1. Introduction
The advent of microscopy provided an expressive progress on the knowledge of the biological world. Particularly important was the development of the electron microscopy at 1930s, making possible to find out a universe of unimaginable dimensions. Its great highlight is the much shorter wavelength of the electron that increases the resolution power of the equipment (Lee, 1993). Currently the electron microscopy is considered a specialized field of science (Bozzola and Russel, 1999). Although electron microscopy is useful to answer important questions about the ultrastructure of biological materials, it is also represent an additional tool that may be used as an ally in several research fields.
The scanning electron microscope (SEM) is useful to analyze microstructural features of solid bodies’ surfaces, such as yeast cells. Besides, it leads to the formation of a three-dimensional image as a direct result of the great depth of field (Lee, 1993). Even samples observed by naked eye may be analyzed at low magnifications with great depth of focus, making possible to obtain images with a pronounced resolution using detectors of secondary electrons. Thus, the electron microscopy may contribute to reveal this nanometer´s world including fungal cell structure and the interaction between fungal cells and their microenviroment.
Most of the ultrastructural studies of
The evolution of intensive care medicine prolongs life expectancy leading populations to high susceptibility for candidal infection. Although ubiquitous in nature,
Yeasts belonging to genus
With reference to morphological characteristics,
For pathogenic yeasts, it is widely accepted that yeast form cells are essential for efficient dissemination through the body, whereas the filamentous forms are required for tissue invasion. For
Most pathogenic
The purpose of this chapter is to sum up some of the recent ultrastructural findings of
2. Ultrastructural features of pathogenic attributes
Multiple characteristics have been proposed to be putative virulence factors related to the pathogenesis of
Recently, França et al. (2010) described a correlation between
Currently, we are employing the SEM to evaluate many events related to pathogenicity of clinical strains of
Precise imaging of yeasts depends on the adequate preservation. To SEM analysis, yeast samples are well fixed by immersion on glutaraldehyde in phosphate buffer at proper concentrations (Hayat, 2000). The use of buffered osmium tetroxide at 1%, for 1 hour, is recommended as post fixation to preserve cellular content and surfaces. In our studies, samples of planktonic cells or biofilms are critical point dried after ethanolic dehydration. In order to preserve the colonies organization some steps were optimized, such as omission of osmium tetroxide, ethanolic dehydration and critical point dried. As a routine in our laboratory colonies are freeze-dried to avoid distortions and to maintain their architecture. Besides, sputtering was performed using a thick layer (50 nm) of gold.
2.1. Adherence patterns in vitro
Adherence is essential for members of the genus
For
As cited previously,
Therefore, ultrastructural investigations of the interface of
We have recently initiated experiments to verify the
In the present study, we compared ultrastructural features related to adhesion of
2.2. Invasion capability
Morphogenesis between yeast and hyphal growth, which facilitates fungal tissue invasion and enables the fungus to evade the defense system of the host is generally accepted as virulence traits of
For non-
Although it is well established that
According to Brown et al. (1999),
In this study, we employed the agar invasion assay to determine the invasive potential of a switch variant strain, exhibiting a crepe morphotype (Figure 2A), obtained from a clinical
Further we analyzed the invaded agar at ultrastructural level, by the employment of fracture technique. Fracture is valuable to reveal internal surfaces and it is performed by dipping the fixed samples in liquid nitrogen and breaking with a sharp scalpel. Growth in YPD shows filamentous forms invading the agar at different planes and angles, as well as yeast cell forms (Figure 3). Thus, SEM may be useful for the detailed analysis of extend and pattern of yeast cells in the course of the invasion process. This is the first report of the employment of SEM to examine the pattern of agar invasion by
2.3. Switching morphotypes: Ultrastructure and morphological types
Phenotypic switching represents an epigenetic state that occurs in a small fraction of the population, is random and reversible. This biological phenomenon is related to the occurrence of spontaneous emergence of colonies with different morphologies that enables the microorganism to undergo rapid microevolution and to adapt to different environments, including various anatomical sites in the human body (reviewed in Soll, 1992). Thus, the switching phenotype event has also being considered a candidal virulence factor (Segal, 2004).
Furthermore, switching has been demonstrated to regulate virulence-associated characteristics in
For fungi this event is defined as the reversible change manifested as altered colony morphology at a rate higher than the somatic mutation rate (reviewed in Soll, 1992).
In yeast, phenotypic switching was originally described in
Ultrastructural investigations revealed a relationship between
SEM was successfully employed for the analyses of whole
The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. After 4 days of colony development, the whole smooth and irregular wrinkled colonies consisted entirely of yeast cells (not shown). The crepe colony phenotype also comprised mainly yeast cells as observed at depressions areas (Figure 5A). Most interesting was the presence of extracellular material forming a biofilm-like colony where many of the cells were almost hidden by this material. It was observed as fibrils, with enlarged structures, connecting neighbouring cells (Figure 5B).
A
2.4. Effect of antifungal compounds on yeast morphology
The therapy of deep fungal infections, particularly those caused by opportunistic pathogens, including
Many studies have addressed the search for natural compounds with antifungal activity. As an example, Duarte et al. (2005) screened 35 medicinal plants commonly used in Brazil for anti-
Ultrastructural investigations of the effect of natural compounds on morphology are limited. However, SEM analysis allowed the observation of irregular budding patterns and pseudohyphae formation in
We employed the scanning electron microscopy to evaluate the effect of baicalein alone and in combination with fluconazole on the morphology of
On the other hand, the data obtained in this study showed that
Untreated cells (control) consisted of blastoconidia and pseudohyphae (not shown). For cells exposed to baicalein alone we observed the presence of elongated cells as well as a great capacity for producing pseudohyphae (Fig. 8A). Cells exposed to baicalein in combination with fluconazole showed an oval shape with profusely flocculent extracellular material connecting yeast cells (Fig. 8B). These data, suggest different inter-species response to baicalein alone as well as to in association with fluconazole.
3. Conclusion
Yeast pathogenicity arises through complex interactions between the organism’s virulence characteristics and the host’s response.
Acknowledgments
The author gratefully acknowledges to all colleagues and students joined to the medical mycology group at State University of Londrina and Technological Federal University of Paraná in Londrina, Paraná-Brazil. Special thanks to the group members: Ana Flávia Leal Specian and Rosapa Serpa that contributed with results obtained during their master thesis work. The authors also thank Osvaldo Capello for technical support. Team´s members using original results made all the figures´ composition. This work was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico –CNPq-Brazil.
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