Mechanisms of Drug Resistance in Fungi and Their Significance in Biofilms - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 45

when compared to the pace of emergence of fungal infections. The components of
the fungal cell wall (CW) such as mannans, glucans, and chitins, and a few of the
enzymes of the ergosterol biosynthetic pathway, are unique to fungal cells
(St Georgiev 2000 ; Munro et al. 2001 ) and have been targeted for the development
of antifungal agents. Among the enzymes of the ergosterol biosynthetic pathway,
squalene epoxidase, P45014DM or CYP51 ( ERG11 ),

14 -reductase ( ERG24 ), and

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7 -isomerase ( ERG2 ) have been the targets of most antifungal agents (Sanglard
et al. 2003 ).
Amongst the known antifungals, azole derivatives like fluconazole (FLC),
ketoconazole (KTC), and itraconazole (ITC) have been the most widely used
triazoles for combating fungal infections. Azoles specifically inhibit the
P45014DM enzyme, which results in the accumulation of 14-methylated sterols
and results in the disruption of membrane structure and function (Vanden Bossche
et al. 1989 ). Fungal cells have adopted several strategies to cope with incoming
drugs (particularly azoles), which are discussed briefly.

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2.1 Target Alteration

One of the most common events associated with the development of drug tolerance
in fungi relates to the azole target protein. The target of azoles, the P45014DM
protein, is modified in resistant cells by the replacement of native amino acids
leading to poor binding of the drug without affecting its function. Investigators
from different groups compared the sequences of the ERG11 (encodes P45014DM
or Erg11 protein) gene of resistant C. albicans strains with the published ERG11
sequence to that of FLC-susceptible strains and identified several point mutations.
Biochemical analysis of these mutations showed that several point mutations in the
protein reduce its affinity for azoles. These mutations, single or in combination, do
not permit normal binding of FLC to target proteins without affecting ergosterol
biosynthesis (Wang et al. 2009 ; Morio et al 2010 ). A comparison of sequence
disparity of the Erg11 protein between susceptible and resistant clinical isolates of
C. albicans revealed several mutations in the resistant isolates predominantly
localized to certain hot spot regions. Such bunching of critical residues, which
probably affects azole binding to target proteins, was subsequently confirmed by
different studies (Wang et al. 2009 ). A few of the Erg11 protein mutations, when
expressed in Saccharomyces cerevisiae, were shown to confer even higher resis-
tance against azole antifungals (Favre et al. 1999 ). Notably, the I-helix stretch of the
Erg11 protein, which is highly conserved in the cytochrome P450 family, does not
show any spontaneous mutations. The exact placement of all the identified muta-
tions in a 3D model of the protein confirmed that these mutations are not randomly
distributed but rather are clustered in select hot spot regions (Marichal et al. 1999 ;
Wang et al. 2009 ).
Mutations in drug target enzymes clearly are an important mechanism resulting
in the emergence of FLC resistant C. albicans strains. However, sequence