Two different types of tandem sequences mediate the overexpression of TinCYP51B in azole-resistant Trichophyton indotineae

Abstract

Trichophyton indotineae is an emerging dermatophyte that causes severe tinea corporis and tinea cruris. Numerous cases of terbinafine- and azole-recalcitrant T. indotineae-related dermatophytosis have been observed in India over the past decade, and cases are now being recorded worldwide. Whole genome sequencing of three azole-resistant strains revealed a variable number of repeats of a 2,404 base pair (bp) sequence encoding TinCYP51B in tandem specifically at the CYP51B locus position. However, many other resistant strains (itraconazole MIC ≥0.25 µg/mL; voriconazole MIC ≥0.25 µg/mL) did not contain such duplications. Whole-genome sequencing of three of these strains revealed a variable number of 7,374 bp tandem repeat blocks harboring TinCYP51B. Consequently, two types of T. indotineae azole-resistant strains were found to host TinCYP51B in tandem sequences (type I with 2,404 bp TinCYP51B blocks and type II with 7,374 bp TinCYP51B blocks). Using the CRISPR/Cas9 genome-editing tool, the copy number of TinCYP51B within the genome of types I and II strains was brought back to a single copy. The azole susceptibility of these modified strains was similar to that of strains without TinCYP51B duplication, showing that azole resistance in T. indotineae strains is mediated by one of two types of TinCYP51B amplification. Type II strains were prevalent among 32 resistant strains analyzed using a rapid and reliable PCR test.

Keywords: TinCYP51B; Trichophyton indotineae; antifungal resistance; dermatophytes; gene duplication; itraconazole; voriconazole.

Fig 1

Identification of types I and II strains in T. indotineae. (A) Types I and II strains were identified by amplification of genomic DNA with primer pairs P1–P2 and P3–P4, respectively. TIMM200114 and TIMM 200115, which are susceptible, were used as negative controls (lanes 1 and 2). Azole-resistant strains TIMM200116, TIMM200118, and TIMM 200119, which are known to harbor 5–7 alleles of CYP51B in as many 2,404 bp blocks in tandem, were used as positive controls for the type I strains (lanes 3–5). Lanes 6–29: Search for types I and II tandem sequences in the T. indotineae strains in Table S1. Lanes 6–20: Strains deemed to be resistant to azoles (ITC MIC ≥ 0.25 µg/mL; VRC MIC ≥ 0.25 µg/mL) marked by an asterisk (*) in Table S1. Strain 216510/17 was type I (lane 16). All other strains were type II. Lanes 21–29: Strains deemed to be susceptible to azoles (ITC MIC ≤ 0.25 µg/mL; VRC MIC ≤ 0.25 µg/mL) marked with a hash (#) in Table S1. Two of the later strains (UKJ 1673/17 and 250063/18) were of type II (lanes 24 and 29). (B) Southern blotting analysis of genomic DNA samples from six T. indotineae strains (TIMM20114, TIMM20118, TIMM20120, TIMM20121, TIMM20122, and TIMM20123). Genomic DNA from each strain was digested with XhoI and separated by electrophoresis on a 0.8% (w/v) agarose gel. Lanes 1–6: genomic DNA samples from TIMM20114, TIMM20118, TIMM20120, TIMM20121, TIMM20122, and TIMM20123, respectively. An internal fragment (about 410 bp) of the TinCYP51B gene was amplified by PCR with P21-P16 primers (Table S2) and used as a hybridization probe. The DNA standard fragment sizes are shown on the left.

Fig 2

PacBio sequencing reveals direct tandem repeats of 7,374 bp blocks in type II strain TIMM20122. (A) Scheme describing the organization of the tandem blocks within TIMM20122. The black boxes correspond to the TinCYP51B open reading frame. Upstream and downstream sequences including neighboring genes are shown by gray and white boxes, respectively. (B) Examples of the reads obtained, including reads covering the 5′ border of the duplicated regions (read m64281e_230107_125321/117836369/ccs), reads covering the 3′ border of the duplicated region (read m64281e_230107_125321/106759815/ccs), and reads localized within the duplicated region demonstrating the presence of at least five repeats in this type II strain (read m64281e_230107_125321/89260242/ccs). Sequences of these three reads are given in the supplementary materials.

Fig 3

Comparison of the 2,404 bp duplicated blocks within type I strains and the 7,374 bp blocks found in type II strains. Type I strains include only the TinCYP51B gene (A). Type II strains contain TinCYP51B (skin-colored boxes) and the neighboring genes TinCHKB and TinFYV4 (blue boxes) (B). The coverage of each type of block is marked with a red arrow and the sequences of the borders of the original block are also shown in red. The conserved nucleotides are marked in bold, revealing the absence of any conservation at the borders of the type II 7,374 bp duplicated blocks. The results of the PacBio sequencing results and southern blotting analysis are consistent. X, XhoI site.

Fig 4

CRISPR/Cas9-mediated genetic modification of the TinCYP51B locus in the type I azole-resistant strain TIMM20118 and the type II azole-resistant strains TIMM20121 and TIMM20122. (A) Schematic representation of the binary vector pAg1-TinCYP51B/R. The nptII cassette is composed of the promoter sequence of Aspergillus nidulans tryptophan C (trpC) gene (PtrpC), nptII, and the termination sequence of A. fumigatus cgrA gene (TcgrA). The two silent mutations on the repair template cannot be cleaved by the RNP nuclease. Plasmid DNA of pAg1-TinCYP51B/R was used as a template for PCR with P15–P18 primers, to amplify the sequence (repair template) indicated by the thick line. (B) Schematic representation of the TinCYP51B locus before and after modification. The cleaved TinCYP51B locus is replaced by the repair template, resulting in the introduction of the desired modification. The replaced genomic DNA cannot be cleaved by the RNP nuclease. (C AND D) Southern blotting analysis. Aliquots of approximately 5 µg of genomic DNA from each strain were digested with XhoI and separated by electrophoresis on 0.8% (w/v) agarose gels. (C) Lane 1, TIMM20114 (azole susceptible); lane 2, 18 MM_25; lane 3, TIMM20118 (parent strain); M, DNA standard fragments (λ-EcoT14I/BglII digest). (D) Lane 1, TIMM20114 (azole susceptible); lanes 2 to 5, 21 MM_16–1, 21 MM_16–2, 21 MM_23–12, and 21 MM_23–13; lane 6, TIMM20121 (parent strain); M, DNA standard fragments. DNA standard fragment sizes are shown on the left. An internal fragment (about 410 bp) of the TinCYP51B gene was amplified by PCR with P21–P16 primers (Table S2) and used as a hybridization probe.

Fig 5

Evaluation of ITC and VRC susceptibility in the genetically modified T. indotineae strains by the serial dilution drug susceptibility assays and Etests. For the serial dilution drug susceptibility assays, spores from each strain were spotted at different dilutions on SDA plates, as previously described (4, 12). The plates were incubated at 28°C for 3 to 5 d.