Growth, Differentiation and Sexuality

74 J.P. Latgé and R. Calderone
being composed of melanin. During the first
stages of germination (swelling), polysaccharide
hydrolysis occurs to soften the cell wall, along with
the de novo synthesis of a new electron lucent
inner layer. Hyphae which emerge have a single
mono-layered cell wall. A similar situation exists
in yeast, although biosynthetic and lytic sites are
differently located (Cabib et al. 2001). Cell wall
biosynthesis appears then as a dynamic, essential
and timely process which is correlated with growth
(Latgé et al. 2005). Polysaccharide components of
the cell wall are unique to fungi and, consequently,
putative inhibitors of the biosynthetic pathways
responsible for cell wall construction are therefore
unlikely to have secondary toxic effects, as is the
case for some existing anti-fungal drugs such as
amphotericin B. The recent initiatives by the pharmaceutical
industry with the echinocandins, drugs
inhibiting β1,3 glucan synthase, has validated the
enormous potential of the cell wall as a source of
targets for the development of lead anti-fungal targets
(Denning 2003; Walsh et al. 2004). Inhibitors
of cell wall polysaccharide synthesis have not been
developed in plant pathogenic fungi. However, in
these fungi, inhibitors of the synthesis of cell wall
pigment are powerful fungicides (Yamaguchi and
Kubo 1992).
These reasons are sufficient to investigate the
polymer organisation of the fungal cell wall and to
characterize proteins/genes involved in the biosynthesis
of the constitutive polymers of the cell wall.
In this chapter, we will review our current understanding
of the structural organisation of the cell
wall and of the enzymes responsible for the biosynthesis
of cell wall components. This analysis takes
into account recent genomic data from the comparison
of yeast and mould genome sequences as
well as chemical data on the cell wall of these fungi
to identify general core pathways common to all
fungi.
II. Cell Wall: Composition
and Organisation
A. Techniques to Study Cell Wall
The cell wall is a highly insoluble material. Chemicalanalysisofcellwallcomponentsrequiresfirst
their solubilization. Different techniques, using
mostly chemicals, have been used to solubilize
cell wall fractions (Fleet 1985, 1991; Perez and
Fig. 5.2. An example of a protocol for the extraction of
fungal cell wall components
Ribas 2004). Numerous protocols are given in
the literature, without any comparative efficiency
in the qualitative or quantitative recovery of the
different constitutive polymers. Although some
of the chemical methods are recommended for
specific polymer extractions (for example, long
N-mannans in yeast are easily recovered by
Fehling’s solution or cetavlon precipitation; Fleet
1985, 1991), no consensus exists today on the
best methods to use and their most appropriate
sequence to analyse cell walls. In spite of this, the
protocolgiveninFig.5.2hasgainedthemost
acceptance for cell wall extraction of most fungi.
Sodium hydroxide treatment (sometimes prior or
after acetic, hydrofluoric (HF) or hydrochloric acid
hydrolysis) is usually needed to initially solubilize
cell wall. Further fractionation of the alkali-soluble
and insoluble extracts must then be achieved with
specific recombinant endo-glycosylhydrolases
available to date to cleave all cell wall polysaccharides
except mannan chains, which can be
cleaved only internally by chemical acetolysis
(Latgé et al. 1994). Carbohydrate linkages in the
different fractions are then identified using specific
carbohydrate chemistry methodologies (Fontaine
et al. 2000).
B. Composition and Fungal Evolution
The major component of the cell wall is polysaccharide,
which accounts for > 90% of the cell
wall.Threebasiccomponentsrepresentthemajor
polysaccharides of the cell wall: glucans, mannans
and chitin. Other carbohydrates less frequently
found are galactose, galactosamine, glucosamine,
xylose, fucose and hexuronic acid. A survey of the
composition of the cell wall of all fungal species
shows that the study of galactose or D-glucosamine