Growth, Differentiation and Sexuality

Characterization of the chs4 mutant indicated
that it produces functional but highly zymogenic
chitin synthase III activity (Choi et al. 1994; Trilla
et al. 1997). These results suggest that Chs4p could
be a direct activator of chitin synthase III activity
at the membrane site. CHS4 was also identified
in a synthetic lethality screening with the Cdc12p
septin, and was shown to interact with Cdc10p
septin through an anchor protein named Bni4p,
which plays a more general role in the assembly of
the septum machinery (DeMarini et al. 1997; see
also Wendland and Walther, Chap. 6, this volume).
The current model indicates that a complex containing
Chs3p/Chs4p is positioned at the septum
site through its interaction with the Bni4p/septin
complex (DeMarini et al. 1997; Sanz et al. 2004).
In Aspergillus fumigatus, where no homologues of
Bni4p are found, localisation of CHS at the septum
may be under a different regulation. One way for Aspergillus
to bypass this mechanism is via a myosin–
chitin linkage different to that in yeasts: at least one
gene in the CHS families of moulds has a consensus
domain which is homologous to kinesin or myosin
motor-like domains (Horiuchi et al. 1999; Takeshita
et al. 2002; Latgé et al. 2005). The product of the
CHS4 gene is required for chitin synthesis during
mating but not for proper sporulation, because chs4
mutants form completely mature ascospores (Trilla
et al. 1997). S. cerevisiae contains a CHS4 homologue
(SHC1) which is required for the synthesis
of the ascospore’s chitosan layer, and therefore for
spore maturation (Sanz et al. 2002). Both genes
are functionally redundant in activating CSIII, but
Shc1p lacks the localisation domain which allows
Chs4p to direct Chs3p to the septum. A. fumigatus
and N. crassa contain three CHS4 homologues but
their differential regulation, for example, during
sporulation, has not been investigated. C. albicans
contains only one CHS4-like gene, possibly related
to the lack of sporulation in this organism (Roncero
2002).
3. Inhibition
The peptide nucleoside antibiotics polyoxins and
nikkomycins are strong competitive inhibitors of
chitin synthase and are substrate analogues of
UDP-N-GlcNAc (Gaughran et al. 1994; Munro and
Gow 2001). Polyoxin and nikkomycin are highly
active in vitro but only poorly active in vivo.
Reasons for this are the following: (1) as peptidyl
nucleosides, these compounds are susceptible to
cleavage by peptidases of microbial or host origin,
Fungal Cell Wall 83
and (2) nikkomycins/polyoxins must traverse the
cell membrane and be transported to the active
site of chitin synthase, and are poorly accumulated
by fungal pathogen. Two new amphipatic
compounds without peptide bonds, and with
a well-defined hydrophobic domain facilitating
the crossing of the hydrophobic cell membrane,
have been recently described: they are a series of
novel nikkomycin analogues with a hydrophobic
group at the terminal amino acid (Obi et al. 2000),
and a new class of inhibitors with structures
differing markedly from those of the polyoxins
and nikkomycins (Masubuchi et al. 2000). In spite
of high activities in vitro, none of the inhibitors of
chitin synthesis have been launched yet in clinical
practice.
B. Glucan Synthesis
1. β1,3 Glucan Synthases
a) Synthesis
β1,3glucansaresynthesisedbyaplasmamembrane-bound
glucan synthase complex which uses
UDP-glucose as a substrate, and extrudes linear
β1,3 glucan chains through the membrane into
the periplasmic space (Douglas 2001). The protein
complex contains at least two proteins, a putative
catalytic subunit encoded by the genes FKS1 with
a Mr > 200 kDa and a regulatory subunit encoded
by RH01 with an ∼20 kDa Mr. The reaction adds
onemoleofglucoseforeverymoleofUDP-glucose
hydrolysed to produce chains of increasing length.
The polysaccharide produced in the reaction was
estimated to be 60–80 glucose residues long in
yeasts. In A. fumigatus, 1500 glucose residues are
produced per chain (Beauvais et al. 2001).
UDP-glucose, the substrate of β1,3 glucan
synthase (GS), is synthesised from hexose
phosphate precursors: glucose 6-P is utilised
by the phosphoglucomutase to produce glucose
1P which is then transformed to UDP-glucose
by a uridyltransferase. Genes of this pathway
are present in all genomes sequenced to date
and, in addition, multiple alternative pathways
can be used by fungi to produce intracellularly
the β-glucan synthase substrate (http://www.ge
nome.jp/kegg/pathway/map/map00520.html). The
UDP-glucose is then transported to the plasma
membrane (Munoz et al. 1996; Castro et al. 1999).
In S. cerevisiae,threeFKS genes exist of which
only two are involved in β1,3 glucan synthesis