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Journal of Bacteriology, September 1999, p. 5350-5354, Vol. 181, No. 17
Unité de Chimie des Interfaces,
Université Catholique de Louvain, 1348 Louvain-la-Neuve,
Belgium,1 and Laboratoire de
Biotechnologie des Champignons Filamenteux, Institut National de la
Recherche Agronomique, Faculté des Sciences de Luminy, F-13288
Marseille, France2
Received 15 April 1999/Accepted 22 June 1999
Atomic force microscopy (AFM) has been used to probe, under
physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 ± 1 nm, which is in agreement with earlier
freeze-etching measurements. In contrast, germinating spores had a very
smooth surface partially covered with rough granular structures.
Force-distance curve measurements demonstrated that the changes in
spore surface ultrastructure during germination are correlated with
profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 ± 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular
interactions of living fungal spores at the nanometer scale and offers
new prospects for mapping microbial cell surface properties under native conditions.
Spores have great importance in the
life cycle of fungi, since they are means of vegetative multiplication,
dispersal, and survival (1, 30). The surface of spores of
many fungi, including ascomycetes, basidiomycetes, and zygomycetes, is
covered by a thin layer of regularly arranged rodlets (1, 6, 9,
13, 24, 32). These structures, 5 to 10 nm thick and up to 250 nm
in length, are composed mainly of proteins (31), while
hyphal walls are essentially composed of polysaccharides, such as
glucans and chitin. Germination results in the disruption of the rodlet layer (13).
Understanding spore adhesion (10), spore aggregation
(8), and spore dispersal (1) requires knowledge
of the structure and physicochemical properties of the spore surface at
supramolecular scale. The ultrastructure of fungal spores has been
investigated by transmission electron microscopy combined with
freeze-fracture and surface replica techniques for over 3 decades
(1, 2, 11, 27, 28); however, direct information on the
native spore surface was inaccessible. On the other hand, methods used
so far to measure interaction forces at biological interfaces,
including the osmotic stress method (18), the surface-force
apparatus (19), and the micropipette technique (7,
20), did not offer lateral resolution.
Atomic force microscopy (AFM) (3) has proved useful to
investigate the morphology of biological surfaces (4, 12, 14, 23) and to measure interaction forces between complementary biomolecules (17, 33). In particular, well-ordered
arrangements of isolated bacterial surface proteins have been
visualized at high spatial resolution (15, 21, 22, 25, 29).
However, the application of AFM to direct probing of the surface
ultrastructure of living microbial cells down to the nanometer
scale has not been reported. In this study, we used AFM to
determine, in situ, the ultrastructure and molecular interactions at
the surface of living spores of Phanerochaete chrysosporium
and their changes during germination.
Conidiospores of P. chrysosporium INA-12 (strain
I-398 in the Collection Nationale de Culture de Microorganismes,
Institut Pasteur, Paris, France) were collected from mycelial mats and grown in agitated liquid medium for 8.5 h at 37°C, as described earlier (8). They were washed twice by resuspension in
deionized water (Milli-Q; Millipore) and centrifugation and resuspended in water. The pH of the spore suspension was about 6.5. Spores were
immobilized by mechanical trapping in an Isopore polycarbonate membrane
(Millipore) with a pore size similar to the spore size (16).
After filtering of a spore suspension (10 ml; 106 cells per
ml), the filter was carefully cut (1 by 1 cm) and attached to a steel
sample puck (Digital Instruments, Santa Barbara, Calif.) by using a
small piece of adhesive tape, and the mounted sample was transferred
into the AFM liquid cell. AFM measurements were made at room
temperature (20°C), under water, by using an optical lever microscope
(Nanoscope III; Digital Instruments), applied force maintained below 1 nN, a scan rate of ~2 Hz, and oxide-sharpened microfabricated
Si3N4 cantilevers (Park Scientific Instruments, Mountain View, Calif.) with a spring constant of 0.03 N/m and a probe
curvature radius of typically 20 nm (according to manufacturer specifications). Images were recorded in both height and deflection modes. In height mode, the local height variations are measured while
the probe-sample interaction force is kept constant; in deflection
mode, the deflection of the cantilever is measured while the probe
scans at a constant height. While height images provide quantitative
information on sample surface topography, deflection images often
exhibit higher contrast of the morphological details.
Figure 1a shows an optical
micrograph of spores after 8.5 h of incubation in liquid medium.
While dormant spores are spherical particles about 5 µm in diameter,
germinating spores have a swollen part which is the starting point of
the germ tube.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Direct Probing of the Surface Ultrastructure and
Molecular Interactions of Dormant and Germinating Spores of
Phanerochaete chrysosporium
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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FIG. 1.
(a) Optical micrograph of P. chrysosporium
spores observed after 8.5 h of incubation in agitated liquid
medium. Scale bar = 5 µm. While dormant spores (D) are spherical
particles, germinating spores are swollen and allow identification of a
germinating zone (G). (b and c) AFM images of a dormant spore recorded,
under water, in two different modes: height image (b) and deflection
image (c). Scale bar = 5 µm; z range = 2 µm
(b) and 1 µm (c).
To investigate spores under native conditions by AFM, we achieved cell immobilization by mechanical trapping in porous membranes. Low-resolution AFM images of a dormant spore are shown in Fig. 1b and c. While the height image (Fig. 1b) provides quantitative topographic information, the deflection image (Fig. 1c) reveals fine surface details (12). At imaging forces lower than 1 nN, images of the same area were obtained repeatedly without detaching the spores or altering the surface morphology. In the deflection image (Fig. 1c), the spore is surrounded by artifactual structures resulting from the contact between the AFM probe and the edges of the spore and of the pore.
High-resolution images of dormant spores showed that the surface was uniformly covered with a regular pattern of rodlets (Fig. 2a and b). These structures were several hundred nanometers in length and had a periodicity of 10 ± 1 nm, in excellent agreement with earlier freeze-etching characterization (9). Images obtained by forward and backward scanning were identical, indicating no significant contribution of lateral forces to the apparent topographic contrast. Changing the scanning angle by 90° resulted in the rotation of the rodlet orientation accordingly, demonstrating that the observed patterns are due to real surface relief.
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A force-versus-distance curve recorded between the silicon nitride probe and the surface of a dormant spore is shown in Fig. 2c. Upon approach, no significant deviation from linearity was seen in the contact region, indicating that the sample was not deformed by the probe. Upon retraction, no adhesion forces were detected, reflecting the absence of molecular interactions between probe and surface. 32 × 32 force-distance curves recorded on 1- by 1-µm areas all showed the same features, indicating that the spore surface was homogeneous as regards physicochemical properties.
The ultrastructure of germinating spores was clearly different from that of dormant spores. AFM images (Fig. 3a to d) revealed that part of the surface was coated with rough granular material (rms roughness on 250- by 250-nm areas of height images = 3.4 ± 1.2 nm), apparently resulting from the fragmentation of the rodlet layer, while the underlying surface was very smooth (rms roughness = 0.4 ± 0.1 nm) and showed ring-like structures (Fig. 3c and d). Images recorded on the germinating zone (Fig. 1a) were uniformly smooth, while those obtained on other zones showed that the rough material represented a high surface fraction. The change in cell surface ultrastructure is consistent with earlier electron microscopy observations of the same spores: a rodlet layer was detected at the surface of dormant spores, while the wall of germinating spores became fragmented and residual rodlets were disseminated on the new spore surface (9). In the same way, dormant sporangiospores of Syncephalastrum racemosum had a regular pattern of rodlets at their surface (13); during germination, these rodlets were displaced, revealing a very smooth germ tube. The granular material is thus attributed to residues of loosely bound rodlets, while smooth zones with rings would reflect the new cell wall material.
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Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination went along with profound modifications of the physicochemical properties (Fig. 3e and f). In contrast with dormant spores, germinating spores revealed a curvature upon approach, reflecting sample softness and/or repulsive surface forces. These might be due to electrostatic interactions; however, silicon nitride surfaces were shown to be close to electrical neutrality over a wide pH range (pH 6 to 8.5) (26). The heterogeneous surface morphology of germinating spores was directly correlated with differences in adhesion forces revealed by retraction curves: no adhesion forces were measured between the probe and the granular material (Fig. 3e), while strong adhesion forces, of 9 ± 2 nN (n = 50) magnitude, were measured on smooth zones (Fig. 3f).
These results represent the first direct (i.e., under physiological conditions) characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale. They demonstrate that dramatic changes in cell surface ultrastructure and physicochemical properties occur during germination of P. chrysosporium spores and provide direct indications as to the functions of fungal surfaces. It has been suggested that rodlet layers play a role in spore dissemination and protection against lytic enzymes (1, 5). Our finding that rodlet layers at the surface of dormant spores of P. chrysosporium are rigid and endowed with nonadhesive properties points to their role as protection and dispersal structures. Polysaccharides at the germ tube surface have been proposed to mediate spore aggregation (6, 13). For P. chrysosporium, an increase in the surface polysaccharide concentration during germination was demonstrated by surface analysis with X-ray photoelectron spectroscopy and suggested to be involved in spore aggregation (8). The observation of strong adhesion forces on the smooth zones of germinating spores may thus be attributed to the presence of polysaccharides and provides evidence for their role in the aggregation process. Molecular interactions could be investigated further by using chemically modified probes.
The approach presented here offers promise as an application for the nanoscale characterization of the surface properties of prokaryotic, animal, and plant cells in the native state. In particular, probes functionalized with relevant chemical functions or biochemical moieties (33) could be used to map the distribution of specific cell surface components. This would provide new insight into the molecular mechanisms of biointerfacial phenomena such as cell aggregation, cell adhesion, molecular recognition, and intercellular communication.
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ACKNOWLEDGMENTS |
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The support of the National Foundation for Scientific Research (FNRS), of the Foundation for Training in Industrial and Agricultural Research (FRIA), and of the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme) is gratefully acknowledged.
We thank P. Grange for the use of the atomic force microscope and K. Lee for programming associated with the force measurements.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité de Chimie des Interfaces, Université Catholique de Louvain, Place Croix du Sud 2/18, 1348 Louvain-la-Neuve, Belgium. Phone: (32) 10 47 35 89. Fax: (32) 10 47 20 05. E-mail: dufrene{at}cifa.ucl.ac.be.
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Journal of Bacteriology, September 1999, p. 5350-5354, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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