Interactions Between Macrophages and Cell Wall Oligosaccharides of Candida albicans
The fungal cell wall is the armour that protects the cell from changes in the external environment. The wall of Candida albicans, an opportunistic human pathogen, is also the immediate point of contact with the host immune system and contains most of the
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1. Introduction Recent years have seen considerable research activity focussed on the identification of the PAMPs (pathogen-associated molecular patterns) of fungi and their cognate PRRs (pattern recognition receptors; 1). The identification of these ligands and their receptors has the potential to provide the means to control the nature of immune recognition responses to fungi by adjunct immunotherapy. One approach has been the use of mutants with defined alterations in cell wall composition as tools to investigate which cell wall components stimulate specific immune responses (2–5). A complementary approach has been to purify specific cell wall components of a fungus and to present these to immune cells and observe their
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_16, © Springer Science+Business Media, LLC 2012
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immune reactivity. This chapter provides some guidelines as to how to prepare such molecules for experiments. The major components of the C. albicans cell wall are covalently bonded to each other. Therefore, the extraction of pure wall components requires relatively harsh treatments and care must be taken to check the purity of the resulting purified molecules. The C. albicans cell wall is similar, but by no means identical, in structure to that of Saccharomyces cerevisiae (6). Consequently, many of the purification protocols originated from work in this rather distantly related yeast. The outer wall of C. albicans is enriched with highly glycosylated mannoproteins that are more than 80–90% mannose sugar by mass and represent 35–40% of the dry weight of the wall (6). This mannoprotein layer is fibrillar in appearance and is attached via a GPI-remnant to β-1,6 glucan, which is in turn attached to either β-1,3 glucan or chitin (1, 6). These two latter molecules form the skeletal inner cell wall layer. Chitin, a homopolymer of N-acetyl glucosamine, represents only about 2–4% of the dry weight of the cell wall, while β-1,6 glucan and β-1,3 glucan represent 20 and 40% of the wall, respectively. Chitin is unique in the cell wall in being resistant to both acid and alkali extractions. Purification of this molecule takes advantage of this property. If purified molecules from the cell wall (mannan, glucan, chitin, etc.) are to be used in immunological experiments, it is important to establish the purity of the material generated, both in terms of contamination by other wall components and also by LPS and other bacterial components that may introduce spurious experimental results. It should also be kept in mind that while some cell wall components, such as chitin or β-1,3 glucan, appear to be highly conserved in structure and composition, chitin can adopt a number of different architectures (7) and glucans can have variable levels of branching (8). These differences may influence the way in which the molecules interact with human cells and immune receptors.
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