Growth, Differentiation and Sexuality

94 J.P. Latgé and R. Calderone
(Tada et al. 2003). Two redundant genes, CNA1
and CNA2, encode the catalytic subunit whereas
the regulatory subunit is encoded by CNB1. Both
subunits are required for enzymatic activity.
Surprisingly, it was research efforts focused
on the Ca 2+ -calmodulin-dependent phosphoprotein
phosphatase calcineurin, and the mode of
action of immunosuppressive compounds such as
tacrolimus (FK506) and cyclosporin A (CsA) which
helped to uncover genes encoding subunits of
β1,3 glucan-synthase (Douglas et al. 1994; Douglas
2001). This result was due to the presence of the
proteins Fks1p and Fks2p which are alternate subunits
of the β1,3 glucan-synthase enzyme complex,
with an essential overlapping function (Mazur
et al. 1995). When regulation of FKS2 was studied,
it was discovered that transcription is induced
by Ca 2+ in a calcineurin-dependent manner, and
inhibited by tacrolimus (Zhao et al. 1998). Because
calcineurin is known to be the target of tacrolimus
and CsA, it follows that cells with null alleles in
FKS1 are hypersensitive to tacrolimus or CsA
because they rely on Fks2p for viability.
In C. neoformans, the calcineurin mutants
are defective in growth at 37 ◦ C (Odom et al.
1997; Kraus and Heitman 2003), resulting in an
enhanced susceptibility of a cell integrity pathway
gene (MPK1) mutant to β1,3 glucan synthesis
inhibitors (Kraus et al. 2003). Further, the same
authors showed that in a cnb1 mutant, activation of
FKS1 occurs in cells in an Mpk1-dependent manner.
Thus, in C. neoformans which has a unique FKS
gene (Thompson et al. 1999), both the cell integrity
and calcineurin pathways interact to regulate FKS1
transcription, and hence cell wall glucan synthesis.
Calcineurin is also critical to the morphogenesis
of C. albicans (Bader et al. 2003; Blankenship
et al. 2003; Sanglard et al. 2003). The function of the
calcineurin CNA1 in cell wall formation was suggested
by the sensitivity of the mutant to cell wallperturbing
agents such as SDS, Calcofluor white
and Congo red, and by evidence that the activation
of FKS2, a glucan synthase subunit-encoding
gene, is regulated by upstream events which are
calcineurin-dependent (Sanglard et al. 2003).
That immunosuppressants such as rapamycin
which target TOR, and FK506 (tacrolimus) and
cyclosporine which target calcineurin might have
anti-fungal properties has been theorized based
upon the important cell functions described above.
The inhibitory activity of the immunosuppressants
has also been evaluated in vitro against A.
fumigatus, C. albicans and C. neoformans. Ofthe
three drugs, FK506 was the most inhibitory and,
in agreement with its connection with glucan
synthase, FK506 in combination with inhibitors of
β1,3glucansynthaseresultedinthebestsynergy
(Cruz et al. 2000; del Poeta et al. 2000; Steinbach
et al. 2004).
VI. Perspectives
In spite of the essential role of the cell wall for fungi,
very little is known in this field and most of our
present knowledge is based on the seminal studies
of Enrico Cabib for chitin and β1,3 glucan synthases.
Encoding genes for these transmembrane
enzymes and their enzymatic activities have been
characterized but none of these enzyme activities
have been demonstrated in vitro using recombinant
proteins, so that there is a very limited knowledge
on their mode of action. Moreover, an increasing
number of studies point to the association between
polysaccharide synthesis and phospholipid
metabolism. Such relationships should be investigatedinmoredetail.Thiscouldleadtospecificantifungals,
in a similar way to sphingolipids inhibitors.
Whereas the molecular analysis of genes encoding
the β1,3 glucan synthases has led to the discovery of
the anti-fungal echinocandins, no compounds exist
in clinical practice which target chitin synthesis
in the treatment of fungal disease.
Recent comparative chemogenomic analyses of
the yeast and mould cell wall described above have
resulted in a major progress in delimiting the key
components of the cell wall structure. More development
in this area should be forthcoming following
the analysis of the cell wall of ancient fungi such
as the Chytridiomycetes. Data with these fungi remain
very incomplete, since neither the presence of
branches in the glucan nor the chitin–glucan linkages
have been investigated. Genomic analysis of
these fungi should give us some important clues
aboutundiscoveredcellwallgenesessentialforits
construction.
Another area of study is the analysis of glycosyltransferases
which are active in cell wall construction,
since branching and cross-linking between
chitin and β1,3 glucan are responsible for
the formation of a resistant fibrillar skeletal component
of the cell wall of all fungi. Linkages between
these two polysaccharides have been shown
to be essential for the construction of the fibrillar
core of the fungal cell wall, providing strength
and protection against adverse environmental con-