooved surfaces. (a
and b) M. canis following scratches; note
in (a) only the hyphae which closely adhered to the substrate responded (arrows).
(c and d) E. floccosum following scratches
in cellophane membranes. Note in (c)
branches are formed at 90 ° to the main
hypha (arrow). (e) 72 mentagrophytes growing along the edge of a scratch.
Bars = 20/~m.
Infection by hyphae of dermatophytes is generally
cutaneous and restricted to dead cornified layers, and
penetration into the deeper tissues occurs only in
immunocompromised hosts. We report here thigmotropic
behaviour in fungi that are representative of the three
major genera of dermatophytes and suggest that this
behaviour may facilitate the fungi to burrow within these
keratinized layers.
lates obtained from the Regional Mycology Reference
Laboratory, Glasgow. Mucor mucedo LAN Z43- and
Neurospora crassa WT EmA124 were from the University
of Aberdeen culture collection.
The dermatophytes were grown on Sabouraud glucose
agar (SGA) at 30 °C. 34. mucedo and N. crassa were
grown on malt extract agar (MEA) at 25 °C. These solid
media were overlaid with various artificial membranes as
described below.
Materials and methods
Organisms and growth media
Nuclepore membranes
Epidermophyton floccosum, Microsporum canis and
Trichophyton mentagrophytes were all recent clinical iso-
Nuclepore membranes (pore diameters 5, 8 and 10pm)
were autoclaved between moist filter paper and placed
~ 1997 ISHAM, Journal of Medical & Veterinary Mycology 35, 289-293
Contact-sensing by fungal hyphae
\
\
Fig, 2 Scanning electron micrograph of
hyphae penetrating pores. (a) E. floccosum
entering pores on the upper surface of a
Nuclepore membrane (arrows). (b and c)
Show the under surfaces of the
membranes, which have been penetrated
by M. canis from above. Note the intense
branching of hyphae in (c) (arrows).
Bars = 20 #m.
over SGA or MEA plates. Using a sterile loop or toothpick, fragments of mycelium were scraped off from cultures that were growing for 4~5 days, and spread over the
membranes. Plates were incubated at the appropriate
temperatures for 16-24 h. Thereafter the membranes with
the attached mycelium were lifted from the underlying
agar and fixed as described below for examination in the
scanning electron microscope.
washed in 0.1 M phosphate buffer pH 7'2, and post-fixed
in osmium tetroxide for 1.5 h. The membranes were then
washed in distilled water and dehydrated through a
graded series of ethanol solutions. The samples were
then critical point dried from CO2, glued onto aluminium
stubs with colloidal silver adhesive, and sputter-coated
with 20 nm platinum and examined in a Jeol JSM-35CF
scanning electron microscope operating at 12 kV.
Cellophane membranes
Cellophane membranes were scratched using sandpaper
to obtain a topography which was predominantly flat
with a range of grooves of varying depth. The membranes
were then washed in 70% (v:v) ethanol and boiled for
20 rain in distilled water to remove plasticizers. Thereafter
the membranes were autoclaved between moist filter
papers before placing on SGA or MEA plates. The
membranes were inoculated as described above.
Scanning electron microscopy
The membranes with attached mycelium were lifted from
the underlying agar and immersed in 2.5% (v:v) glutaraldehyde and 2.5 mM MgC12 in 0.89 M phosphate buffer,
pH 7-2 for 2 h at room temperature. Thereafter they were
© 1997 ISHAM, Journal of Medical & VeterinaryMycology35, 289-293
Results
The hyphae of all the dermatophytes reorientated
when encountering scratches on Cellophane membranes
(Fig. la-e). Hyphae grew either along the bottom of the
scratches (Fig. 1b and c) or followed the ridge at the top
of the scratch (Fig. le). E. floccosum formed a highly
branched mycelium with hyphae that grew along the
Cellophane grooves (Fig. l d). Branches from the main
hypha often formed perpendicular to the scratch (Fig. lc).
When mycelia were grown on Nuclepore membrane
filters that were placed over SGA, hyphae grew over the
membrane surface and on contacting a lip of a pore grew
through to the other side of the membrane (Figs 2a and
3a). This was observed for all three dermatophytes.
Examination of the under surface of the membrane
Perera et al.
; %,,
Fig. 3 Scanning electron micrographs of
(a) T. mentagrophytes penetrating pores
(arrows). (b) M. mucedo and (c) N. crassa
following scratches on Cellophane
membranes. (d) Shows the underside of
the membrane where a hypha of N. crassa
has penetrated from above. Bars = 20/tm.
showed hyphae which had grown from the top to the
bottom (Fig. 2b). Hyphae which contacted a pore on the
under surface often grew back through to the top surface.
During the process of penetration from the top to the
bottom surface hyphae of M. canis formed a profusion of
apical branches that then grew towards the lower surface
of the membrane (Fig. 2c). The lower surface of the
membrane could be readily distinguished from the upper
surface by the presence of adhering fragments of agar.
M. mucedo and N. crassa were also guided by scratches
on membranes and responded to pores demonstrating
that hyphae of the non-pathogenic fungi also exhibit
thigmotropism (Fig. 3b-d).
Discussion
We have described here thigmotropic responses of hyphae
of the human pathogenic fungi E. floccosum, T. mentagrophytes and M. canis and the saprophytic fungi N. crassa
and M. mucedo. These fungi responded to scratches by
redirecting their growth and following them on contact.
Growth along ridges and scratches is a frequently
observed thigmotropic response in neurones [20] and is
similar to epiphytic growth patterns of certain pathogens
of plants. However, this ridge-following behaviour differs
from the common pattern of contact sensing by the
phytopathogenic fungi such as the Uromyces spp. and
Puccinia hordei, which grow perpendicular to cell junctions [3]. For pathogens which grow on graminaceous
plants, perpendicular growth maximizes the chance of
a germ tube encountering a stoma, as these occur in
staggered rows. However, Rhizoctonia solani [1], which
infects cotton and secondary germ tubes of P. hordei [3]
also grow parallel to the longitudinal axis of epidermal
cells.
A number of possible mechanisms exist by which a cell
can sense a change in surface topography. In Uromyces a
stretch-activated ion channel was isolated when examined
© 1997 ISHAM,Journal of Medical & Veterinary Mycology 35, 289-293
Contact-sensingby fungal hyphae
with patch clamp electrodes [21] which was blocked
effectively by Gd 3 +. Thus it is possible that as the cell
contacts an inductive surface stretching of the plasma
membrane occurs, during which channels open allowing
efflux or influx of specific ions such as Ca -~+. This would
result in a modulation of ion concentrations inside the cell
which may activate a cascade of events that produce a
physiological response including hyphal reorientation or
thigmodifferentiation of appressorria. Such channels have
been reported in a number of fungi including N. crassa,
Saccharomyces cerevisiae and Saprolegnia ferax [22-24].
As organization of actin filaments is highly sensitive to
Ca 2 +~ it further shows the likelihood of such channels
playing a part in redirection of growth.
Although dermatophytes are pathogenic organisms
they are also capable of living as saprophytes in soil.
When they do live as pathogens they are restricted to the
dead cornified layers. In this respect it is not surprising
that contour guidance is also a feature of the growth of
saprophytes such as N. crassa and M. mucedo. The fungal
tip probably evolved for facilitating penetration and
exploitation of insoluble substrates, living or dead.
Thigmotropism may therefore enhance the ability of a
fungus to explore, invade and degrade materials in the
environment.
Acknowledgements
We thank The University of Aberdeen, Medical Research
Endowments scheme for a studentship to T.P. and the
Wellcome Trust and BBSRC for financial support.
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