Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

Abstract

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu³⁹³, Phe³⁹⁷, Phe⁴¹⁵, and His⁴⁴⁰) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Keywords: Trichophyton; antifungal resistance; dermatophytes; reverse genetics approach; squalene epoxidase; terbinafine.

FIG 1

Agarose gel electrophoresis of the TRS-1 (lanes 1 to 4) and TRS-2 (lanes 5 to 8) regions from terbinafine-resistant T. rubrum clinical isolates with the same point mutation in the SQLE gene. (A) Strains with the replacement of Leu³⁹³ with Phe in SQLE. Lanes 1 and 5, TIMM20083; lanes 2 and 6, TIMM20084; lanes 3 and 7, TIMM20093; lanes 4 and 8, TIMM20094. (B) Strains with the replacement of Phe³⁹⁷ with Leu in the SQLE. Lanes 1 and 5, TIMM20085; lanes 2 and 6, TIMM20086; lanes 3 and 7, TIMM20087; lanes 4 and 8, TIMM20092. Leu³⁹⁷ is encoded by TTA in TIMM20085 and TIMM20087 and by CTC in TIMM20086 and TIMM20092.

FIG 2

Introduction of point mutations into the endogenous SQLE gene of A. vanbreuseghemii by gene replacement strategy. (A) Schematic representation of a series of binary AvSQLE-targeting vectors. DNA fragments (SQLEa and SQLEb) containing the 5′ UTR of the AvSQLE gene and the open reading frames encoding wild-type and mutated A. vanbreuseghemii or T. rubrum SQLE proteins (ORF*) as well as the 3′ UTR of the AvSQLE gene were subcloned into the pAg1-AbKu70/T2 upstream (SpeI/ApaI) and downstream (BamHI/KpnI) of the PcFLP/FRT module, respectively (Table 2 and Fig. S1). The nptII cassette is composed of Aspergillus nidulans trpC promoter (P_trpC), E. coli neomycin phosphotransferase gene (nptII), and the A. fumigatus cgrA terminator (T_cgrA). P_ctr4, T. rubrum ctr4 promoter (34); Pcflp, the synthetic flp gene with Penicillium chrysogenum-optimized codon usage (35); T_trp1, Cryptococcus neoformans trp1 terminator (36); FRT, FLP recombinase target sequence; LB and RB, left and right borders, respectively; A, ApaI; B, BamHI; C, ClaI; K, KpnI; Sc, SacI; Sh, SphI; Sp, SpeI. (B) Schematic representation of the AvSQLE locus before and after homologous recombination and excision of the PcFLP/FRT module. Site-specific recombination between the flanking FRT sequences was induced by conditional expression of Pcflp after transformation. All the internal BamHI sites contained in the amplified fragments were inactivated by overlap extension PCR. (C) Southern blotting. Aliquots of approximately 10 μg of total DNA from each mutant strain were digested with BamHI and separated by electrophoresis on 0.8% (wt/vol) agarose gels. 1062Av1401 indicates the parent strain. Lane 1, Av-FRT-1-3 (AvSQLE's control); lane 2, Av-S38A (Leu393Phe); lane 3, Av-1-3 (Leu393Ser); lane 4, Av-S714J6 (Phe397Leu); lane 5, Av-7-5 (Phe397Ile); lane 6, Av-4-1 (Phe397Val); lane 7, Av-S28M (Phe415Val); lane 8, Av-2-4 (His440Tyr); lane 9, Tr-FRT-52-9 (TrSQLE's control); lane 10, Tr-T31C (Leu393Phe); lane 11, Tr-2-3 (Leu393Ser); lane 12, Tr-T719J (Phe397Leu); lane 13, Tr-4-1 (Phe397Ile); lane 14, Tr-75-6 (Phe397Val); lane 15, Tr-T4B (Phe415Val); lane 16, Tr-2-1 (His440Tyr). A 566-bp fragment of the AvSQLE gene was amplified by PCR with the primer pair AvSQLE-F23 and AvSQLE-R21 (Table 4) and used as a hybridization probe. DNA standard fragment sizes are shown on the left.

FIG 3

The growth properties of A. vanbreuseghemii transformants expressing mutated AvSQLE and TrSQLE proteins on solid terbinafine-containing medium. Aliquots of 10 μl of conidial suspensions containing 1 × 10⁵ cells were spotted onto SDA with (+) or without (−) 0.005 μg/ml terbinafine and incubated at 28°C for 3 days. Bar, 1.0 cm.

FIG 4

The growth properties of A. vanbreuseghemii transformants with the substitution Phe³⁹⁷Val in the SQLE protein (A) and T. rubrum clinical isolates harboring the Phe397Val or Phe415Ser substitution in the SQLE protein (TIMM20097 or TIMM20098, respectively) in comparison to a Leu or Val residue, respectively (TIMM20085 and TIMM20082, respectively) (B) on solid medium. Aliquots of 10 μl of conidial suspensions containing 1 × 10⁵ cells were spotted onto SDA and incubated at 28°C for 4 days (A) or 5 days (B). The wild-type T. rubrum CBS118892 was used as control. Bar, 1.0 cm.