Cloning and Characterization of Trichophyton rubrum Genes Encoding Actin, Tri r2, and Tri r4

Abstract

The three structural genes of Trichophyton rubrum encoding actin (3,429 bp) and two antigens, Tri r2 (2,950 bp) and Tri r4 (3,988 bp), were cloned and characterized. They contained six, four, and five exons, respectively. The T. rubrum actin protein sequence revealed extremely high homology to other fungal actins.

Trichophyton rubrum is the most common pathogen causing dermatophytosis, accounting for approximately 80% of the reported cases of onychomycosis (2). Studies regarding the structure, expression, and regulation of the genes of T. rubrum have been relatively limited because of its nonaggressive and non-life-threatening nature. Actin, a major cytoskeletal component, is involved in various cellular processes such as growth, differentiation, motility, endocytosis, and exocytosis (1). The Tri r2 and Tri r4 antigens have been identified as the putative allergens causing the delayed-type hypersensitivity reactions seen in some patients with T. rubrum infections (4). Tri r2 belongs to the class D subtilase subfamily, whereas Tri r4 is a member of the prolyl oligopeptidase family of serine proteinase (4). Thus, we sought to provide new genetic information by cloning and characterizing the ACT. Tri r2, and Tri r4 genes of T. rubrum.

For this purpose, a genomic library was constructed from one isolate of T. rubrum (catalogue number 14001; American Type Culture Collection, Manassas, Va.) and screened with ACT. Tri r2, and Tri r4 gene-specific probes. Briefly, T. rubrum was freshly grown on Sabouraud agar at 28°C for a week and its microscopic morphology was confirmed before harvest. Genomic DNA and total RNA were isolated from T. rubrum mycelia with the QIAGEN RNA/DNA kit (QIAGEN, Valencia, Calif.). The ACT probe was prepared through PCR with primers 5′-GTCTCCATCCAGGCTGTGCTCTCCCTC-3′ and 5′-CGATGATCTTGACCTTCATCGACGATG-3′, which were designed on the basis of the published partial genomic sequence (3). The Tri r2 and r4 probes were obtained by reverse transcription-PCR with primers 5′-ATGGGTTTCATCACCAAAGCCATTCCT-3′ and 5′-CAGGTTCGCGATGGCGGAGCGCACCAA-3′ (Tri r2) and primers 5′-ATGGCAGCAGCCAAATGGTTGATTGCC-3′ and 5′-GTCTAGTAGTCGAAGTAAGAGTGAGCC-3′ (Tri r4). To isolate the recombinant phages positive for ACT. Tri r2, or Tri r4, the genomic library of T. rubrum (5 × 10⁴ PFU), constructed with the Lambda FIX II/XhoI partial fill-in vector kit (Stratagene, La Jolla, Calif.), was hybridized, respectively, with the above three probes in Rapid-Hyb buffer (Amersham Biosciences, Piscataway, N.J.).

Genomic structure analysis of the ACT (3,429 bp), Tri r2 (2,950 bp), and Tri r4 (3,988 bp) genes of T. rubrum showed the presence of six, four, and five exons, respectively. The exon-intron organization of T. rubrum ACT was similar to that of the actin-encoding genes of most filamentous fungi (1, 3). The introns of the ACT. Tri r2, and Tri r4 genes match the fungal consensus sequences described for exon-intron boundaries and splice signals for lariat formation (1, 3) (Table 1). Each gene contained a CAAT motif, pyrimidine stretches at the 5′ untranslated region, and the polyadenylation signal AATAA at the 3′ untranslated region. Interestingly, a putative TATA box was found only in the ACT sequence.

TABLE 1.

Exon-intron structures of ACT, Tri r2, and Tri r4 of T. rubrum^a

Sequence	5′ Boundary	Lariat intermediate	3′ Boundary
Fungal consensus	GTAHG	CTRAC	YAG
ACT intron 1	GTATG	CTAAC	TAG
ACT intron 2	GTGAG	CTGAC	CAG
ACT intron 3	GTAAG	CTGAC	CAG
ACT intron 4	GTAGG	CTAAC	CAG
ACT intron 5	GTATG	CTAAC	TAG
Tri r2 intron 1	GTATA	CTAAC	TAG
Tri r2 intron 2	GTAAG	CTAAT	TAG
Tri r2 intron 3	GTAAG	CTAAC	TAG
Tri r4 intron 1	GTAAG	TTGAC	CAG
Tri r4 intron 2	GTAAG	CTGAC	TAG
Tri r4 intron 3	GTGAG	CTTAC	CAG
Tri r4 intron 4	GTAAG	CTAAC	TAG

^aH is A, C, or T; Y is C or T; and R is A or G according to the International Union of Biochemistry and Molecular Biology codes. Adenosines at the branch site conserved among the filamental fungi are underlined.

The 375-amino-acid actin deduced from both the genomic and cDNA sequences had a predicted molecular mass of 42.0 kDa and an isoelectric point of 5.63, resembling other fungal actins previously described (1). In a BLAST search analysis, the T. rubrum actin protein sequence showed extremely high identity and similarity scores with respect to other fungal actins (Table2). The following three signatures characteristic of actins were identified in T. rubrum actin: ⁵³YVGDEAQSKRG⁶³, ³⁵⁶WISKQEYDE³⁶⁴, and ¹⁰⁴LLTEAPINPKSNR¹¹⁶ (1). The T. rubrum ACT genomic sequence showed 97 to 99% identity to partial ACT sequences of T. verrucosum (GenBank accession no. AF152232), T. violaceum (GenBank accession no. AF152233), T. mentagrophytes (GenBank accession no. AJ430623), and T. schoenleinii (GenBank accession no. AJ430622). The T. rubrum Tri r2 genomic sequence exhibited 90% identity to Tri m2 (GenBank accession no. AJ430841) and Tri m2 (GenBank accession no. AJ430840). Finally, T. rubrum Tri r4 showed 95% identity to Tri s4 (GenBank accession no. AJ430626).

TABLE 2.

Amino acid sequence homology of T. rubrum actin to other fungal actins

Organism	GenBank accession no.	Identity (%)	Similarity (%)
Paracoccidioides brasiliensis	AAR15701	97	98
Acremonium chrysogenum	AAF00008	96	99
Neurospora crassa	CAC28718	96	99
Gaeumannomyces graminis	AAR01976	96	99
Botryotinia fuckeliana	CAA04009	96	99
Exophiala dermatitidis	AAL68896	96	98
Humicola grisea var. thermoidea	BAA74960	96	99
Emericella nidulans	P20359	96	98
Penicillium chrysogenum	Q9URS0	95	98
Colletotrichum gloeosporioides f. sp. malvae	AAD41038	94	97
Thermomyces lanuginosus	P10365	92	97
Schizosaccharomyces pombe	NP_595618	91	96
Saccharomyces cerevisiae	NP_116614	91	96
Candida glabrata	P60009	91	96
Saccharomyces pastorianus	CAA24599	91	96
Saccharomyces bayanus	JS0702	91	96
Absidia glauca	P26197	90	96

In summary, we report the complete genomic sequences and organization of three genes (ACT. Tri r2, and Tri r4) of T. rubrum, the most common pathogen causing fungal infectious disease. Our data may form the basis for the development of molecular diagnosis of dermatophytosis in the future.

Nucleotide sequence accession numbers.

The nucleotide sequences of ACT (3,429 bp), Tri r2 (2,950 bp), and Tri r4 (3,988 bp) were deposited in the GenBank database and assigned accession numbers AY525329, AY525330, and AY525331, respectively.