Keratin hydrolysis by dermatophytes

Keratin hydrolysis by dermatophytes Abstract Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes’ extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and ‘-omic’ studies that are helping to dissect the complex process of keratin breakdown and utilization. Dermatophyte, Keratin hydrolysis, Protease, Peptidase, Sulfitolysis, Keratinase Introduction Dermatophytes comprise 52 species of keratin-degrading ascomycetes of the genera Trichophyton, Microsporum, Epidermophyton, Arthroderma, Lopophyton, Nannizia, Ctenomyces, Guarromyces, and Paraphyton.1,2 The principal etiologic agents of human dermatophytosis globally are T. rubrum, T. interdigitale, T. mentagrophytes, E. floccosum, and M canis (anamorph of A. otae). Of these, T. rubrum is isolated in 50–80% of cases, mainly from onychomycosis and tinea pedis.1–4 Dermatophytes are keratinophilic, capable of infecting keratinous tissues, including skin, nails, and hair, of humans and other vertebrates.1 Keratinolytic enzymes, including proteases and peptidases, are important virulence factors of dermatophytes and are essential for their ability to infect keratinous tissues.4 The keratin superfamily of proteins contains >60 members which include acidic (type I, PI 4.8–5.4), and basic to neutral (type II, PI 6.5–8.5) proteins, normally containing a central rod-like domain of alpha-helical secondary structure and less organized terminal domains. Hard keratins, typical of nails and hair, contain regions with highly ordered protein filaments, extensively cross-linked by intermolecular disulfide bonds (∼18% cysteine). Soft keratins, typical of the stratum corneum, contain relatively disordered protein filaments with fewer disulfide linkages (∼2–4% cysteine),5,6 as well as other linkages, for example, isopeptide (gamma-glutamyl–epsilon-lysine) bonds.7,8 Over 144 different proteins have been detected in human nails including keratins, cytoplasmic and junctional proteins.9 In the skin, proteins associated with keratin include elastin, collagen, fibronectin, laminin,10 filaggrin,11 and the small proline rich proteins loricrin and involucrin,12,13 among others. Here, we summarize the current state of the art of keratin biodegradation by dermatophytes and its links with virulence, focusing specifically on sulfitolysis and proteolysis, and build on previous reviews on the subject.14–16 Search strategy and selection criteria All publications cited within this review were identified via PubMed searches up to and including March 2017, and references cited therein. Search terms used included but were not limited to dermatophyte, Trichophyton, Microsporum, Epidermophyton, Arthroderma, protease, peptidase, proteinase, protein hydrolysis, keratin hydrolysis, keratinase, and sulfitolysis/sulphitolysis, and combinations thereof. Descriptions of the classes of ‘protease/proteinase/peptidase’ were based on those stated within the cited references and/or via definitions in the MEROPS database (MEROPS the peptidase database; https://www.ebi.ac.uk/merops/).17 Gene/protein homology searches were conducted using the relevant BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using nucleotide, protein, genome or GEO datasets. Cited nucleotide and/or protein sequences were from peer-reviewed scientific publications where possible and appropriate accession numbers were cited. Genomic analysis of proteases and peptidases In a comparison of dermatophyte genomes with those of other fungi, proteases constituted one of four over-represented functional categories.18 Approximately 20% of the 100 most expressed secreted proteins of Trichophyton benhamiae (previously Arthroderma benhamiae) were proteases, during growth both in vivo and on keratin in vitro.19 The closely related dermatophytes T. verrucosum and T. benhamiae were found to possess 235 predicted proteinase-encoding genes (87 with signal peptides), none of which were unique to either species. Enzyme families represented included 18 metallopeptidases and 14 serine peptidases accompanied by cysteine and aspartic peptidases. Whether all these genes are expressed is not known, although an M14 metallocarboxypeptidase, McpA, identified in this study had been isolated and characterised previously from T. rubrum.20 Proteolysis-related genes/proteins for which functional characterization has been conducted are described in Supplementary Table S1. Analysis of the secretome of T. benhamiae following growth on keratin, identified significantly fewer proteinases than was predicted from the genome. Proteinases identified included subtilisin-like serine proteases (Sub3, Sub4, and Sub7), metalloproteinases (fungalysins) (Mep1, Mep3, and Mep4), leucine aminopeptidases (Lap1 and Lap2) and dipeptidyl peptidases (DppIV and DppV). Interestingly, upon co-culture with keratinocytes, of the proteinases identified from the secretome only the expression of the genes encoding DppV and a putative S1 carboxypeptidase (Acc no ARB_06019) were upregulated.21 Two S10 serine carboxypeptidases (ScpA and ScpB) of T. rubrum contained predicted C-terminal Glycosylphosphatidylinositol (GPI) anchors and concomitant omega sites (PredGPI22), suggesting a cell surface location. Both enzymes were detected in membrane extracts.20 A BLAST search of the T. rubrum ScpA protein revealed >90% identity with proteins from T. violaceum, T. interdigitale, T. benhamiae and other dermatophytes (Table S1). A similar search of the T. rubrum ScpB protein revealed >90% identity with proteins from T. benhamiae, T. interdigitale, and T. equinum (Table S1), all of which were predicted to contain GPI anchors.19 Interestingly, most dermatophytes also produced examples of S10 serine carboxypeptidases that did not contain predicted GPI anchors, such as ScpC of T. rubrum.20 One M36 fungalysin identified in T. equinum and T. tonsurans was predicted to contain a GPI anchor unique among dermatophyte fungalysins.18 Analysis of the T. benhamiae secretome predicted that 22 proteases were likely to be GPI-anchored, including fungalysins, deuterolysins, subtilisins, and a range of peptidases.19 Analysis of the genome of T. rubrum var. raubitschekii IGIB-SBL-CI1 identified 16 enzymes belonging to the subfamilies S8A (12 genes; subtilisins), S8B (one gene; kexin) and S53 (three genes; sedolisin),23 indicating a need for further investigations of dermatophyte genomics. The dermatophyte proteases described in Table S1 are representative of published data and three examples from different species are given for each. In addition to the proteases described in Table S1, there remain many predicted/putative proteases that have not been characterised and hypothetical proteins with regions of homology to known protease families.17 For example, the T. benhamiae genome contains 9 predicted A1 aspartic proteases and 2 C40 peptidases. Of the 29 predicted metalloproteases (M10B, M12B, M14, M19, M20, M28, M35, M36, and M43 families) and 35 serine proteases (S1, S8, S9, S10, S28, S33, S41, and S53 families), only 13 and 20, respectively, have been confirmed by mass spectrometry,19 but not confirmed as proteases experimentally. It is possible that some of these genes are not expressed or are pseudogenes, such as the scpD homologue found in T. rubrum CHUV1673-05 which is interrupted by four stop codons.20 Preliminary sulfitolysis Although dermatophytes produce several proteases, most are not capable of degrading native keratin until the hydrolysis of cross-linking disulfide bonds by sulfitolysis has occurred (Fig.1).24–26 L-cysteine acts as the substrate for sulfite production by T. benhamiae27 and there is sufficient free cysteine in keratin to support sulfite formation.28 In T. benhamiae, cysteine is oxidised to cysteine sulfinic acid (cysteine sulfinate) by cysteine dioxygenase (Cdo1), leading, ultimately, to sulphite production.27 Cysteine dioxgenase was also identified in T. mentagrophytes and the recombinant enzyme produced cysteine sulfinic acid from cysteine.28 It is hypothesized that in dermatophytes, cysteine sulfinic acid undergoes transamination by glutamate-oxaloacetate transaminase (aspartate aminotransferase [AspAT]) to generate β-sulfinylpyruvate, which spontaneously decomposes to pyruvate and sulfite, as occurs in mice.29 Sulfite is secreted from the cell via the sulfite efflux pump Ssu1 (an integral membrane protein with 10 transmembrane domains), which belongs to the tellurite-resistance/dicarboxylate transporter (TDT) family.30 Extracellular sulfite could then reduce the disulfide bonds in keratin, facilitating proteolysis (Fig. 1 and Table S1).27 In an analogous system in T. mentagrophytes, cystine induced the expression and activation of Cdo1.31 The sulphite efflux pump Ssu1 is also produced by T. rubrum.30 Targeted gene knockouts of Cdo1 and Ssu1 in T. benhamiae resulted in a strain hypersensitive to cysteine and unable to grow on hair and nails, demonstrating a role for these enzymes in cysteine detoxification.27 In an alternative branch of the pathway, cysteine sulfinic acid can be metabolised to hypotaurine or taurine, via cysteine sulfinic acid decarboxylase and hypotaurine dehydrogenase, but this has no known role to play in keratin hydrolysis.15 Figure 1. View largeDownload slide Keratin sulfitolysis by dermatophytes, modified from24. Figure 1. View largeDownload slide Keratin sulfitolysis by dermatophytes, modified from24. Keratin hydrolysis: endoproteases The main groups of proteases produced by dermatophytes are shown in Figure 2 and described in Table S1. The major dermatophyte endoproteinases are subtilisins (S8A serine protease family) and fungalysins (M36 metallopeptidase family).17 These and other endoproteinases have undergone family expansions in the dermatophytes in comparison to most other pathogenic fungi.18,19 Figure 2. View largeDownload slide Keratin proteolysis by dermatophytes. Figure 2. View largeDownload slide Keratin proteolysis by dermatophytes. Growth of dermatophytes on keratin in vitro and in infection models typically results in overexpression of subtilisin genes, though the individual enzyme/s affected may vary according to the conditions and the fungal species involved. An enzyme later identified as Sub332 was isolated from M. canis grown in vitro and in/on M. canis hyphae (but not spores) in hairs of naturally infected cats33 and experimentally infected guinea pigs.34 The purified enzyme hydrolysed keratin azure, even in the absence of reducing agents, and a chymotrypsin/subtilisin test substrate, as did recombinant Sub3,35 and activity was inhibited by phenylmethylsulfonyl fluoride (PMSF).33,36 In M. canis, high keratinolytic activity was associated with increased severity of infections in guinea pigs, a correlation not seen for other hydrolytic enzymes.37 Subtilisins Sub1, Sub2, and Sub3 were cloned from M. canis IHEM15221, and mRNA of all three genes was detected by polymerase chain reaction (PCR) in the hairs of experimentally infected guinea pigs.32 All three genes contained the catalytic triad Asp/His/Ser characteristic of S8 subtilisins (IPR023828)38 as well as prepro- and signal peptide sequences (IPR010259) characteristic of secreted proteins.32 Also, sub1 and sub3, but not sub2, were transcribed in M. canis IHEM21239 arthroconidia, suggesting a role for the two proteases in the early stages of infection.39 The adherence of M. canis arthroconidia to reconstructed interfollicular feline epidermis was significantly reduced in the presence of serine protease inhibitors and by monoclonal antibodies to Sub3, suggesting that subtilisins, including Sub3, have a role in adherence,39 as postulated for the serine proteases of other fungi, including Candida spp.40 Experiments with an RNA-silenced-Sub3 strain of M. canis IHEM22957 also suggested that Sub3 had an important role in adherence, but was not necessary for the invasion of keratinized tissues.41 A role for Sub3 in adherence was also demonstrated with epidermis from humans and a range of animals, although the addition of recombinant Sub3 had no effect on adherence of this strain, indicating that Sub3 may be cell-associated.42 The propeptide of Sub3 acts as a noncompetitive inhibitor of Sub3 activity, but could not prevent adherence of M. canis to feline epidermis, probably being inactivated by cell surface-associated protease/s.43 For T. rubrum, serine protease activity was detected in crude culture supernatants44 and in isolated keratinases.45–47 Genes (sub1–7) encoding putative subtilisin serine proteases (S8 family) were identified in T. rubrum and phylogenetic analysis revealed sub2 as the likely ancestral gene.25 Extracellular Sub3 and Sub4 were produced when T. rubrum was grown on soy protein as the sole carbon and nitrogen source, and recombinant Sub3 and Sub4 could hydrolyse non-reduced and reduced keratin azure, but were less active against non-keratin proteins.25 Expressed sequence tags for sub1, sub5, and sub6, but not sub2, sub3, sub4 and sub7 were identified following growth of T. rubrum BMU01672 on YPG medium.48 Overexpression of subtilisin genes has been demonstrated in T rubrum strains grown on keratin for sub1, sub3, and sub 649 and separately for sub3 and sub550,51 and sub3 and sub4.58,59 In contrast, when T. rubrum ATCC52021 was grown on keratin, no such upregulation was detected.54 Real-time PCR analysis revealed high levels of sub7 expression following growth on human nail, whereas both sub7 and sub5 expression was elevated following growth on human stratum corneum.55 Elevated expression of sub1 was observed when T. rubrum CBS118892 was co-cultured with the HaCaT human keratinocyte cell line for 24 h.56 In a T. benhamiae LAU2354-2 guinea pig dermal infection model, upregulation of expression of sub6 by was detected, along with expression of sub1-3 and sub7, but not sub4, whereas growth in vitro on keratin was accompanied by strong upregulation of sub3 and sub4.57 In a later study, the genes for sub6, sub7, sub8, and sub10 were among the most highly expressed in vivo, whereas sub3 and sub4 were among the most highly expressed in vitro.19 Sub6, identified as the T. rubrum antigen/allergen Tri r 2,58,59 appears to have a role in dermatophyte virulence. Disruption of sub6 by site-directed mutagenesis in T. mentagrophytes ATCC28185 increased in vitro proteolytic activity and altered levels of expression of several protease genes. It also resulted in delayed onset of clinical symptoms, lower-grade lesions and reduced inflammation in infected guinea pigs. The reduced pathogenicity of the manipulated strain seemed to result in part from changes in the pattern of cytokine production.60 Of the enzymes secreted by T. rubrum during growth on keratin, Sub6 was the only subtilisin recovered from the nail beds of onychomycotic patients by Mehul and co-workers.61 Analysis of proteinase secretion by T. rubrum growing on human skin demonstrated that Sub3, Sub4, and Mep4 were probably the main proteinases responsible for the invasion of skin during infection.52 However, gene expression in cultures grown on excised human skin differed from that in cultures grown on media containing keratin, elastin, and collagen. The dermatophyte zinc-dependent metalloproteases (M36 family; fungalysins) were first identified in M. canis as three genes (mep1–3) homologous to the single Aspergillus fumigatus Mep gene.14,62,63 And mep2 and mep3 were expressed in M. canis infected guinea pigs, indicating a potential role in virulence/infection.63 Only one mep was identified in the geophile A. gypseum64, with significant homology to mep2 of T. rubrum and mep3 of T. tonsurans. This family was subsequently expanded to five genes (mep1–5) in T. rubrum, T. mentagrophytes and M. canis, all with high sequence homology and containing the characteristic HEXXE amino acid motif.65 When grown in vitro on soy protein, Meps accounted for 19–36% of total secreted protein and their activity was inhibited by the metalloprotease inhibitor o-phenanthroline.66 A comparison of the pathogenic potential of five metalloprotease genes from T. mentagrophytes led to the proposal that Mep4 and Mep5 were most likely to affect pathogenicity, determined in a guinea pig model and a keratin degradation test,67 whereas expression of only mep4 was significantly upregulated following growth in vitro on keratin, collagen, elastin or human skin sections.50,52 When T. rubrum was cultured with human keratinocytes (HaCaT), elevated expression of mep4 and subtilisin genes was observed,56 whereas in a feline M. canis skin infection model there was no evidence of mep expression during adhesion or early stages of invasion.68 The expression of protease genes, and production of the encoded proteases, is further complicated by the fact that keratin hydrolysis in vitro and infection of keratinous tissues are two distinct processes that require the production of different proteases and associated genes/proteins, a fact that has only recently become better understood. Keratin hydrolysis: exoproteases The action of endoproteases on keratin releases free peptides on which exoproteases may act.69,70 The extracellular aminopeptidases Lap1 and Lap2 (M28A family) and the dipeptidyl peptidases DppIV and DppV (S9 family) of T. rubrum grown on keratin were characterised by Monod and colleagues.71 DppV was originally identified as the T. tonsurans allergen Tri t 472, Tri r 4 of T. rubrum59 and Tri m 4 of T. mentagrophytes.73 Interestingly, when T. benhamiae was co-cultured with keratinocytes, expression of DppV was upregulated, but the expression of the other exoproteases described above was not.21 The leucine aminopeptidases Lap1 and Lap2 hydrolyse peptides from the N-terminus until they reach X-Pro or X-Ala sequences, which act as a stop.68 Transcriptional profiling of T. rubrum revealed that expression of lap1 and lap2 was upregulated during growth on medium containing keratin, but not elastin.49 Western blotting of culture supernatant of T. rubrum detected the presence of Lap1 and Lap2 and recombinant Lap1 and Lap2 hydrolysed leucine from the Leu-aminoacyl-4-methylcoumaryl-7-amide (AMC) substrate. Neither Lap hydrolysed the Gly-Pro-AMC DppIV substrate.71 Species/strain specific differences exist in Lap1 and Lap2 production between different strains of T. rubrum and T. violaceum.74 Secretome analysis of T. benhamiae proteins following keratin hydrolysis identified Lap1 and Lap2.21 An earlier study demonstrated that production of these enzymes was increased during proteolysis in vitro, but not in a guinea pig infection model.57 In a later study, the genes for Lap1 and Lap2 were among the most highly expressed by the same T. benhamiae strain during in vitro growth on keratin but not in an in vivo guinea pig infection model, whereas expression of lap2 alone was upregulated following growth on soy protein.19 Interestingly, when grown at neutral pH in soy protein medium, the major Lap produced by M. canis was Lap1, whereas Lap2 was the major Lap produced by T. benhamiae, T. rubrum, and T. violaceum.74,75 Dipeptidyl peptidase IV removes N-terminal dipeptides (Xaa-Yaa-|-Zaa) sequentially from polypeptides having unsubstituted N-termini preferentially when the penultimate residue (Yaa) is proline, and in some cases alanine, and when Zaa is neither proline nor hydroxyproline. Dipeptidyl peptidase V removes N-terminal dipeptides sequentially from polypeptides having unsubstituted N-termini preferentially when the penultimate residue (Yaa) is alanine. Recombinant DppIV from T. rubrum hydrolyzed Gly-Pro-AMC and Lys-Ala-AMC, but with a preference for X-Pro sequences. Recombinant DppV from T. rubrum CHUV 862-00 hydrolysed Lys-Ala-AMC, but not Gly-Pro-AMC; both enzymes were active between pH 6.5 and 10.5. Neither DppIV or DppV could hydrolyze tripeptides.71 The genes for DppIV and DppV were detected in four strains each of T. tonsurans and T. equinum and demonstrated 99.88 and 99.96% identity, respectively.76 Analysis of the secretome of T. benhamiae LAU2354 (CBS112371) following growth on keratin revealed the presence of DppIV and DppV,21 consistent with the data of Staib and colleagues.57 Expression of dppV was upregulated during co-culture of T. benhamiae with HaCaT keratinocytes, whereas lap1, lap2, and dppIV were not.21 A study of secreted proteins of two strains of T. rubrum and one of T. violaceum grown on soy protein detected DppIV from all three isolates, but DppV only from T. violaceum,74 indicating possible species/strain specificities in Dpp secretion. Transcription of the genes for DppIV and DppV were detectable in vitro in arthroconidia of three isolates of M. canis and dppIV was transcribed during adherence of these isolates and during invasion by two of the isolates in an ex vivo model of cat skin infection, indicating a possible role of these enzymes in infection,68 as observed for DppIV of Aspergillus fumigatus during colonization of collagen and elastin.77 Expression of the DppV gene was induced several-fold in a T. mentagrophytes clinical isolate following growth on medium containing, as the sole source of C and N, keratin, elastin, or blood plasma, but not human skin.73 It is hypothesized that the activity of DppIV and DppV provide access for the Laps to the next amino acids in peptides, potentially acting synergistically as demonstrated in Aspergillus oryzae and lactic acid bacteria.21,78,79 Carboxypeptidases hydrolyse C-terminal amino acids from peptides and this activity was detected in T. rubrum, T. benhamiae, and M. gypseum.69 Three serine carboxypeptidases (ScpA, ScpB, and ScpC) of the S10 family were produced by T. rubrum in vitro;53T. benhamiae expressed the genes for these carboxypeptidases in vitro and during a guinea pig infection.57 ScpA and ScpB of dermatophytes are unusual, in that, unlike their Aspergillus spp. homologues, they are not secreted, but are membrane-associated with GPI anchors, whereas ScpC does not have a GPI anchor, but may be vacuolar.20 All of the genomes of seven dermatophyte species examined (T. rubrum T. tonsurans, T. equinum, T. verrucosum, M. canis, M. gypseum, and T. benhamiae) possessed 11 S10 carboxypeptidase genes, except T. verrucosum, which had 12 carboxypeptidase genes.18 Trichophyton rubrum secreted two zinc-dependent metallocarboxypeptidases, McpA and McpB, (M14A family), when grown on protein. Recombinant McpA and ScpA of T. rubrum efficiently hydrolyzed N-(2-furanacryloyl)-L-phenylalanyl-L-phenylalanine (FAPP), a known carboxypeptidase A substrate.53 Expression of mcpA was increased when T. benhamiae CBS112371 was grown on keratin-soy medium and in a guinea pig infection model,57 whereas Tran et al. found in the same strain that mcpA was one of the most highly expressed during in vitro growth on keratin, but not in guinea pigs.19 Analysis of the same seven dermatophytes described above revealed the presence of four M14 metallocarboxypeptidase genes in the genome of all isolates, except T. benhamiae, which possessed five such genes.18 Analysis of the genome of T. rubrum var. raubitschekii isolated from a patient with onychomycosis, revealed 12 carboxypeptidase genes.23 Microenvironment pH and protease activity The pH of healthy skin and nails is mildly acidic;80 however, the metabolism of amino acids released during keratin breakdown results in a shift to an alkaline pH.14 The dermatophyte keratinases include some which exert optimal activity at the mildly acidic pH prevailing in the early stages of infection, and others with maximal activity at higher pH values found later during keratin breakdown (reviewed by Peres et al. 201051). Culture pH has been shown to affect gene expression and the pattern of protease production in T. rubrum.81 Both M. canis and T. benhamiae generated more endoprotease activity at neutral pH compared to pH 4.0, as was the case for most exoprotease activity of M. canis.75 The proteases secreted at acidic pH by T. benhamiae and M. canis include aspartic proteases and sedolisins/serine carboxypeptidases.75 Dipeptidyl peptidases may have pH-specific roles, as DppIV was one of the major proteases produced by T. benhamiae and M. canis at neutral pH, whereas DppV was more prevalent at acidic pH.75 T. rubrum genes involved in proteolysis and differentially expressed at acidic pH (5.0) included an S10 serine carboxypeptidase ScpB,20 dipeptidases, amino acid permeases, and a major facilitator superfamily di/tripeptide transporter, whereas genes differentially expressed at alkaline pH (8.0) included an amino acid permease and an arginine transporter.51,52 Peptide/Amino acid permeases Information on amino acid and peptide uptake by dermatophytes is relatively scarce, but the expression of 60 genes involved in transport functions were differentially regulated when T. rubrum was grown in the presence of human skin sections, albeit none specifically identified as amino acid/peptide transporters82 and 66 putative transporter genes were identified following growth on keratin, one of which demonstrated significant identity with the Aspergillus fumigatus LysP amino acid/peptide permease (acc. no. AAC98709).83 Additionally, dermatophyte genomes contain genes for ABC transporters that may be involved in amino acid/peptide uptake but have yet to be functionally characterized.84 The TruMDR2 gene, encoding an ABC transporter involved in antifungal resistance,85 in T. rubrum was disrupted, and this correlated with decreased ability of the mutant to grow on nail, indicating a possible role in keratin hydrolysis and uptake.83T. rubrum genes differentially expressed at acidic pH (5.0) included genes with homology to oligopeptide/amino acid permeases, for example, ptr2 and sec24, and a major facilitator superfamily di/tripeptide transporter, whereas genes differentially expressed at alkaline pH (8.0) included those with homology to an amino acid permease and an arginine transporter.51,52 Finally, multiple dermatophyte species also contain genes with homology to the amino acid permease gap1 of Saccharomyces cerevisiae and the proline permease prnB of Aspergillus nidulans that may be under control of Dnr1/AreA.86 Regulation of gene expression The regulation of gene expression in dermatophytes has received little attention. The PacC gene of T. rubrum ATCC MYA-3108 is homologous to the PacC/Rim101p family of pH-responsive transcription factors and expression of pacC is upregulated under alkaline conditions. Disruption of pacC decreased the ability of the mutant to grow on human nail, but not Sabouraud medium, and the secretion of keratinolytic proteases was decreased in a pacC mutant, indicating the probable importance of PacC in keratin hydrolysis and pathogenicity.87 Disruption of the T. rubrum PacC gene had no effect on transcription of the S10 carboxypeptidase ScpB gene.81 Interestingly, expression of pacC was downregulated when heat shock protein 90 (Hsp90) gene expression was chemically inhibited in T. rubrum CBS118892 (ATCC MYA-4607). Hsp90 inhibition also decreased growth of T. rubrum CBS118892 on human nail, but not skin, indicating a probable role of Hsp90 in keratin degradation and pathogenicity via PacC.88 Yamada and colleagues,86 isolated a global nitrogen regulatory gene, dnr1, from M. canis characteristic of the GATA family of transcription factors, and a homologue of the Aspergillus nidulans gene areA.89 These transcription factors activate the expression of genes, including those encoding proteases,90 and play a role in fungal virulence.91 When dnr1 was disrupted, the dnr1− mutants showed reduced growth on ammonia, but could use many amino acids for growth, suggesting a role in the regulation of nitrogen metabolism. The dnr1− mutants showed weak growth on keratin, suggesting that the dnr1 gene might have a role in the regulation of protease production and possibly amino acid uptake.19 Homologues of dnr1/areA have subsequently been identified in T. rubrum (82% identity; 664/808 aa), T. verrucosum (82% identity; 662/808 aa), T. benhamiae (80% identity; 655/820 aa), and T. mentagrophytes (83% identity; 666/805 aa).92 The transcriptional regulator StuA (an APSES transcription factor) has been partially characterized in T. benhamiae. APSES transcription factors have been associated with virulence in plant pathogenic fungi.93T. benhamiae stuA deletion mutants were unable to grow on hair and nail, unlike wild-type and stuA complemented strains, indicating a probable role in keratin degradation and pathogenicity.94 It is probable that other dermatophyte transcription factors remain to be discovered. Discussion The remarkable complexity of keratin biodegradation by dermatophytes begins with sulfitolysis, which involves the intracellular generation of sulfite from cysteine catabolism via the enzymes cysteine dioxygenase (Cdo1) It is hypothesized that in dermatophytes, cysteine sulfinic acid undergoes transamination by glutamate-oxaloacetate transaminase (AspAT; aspartate aminotransferase) to generate β-sulfinylpyruvate, which spontaneously decomposes to pyruvate and sulfite, as occurs in mice.25 The sulphite is exported via the sulphite efflux pump Ssu1 so that it can degrade the extensive disulphide bridges found in the keratin proteins (Fig. 1).27 However, sulfitolysis may not be essential in all cases as some secreted subtilisins can degrade keratin without prior reduction (Sub3 and Sub4 hydrolysis of keratin azure).21,32 The initial stages of keratin proteolysis are performed by endoproteases, of which dermatophytes have expanded families compared with other filamentous fungi, especially S8 family subtilisins and M36 family fungalysins (Fig. 2).18 The peptides generated by endoprotease activity are further hydrolysed by exoproteases, followed by uptake of tripeptides, dipeptides and amino acids for use in central metabolism. The expansion of protease families within the dermatophytes makes them well placed in nature to be able to utilise keratin and infect and invade keratinised tissues. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgments D.K.M. and C.S.S. contributed equally to the preparation of this manuscript. Declaration of interest Derry K Mercer is an employee of NovaBiotics Ltd. Colin Stewart is a former employee of NovaBiotics Ltd. References 1. Borman AM , Summerbell RC . Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses . In: Jorgensen JH , Pfaller MA , Carroll KC , Funke G , Landry ML , Richter SS , Warnock DW , eds. Manual Of Clinical Microbiology, vol 2 . Washington, DC : ASM Press , 2015 : 2128 – 2152 . Google Scholar CrossRef Search ADS 2. de Hoog GS , Dukik K , Monod M et al. Toward a novel multilocus phylogenetic taxonomy for the dermatophytes . Mycopathologia . 2017 ; 182 : 5 – 31 . Google Scholar CrossRef Search ADS PubMed 3. Borman AM , Campbell CK , Fraser M , Johnson EM . Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades . Med Mycol . 2007 ; 45 : 131 – 141 . Google Scholar CrossRef Search ADS PubMed 4. Nenoff P , Kruger C , Ginter-Hanselmayer G , Tietz HJ . Mycology, an update. Part 1: Dermatomycoses: causative agents, epidemiology and pathogenesis . J Dtsch Dermatol Ges . 2014 ; 12 : 188 – 209 . Google Scholar PubMed 5. Moll R , Divo M , Langbein L . The human keratins: biology and pathology . Histochem Cell Biol . 2008 ; 129 : 705 – 733 . Google Scholar CrossRef Search ADS PubMed 6. Bragulla HH , Homberger DG . Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia . J Anat . 2009 ; 214 : 516 – 559 . Google Scholar CrossRef Search ADS PubMed 7. Bruce Fraser RD , Parry DA . The role of disulfide bond formation in the structural transition observed in the intermediate filaments of developing hair . J Struct Biol . 2012 ; 180 : 117 – 124 . Google Scholar CrossRef Search ADS PubMed 8. Gong H , Zhou H , McKenzie GW et al. An updated nomenclature for keratin-associated proteins (KAPs) . Int J Biol Sci. 2012 ; 8 : 258 – 264 . Google Scholar CrossRef Search ADS PubMed 9. Rice RH , Xia Y , Alvarado RJ , Phinney BS . Proteomic analysis of human nail plate . J Proteome Res . 2010 ; 9 : 6752 – 6758 . Google Scholar CrossRef Search ADS PubMed 10. Woodley DT , O’Keefe EJ , Prunieras M . Cutaneous wound healing: a model for cell-matrix interactions . J Am Acad Dermatol . 1985 ; 12 : 420 – 433 . Google Scholar CrossRef Search ADS PubMed 11. McLean WH. Filaggrin failure: from ichthyosis vulgaris to atopic eczema and beyond . Br J Dermatol . 2016 ; 175 : 4 – 7 . Google Scholar CrossRef Search ADS PubMed 12. Eckert RL , Crish JF , Efimova T et al. Regulation of involucrin gene expression . J Invest Dermatol . 2004 ; 123 : 13 – 22 . Google Scholar CrossRef Search ADS PubMed 13. Nithya S , Radhika T , Jeddy N . Loricrin - an overview . J Oral Maxillofac Pathol . 2015 ; 19 : 64 – 68 . Google Scholar CrossRef Search ADS PubMed 14. Monod M. Secreted proteases from dermatophytes . Mycopathologia . 2008 ; 166 : 285 – 294 . Google Scholar CrossRef Search ADS PubMed 15. Kasperova A , Kunert J , Raska M . The possible role of dermatophyte cysteine dioxygenase in keratin degradation . Med Mycol . 2013 ; 51 : 449 – 454 . Google Scholar CrossRef Search ADS PubMed 16. Monod M , Mignon B , Staib S . Dermatophytes as saprophytes and pathogens . In: Sullivan DJ , Moran GP , eds. Human Pathogenic Fungi: Molecular Biology and Pathogenic Mechanisms . Norfolk, UK : Caister Academic Press , 2014 : 223 – 252 . 17. Rawlings ND , Barrett AJ , Finn R . Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors . Nucleic Acids Res . 2016 ; 44 : D343 – 350 . Google Scholar CrossRef Search ADS PubMed 18. Martinez DA , Oliver BG , Graser Y et al. Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection . MBio . 2012 ; 3 : e00259 – 12 . Google Scholar CrossRef Search ADS PubMed 19. Tran VD , De Coi N , Feuermann M et al. RNA Sequencing-based genome reannotation of the dermatophyte Arthroderma benhamiae and characterization of its secretome and whole gene expression profile during infection . mSystems . 2016 ; 1 : e00036 – 16 . Google Scholar CrossRef Search ADS PubMed 20. Zaugg C , Jousson O , Lechenne B , Staib P , Monod M . Trichophyton rubrum secreted and membrane-associated carboxypeptidases . Int J Med Microbiol . 2008 ; 298 : 669 – 682 . Google Scholar CrossRef Search ADS PubMed 21. Burmester A , Shelest E , Glockner G et al. Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi . Genome Biol . 2011 ; 12 : R7 . Google Scholar CrossRef Search ADS PubMed 22. Pierleoni A , Martelli PL , Casadio R . PredGPI: a GPI-anchor predictor . BMC Bioinformatics . 2008 ; 9 : 392 . Google Scholar CrossRef Search ADS PubMed 23. Latka C , Dey SS , Mahajan S et al. Genome sequence of a clinical isolate of dermatophyte, Trichophyton rubrum from India . FEMS Microbiol Lett . 2015 ; 362 : fnv039 . Google Scholar CrossRef Search ADS PubMed 24. Blyskal B. Fungi utilizing keratinous substrates . Int Biodeterior Biodegradation . 2009 ; 63 : 631 – 653 . Google Scholar CrossRef Search ADS 25. Jousson O , Lechenne B , Bontems O et al. Secreted subtilisin gene family in Trichophyton rubrum . Gene . 2004 ; 339 : 79 – 88 . Google Scholar CrossRef Search ADS PubMed 26. Kunert J. Keratin decomposition by dermatophytes: evidence of the sulphitolysis of the protein . Experientia . 1972 ; 28 : 1025 – 1026 . Google Scholar CrossRef Search ADS PubMed 27. Grumbt M , Monod M , Yamada T , Hertweck C , Kunert J , Staib P . Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump . J Invest Dermatol . 2013 ; 133 : 1550 – 1555 . Google Scholar CrossRef Search ADS PubMed 28. Kasperova A , Kunert J , Horynova M et al. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes . Mycoses . 2011 ; 54 : e456 – e462 . Google Scholar CrossRef Search ADS PubMed 29. Griffith OW. Cysteinesulfinate metabolism. altered partitioning between transamination and decarboxylation following administration of beta-methyleneaspartate . J Biol Chem . 1983 ; 258 : 1591 – 1598 . Google Scholar PubMed 30. Lechenne B , Reichard U , Zaugg C et al. Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes . Microbiology . 2007 ; 153 : 905 – 913 . Google Scholar CrossRef Search ADS PubMed 31. Kasperova A , Cahlikova R , Kunert J , Sebela M , Novak Z , Raska M . Exposition of dermatophyte Trichophyton mentagrophytes to L-cystine induces expression and activation of cysteine dioxygenase . Mycoses . 2014 ; 57 : 672 – 678 . Google Scholar CrossRef Search ADS PubMed 32. Descamps F , Brouta F , Monod M et al. Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo . J Invest Dermatol . 2002 ; 119 : 830 – 835 . Google Scholar CrossRef Search ADS PubMed 33. Mignon BR , Nikkels AF , Pierard GE , Losson BJ . The in vitro and in vivo production of a 31.5-kD keratinolytic subtilase from Microsporum canis and the clinical status in naturally infected cats . Dermatology . 1998 ; 196 : 438 – 441 . Google Scholar CrossRef Search ADS PubMed 34. Mignon BR , Leclipteux T , Focant C , Nikkels AJ , Pierard GE , Losson BJ . Humoral and cellular immune response to a crude exo-antigen and purified keratinase of Microsporum canis in experimentally infected guinea pigs . Med Mycol . 1999 ; 37 : 123 – 129 . Google Scholar CrossRef Search ADS PubMed 35. Descamps F , Brouta F , Vermout S , Monod M , Losson B , Mignon B . Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis . FEMS Immunol Med Microbiol . 2003 ; 38 : 29 – 34 . Google Scholar CrossRef Search ADS PubMed 36. Mignon B , Swinnen M , Bouchara JP et al. Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats . Med Mycol . 1998 ; 36 : 395 – 404 . Google Scholar CrossRef Search ADS PubMed 37. Viani FC , Dos Santos JI , Paula CR , Larson CE , Gambale W . Production of extracellular enzymes by Microsporum canis and their role in its virulence . Med Mycol . 2001 ; 39 : 463 – 468 . Google Scholar CrossRef Search ADS PubMed 38. Rawlings ND , Barrett AJ . Families of serine peptidases . Methods Enzymol . 1994 ; 244 : 19 – 61 . Google Scholar CrossRef Search ADS PubMed 39. Baldo A , Tabart J , Vermout S et al. Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes . J Med Microbiol . 2008 ; 57 : 1152 – 1156 . Google Scholar CrossRef Search ADS PubMed 40. Portela MB , Souza IP , Abreu CM et al. Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization . J Oral Pathol Med . 2010 ; 39 : 753 – 760 . Google Scholar CrossRef Search ADS PubMed 41. Baldo A , Mathy A , Tabart J et al. Secreted subtilisin Sub3 from Microsporum canis is required for adherence to but not for invasion of the epidermis . Br J Dermatol. 2010 ; 162 : 990 – 997 . Google Scholar CrossRef Search ADS PubMed 42. Bagut ET , Baldo A , Mathy A et al. Subtilisin Sub3 is involved in adherence of Microsporum canis to human and animal epidermis . Vet Microbiol . 2012 ; 160 : 413 – 419 . Google Scholar CrossRef Search ADS PubMed 43. Baldo A , Monod M , Mathy A et al. Mechanisms of skin adherence and invasion by dermatophytes . Mycoses . 2012 ; 55 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed 44. Meevootisom V , Niederpruem DJ . Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum . Sabouraudia . 1979 ; 17 : 91 – 106 . Google Scholar CrossRef Search ADS PubMed 45. Apodaca G , McKerrow JH . Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum . Infect Immun . 1989 ; 57 : 3072 – 3080 . Google Scholar PubMed 46. Asahi M , Lindquist R , Fukuyama K , Apodaca G , Epstein WL , McKerrow JH . Purification and characterization of major extracellular proteinases from Trichophyton rubrum . Biochem J . 1985 ; 232 : 139 – 144 . Google Scholar CrossRef Search ADS PubMed 47. Sanyal AK , Das SK , Banerjee AB . Purification and partial characterization of an exocellular proteinase from Trichophyton rubrum . Sabouraudia . 1985 ; 23 : 165 – 178 . Google Scholar CrossRef Search ADS PubMed 48. Wang L , Ma L , Leng W et al. Analysis of the dermatophyte Trichophyton rubrum expressed sequence tags . BMC Genomics . 2006 ; 7 : 255 . Google Scholar CrossRef Search ADS PubMed 49. Bitencourt TA , Macedo C , Franco ME et al. Transcription profile of Trichophyton rubrum conidia grown on keratin reveals the induction of an adhesin-like protein gene with a tandem repeat pattern . BMC Genomics . 2016 ; 17 : 249 . Google Scholar CrossRef Search ADS PubMed 50. Maranhao FC , Paiao FG , Martinez-Rossi NM . Isolation of transcripts over-expressed in human pathogen Trichophyton rubrum during growth in keratin . Microb Pathog . 2007 ; 43 : 166 – 172 . Google Scholar CrossRef Search ADS PubMed 51. Peres NT , Sanches PR , Falcao JP et al. Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum . BMC Microbiol . 2010 ; 10 : 39 . Google Scholar CrossRef Search ADS PubMed 52. Leng W , Liu T , Wang J , Li R , Jin Q . Expression dynamics of secreted protease genes in Trichophyton rubrum induced by key host's proteinaceous components . Med Mycol . 2009 ; 47 : 759 – 765 . Google Scholar CrossRef Search ADS PubMed 53. Zaugg C , Monod M , Weber J et al. 2009 . Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins . Eukaryot Cell . 2009 ; 8 : 241 – 250 . Google Scholar CrossRef Search ADS PubMed 54. Baeza LC , Bailao AM , Borges CL , Pereira M , Soares CM , Mendes Giannini MJ . cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin . Microbes Infect . 2007 ; 9 : 1415 – 1421 . Google Scholar CrossRef Search ADS PubMed 55. Chen J , Yi J , Liu L et al. Substrate adaptation of Trichophyton rubrum secreted endoproteases . Microb Pathog . 2010 ; 48 : 57 – 61 . Google Scholar CrossRef Search ADS PubMed 56. Komoto TT , Bitencourt TA , Silva G , Beleboni RO , Marins M , Fachin AL . Gene expression response of Trichophyton rubrum during coculture on keratinocytes exposed to antifungal agents . Evid Based Complement Alternat Med . 2015 ; 2015 : 180535 . Google Scholar CrossRef Search ADS PubMed 57. Staib P , Zaugg C , Mignon B et al. Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection . Microbiology . 2010 ; 156 : 884 – 895 . Google Scholar CrossRef Search ADS PubMed 58. Woodfolk JA , Sung SS , Benjamin DC , Lee JK , Platts-Mills TA . Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2 . J Immunol . 2000 ; 165 : 4379 – 4387 . Google Scholar CrossRef Search ADS PubMed 59. Woodfolk JA , Wheatley LM , Piyasena RV , Benjamin DC , Platts-Mills TA . Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity: sequence homology to two families of serine proteinases . J Biol Chem . 1998 ; 273 : 29489 – 29496 . Google Scholar CrossRef Search ADS PubMed 60. Shi Y , Niu Q , Yu X et al. 2016 . Assessment of the function of SUB6 in the pathogenic dermatophyte Trichophyton mentagrophytes . Med Mycol . 2016 ; 54 : 59 – 71 . Google Scholar PubMed 61. Mehul B , Gu Z , Jomard A , Laffet G , Feuilhade M , Monod M . Sub6 (Tri r 2), an onychomycosis marker revealed by proteomics analysis of Trichophyton rubrum secreted proteins in patient nail samples . J Invest Dermatol . 2016 ; 136 : 331 – 333 . Google Scholar CrossRef Search ADS PubMed 62. Brouta F , Descamps F , Fett T , Losson B , Gerday C , Mignon B . Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis . Med Mycol . 2001 ; 39 : 269 – 275 . Google Scholar CrossRef Search ADS PubMed 63. Brouta F , Descamps F , Monod M , Vermout S , Losson B , Mignon B . Secreted metalloprotease gene family of Microsporum canis . Infect Immun . 2002 ; 70 : 5676 – 5683 . Google Scholar CrossRef Search ADS PubMed 64. Kano R , Yamada T , Makimura K , Yamaguchi H , Watanabe S , Hasegawa A . Metalloprotease gene of Arthroderma gypseum . Jpn J Infect Dis . 2005 ; 58 : 214 – 217 . Google Scholar PubMed 65. Jongeneel CV , Bouvier J , Bairoch A . A unique signature identifies a family of zinc-dependent metallopeptidases . FEBS Lett . 1989 ; 242 : 211 – 214 . Google Scholar CrossRef Search ADS PubMed 66. Jousson O , Lechenne B , Bontems O et al. Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum . Microbiology . 2004 ; 150 : 301 – 310 . Google Scholar CrossRef Search ADS PubMed 67. Zhang X , Wang Y , Chi W et al. Metalloprotease genes of Trichophyton mentagrophytes are important for pathogenicity . Med Mycol . 2014 ; 52 : 36 – 45 . Google Scholar PubMed 68. Mathy A , Baldo A , Schoofs L ,et al. Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats . Vet Microbiol . 2010 ; 146 : 179 – 182 . Google Scholar CrossRef Search ADS PubMed 69. Danew P , Friedrich E , Mannsfeldt HG . Peptidase activity of skin-pathogenic fungi. I. Determination of leucineaminopeptidase, arylamidase, carboxypeptidase and acylase activity in Trichophyton rubrum and Microsporum gypseum . Dermatol Monatsschr . 1971 ; 157 : 232 – 238 [in German ]. Google Scholar PubMed 70. De Bersaques J , Dockx P . Proteolytic and leucylnaphthylamidase activity in some dermatophytes: preliminary results . Arch Belg Dermatol Syphiligr . 1973 ; 29 : 135 – 140 . Google Scholar PubMed 71. Monod M , Lechenne B , Jousson O et al. Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum . Microbiology . 2005 ; 151 : 145 – 155 . Google Scholar CrossRef Search ADS PubMed 72. Woodfolk JA , Slunt JB , Deuell B , Hayden ML , Platts-Mills TA . Definition of a Trichophyton protein associated with delayed hypersensitivity in humans: evidence for immediate (IgE and IgG4) and delayed hypersensitivity to a single protein . J Immunol . 1996 ; 156 : 1695 – 1701 . Google Scholar PubMed 73. Kaufman G , Berdicevsky I , Woodfolk JA , Horwitz BA . Markers for host-induced gene expression in Trichophyton dermatophytosis . Infect Immun . 2005 ; 73 : 6584 – 6590 . Google Scholar CrossRef Search ADS PubMed 74. Giddey K , Monod M , Barblan J et al. Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions . J Proteome Res . 2007 ; 6 : 3081 – 3092 . Google Scholar CrossRef Search ADS PubMed 75. Sriranganadane D , Waridel P , Salamin K et al. Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH . Proteomics . 2011 ; 11 : 4422 – 4433 . Google Scholar CrossRef Search ADS PubMed 76. Preuett BL , Schuenemann E , Brown JT , Kovac ME , Krishnan SK , Abdel-Rahman SM . Comparative analysis of secreted enzymes between the anthropophilic-zoophilic sister species Trichophyton tonsurans and Trichophyton equinum . Fungal Biol . 2010 ; 114 : 429 – 437 . Google Scholar CrossRef Search ADS PubMed 77. Rementeria A , Lopez-Molina N , Ludwig A et al. Genes and molecules involved in Aspergillus fumigatus virulence . Rev Iberoam Micol . 2005 ; 22 : 1 – 23 . Google Scholar CrossRef Search ADS PubMed 78. Byun T , Kofod L , Blinkovsky A . Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis . J Agric Food Chem . 2001 ; 49 : 2061 – 2063 . Google Scholar CrossRef Search ADS PubMed 79. O’Cuinn G , FitzGerald R , Bouchier P , McDonnell M . Generation of non-bitter casein hydrolysates by using combinations of a proteinase and aminopeptidases . Biochem Soc Trans . 1999 ; 27 : 730 – 734 . Google Scholar CrossRef Search ADS PubMed 80. Murdan S. The nail: Anatomy, physiology, diseases and treatment . In: Murthy SN , Maibach HI eds. Topical Nail Products and Ungual Drug Delivery . Boca Raton, FL, USA : CRC Press , 2013 : 1 – 35 . 81. Silveira HC , Gras DE , Cazzaniga RA , Sanches PR , Rossi A , Martinez-Rossi NM . Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling . Microb Pathog . 2010 ; 48 : 91 – 96 . Google Scholar CrossRef Search ADS PubMed 82. Liu T , Xu X , Leng W , Xue Y , Dong J , Jin Q . Analysis of gene expression changes in Trichophyton rubrum after skin interaction . J Med Microbiol . 2014 ; 63 : 642 – 648 . Google Scholar CrossRef Search ADS PubMed 83. Maranhao FC , Paiao FG , Fachin AL , Martinez-Rossi NM . Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis . J Med Microbiol . 2009 ; 58 : 163 – 168 . Google Scholar CrossRef Search ADS PubMed 84. Gadzalski M , Ciesielska A , Staczek P . Bioinformatic survey of ABC transporters in dermatophytes . Gene . 2016 ; 576 : 466 – 475 . Google Scholar CrossRef Search ADS PubMed 85. Fachin AL , Ferreira-Nozawa MS , Maccheroni W , Martinez-Rossi NM . Role of the ABC transporter TruMDR2 in terbinafine, 4-nitroquinoline N-oxide and ethidium bromide susceptibility in Trichophyton rubrum . J Med Microbiol . 2006 ; 55 : 1093 – 1099 . Google Scholar CrossRef Search ADS PubMed 86. Yamada T , Makimura K , Abe S . Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte , Microsporum canis. Med Mycol. 2006 ; 44 : 243 – 252 . Google Scholar CrossRef Search ADS 87. Ferreira-Nozawa MS , Silveira HC , Ono CJ , Fachin AL , Rossi A , Martinez-Rossi NM . The pH signaling transcription factor PacC mediates the growth of Trichophyton rubrum on human nail in vitro . Med Mycol . 2006 ; 44 : 641 – 645 . Google Scholar CrossRef Search ADS PubMed 88. Jacob TR , Peres NT , Martins MP et al. Heat shock protein 90 (Hsp90) as a molecular target for the development of novel drugs against the dermatophyte Trichophyton rubrum . Front Microbiol . 2015 ; 6 : 1241 . Google Scholar CrossRef Search ADS PubMed 89. Kudla B , Caddick MX , Langdon T et al. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans: mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger . EMBO J . 1990 ; 9 : 1355 – 1364 . Google Scholar PubMed 90. Marzluf GA. Genetic regulation of nitrogen metabolism in the fungi . Microbiol Mol Biol Rev . 1997 ; 61 : 17 – 32 . Google Scholar PubMed 91. Hensel M , Arst HN Jr. , Aufauvre-Brown A , Holden DW . The role of the Aspergillus fumigatus areA gene in invasive pulmonary aspergillosis . Mol Gen Genet . 1998 ; 258 : 553 – 557 . Google Scholar CrossRef Search ADS PubMed 92. Yamada T , Makimura K , Hisajima T , Ishihara Y , Umeda Y , Abe S . Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte , Trichophyton mentagrophytes. FEMS Microbiol Lett. 2009 ; 298 : 208 – 217 . Google Scholar CrossRef Search ADS 93. Zhao Y , Su H , Zhou J , Feng H , Zhang KQ , Yang J . The APSES family proteins in fungi: characterizations, evolution and functions . Fungal Genet Biol . 2015 ; 81 : 271 – 280 . Google Scholar CrossRef Search ADS PubMed 94. Krober A , Etzrodt S , Bach M ,. The transcriptional regulators SteA and StuA contribute to keratin degradation and sexual reproduction of the dermatophyte Arthroderma benhamiae . Curr Genet . 2017 ; 63 : 103 – 116 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Keratin hydrolysis by dermatophytes

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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1369-3786
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1460-2709
D.O.I.
10.1093/mmy/myx160
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Abstract

Abstract Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes’ extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and ‘-omic’ studies that are helping to dissect the complex process of keratin breakdown and utilization. Dermatophyte, Keratin hydrolysis, Protease, Peptidase, Sulfitolysis, Keratinase Introduction Dermatophytes comprise 52 species of keratin-degrading ascomycetes of the genera Trichophyton, Microsporum, Epidermophyton, Arthroderma, Lopophyton, Nannizia, Ctenomyces, Guarromyces, and Paraphyton.1,2 The principal etiologic agents of human dermatophytosis globally are T. rubrum, T. interdigitale, T. mentagrophytes, E. floccosum, and M canis (anamorph of A. otae). Of these, T. rubrum is isolated in 50–80% of cases, mainly from onychomycosis and tinea pedis.1–4 Dermatophytes are keratinophilic, capable of infecting keratinous tissues, including skin, nails, and hair, of humans and other vertebrates.1 Keratinolytic enzymes, including proteases and peptidases, are important virulence factors of dermatophytes and are essential for their ability to infect keratinous tissues.4 The keratin superfamily of proteins contains >60 members which include acidic (type I, PI 4.8–5.4), and basic to neutral (type II, PI 6.5–8.5) proteins, normally containing a central rod-like domain of alpha-helical secondary structure and less organized terminal domains. Hard keratins, typical of nails and hair, contain regions with highly ordered protein filaments, extensively cross-linked by intermolecular disulfide bonds (∼18% cysteine). Soft keratins, typical of the stratum corneum, contain relatively disordered protein filaments with fewer disulfide linkages (∼2–4% cysteine),5,6 as well as other linkages, for example, isopeptide (gamma-glutamyl–epsilon-lysine) bonds.7,8 Over 144 different proteins have been detected in human nails including keratins, cytoplasmic and junctional proteins.9 In the skin, proteins associated with keratin include elastin, collagen, fibronectin, laminin,10 filaggrin,11 and the small proline rich proteins loricrin and involucrin,12,13 among others. Here, we summarize the current state of the art of keratin biodegradation by dermatophytes and its links with virulence, focusing specifically on sulfitolysis and proteolysis, and build on previous reviews on the subject.14–16 Search strategy and selection criteria All publications cited within this review were identified via PubMed searches up to and including March 2017, and references cited therein. Search terms used included but were not limited to dermatophyte, Trichophyton, Microsporum, Epidermophyton, Arthroderma, protease, peptidase, proteinase, protein hydrolysis, keratin hydrolysis, keratinase, and sulfitolysis/sulphitolysis, and combinations thereof. Descriptions of the classes of ‘protease/proteinase/peptidase’ were based on those stated within the cited references and/or via definitions in the MEROPS database (MEROPS the peptidase database; https://www.ebi.ac.uk/merops/).17 Gene/protein homology searches were conducted using the relevant BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using nucleotide, protein, genome or GEO datasets. Cited nucleotide and/or protein sequences were from peer-reviewed scientific publications where possible and appropriate accession numbers were cited. Genomic analysis of proteases and peptidases In a comparison of dermatophyte genomes with those of other fungi, proteases constituted one of four over-represented functional categories.18 Approximately 20% of the 100 most expressed secreted proteins of Trichophyton benhamiae (previously Arthroderma benhamiae) were proteases, during growth both in vivo and on keratin in vitro.19 The closely related dermatophytes T. verrucosum and T. benhamiae were found to possess 235 predicted proteinase-encoding genes (87 with signal peptides), none of which were unique to either species. Enzyme families represented included 18 metallopeptidases and 14 serine peptidases accompanied by cysteine and aspartic peptidases. Whether all these genes are expressed is not known, although an M14 metallocarboxypeptidase, McpA, identified in this study had been isolated and characterised previously from T. rubrum.20 Proteolysis-related genes/proteins for which functional characterization has been conducted are described in Supplementary Table S1. Analysis of the secretome of T. benhamiae following growth on keratin, identified significantly fewer proteinases than was predicted from the genome. Proteinases identified included subtilisin-like serine proteases (Sub3, Sub4, and Sub7), metalloproteinases (fungalysins) (Mep1, Mep3, and Mep4), leucine aminopeptidases (Lap1 and Lap2) and dipeptidyl peptidases (DppIV and DppV). Interestingly, upon co-culture with keratinocytes, of the proteinases identified from the secretome only the expression of the genes encoding DppV and a putative S1 carboxypeptidase (Acc no ARB_06019) were upregulated.21 Two S10 serine carboxypeptidases (ScpA and ScpB) of T. rubrum contained predicted C-terminal Glycosylphosphatidylinositol (GPI) anchors and concomitant omega sites (PredGPI22), suggesting a cell surface location. Both enzymes were detected in membrane extracts.20 A BLAST search of the T. rubrum ScpA protein revealed >90% identity with proteins from T. violaceum, T. interdigitale, T. benhamiae and other dermatophytes (Table S1). A similar search of the T. rubrum ScpB protein revealed >90% identity with proteins from T. benhamiae, T. interdigitale, and T. equinum (Table S1), all of which were predicted to contain GPI anchors.19 Interestingly, most dermatophytes also produced examples of S10 serine carboxypeptidases that did not contain predicted GPI anchors, such as ScpC of T. rubrum.20 One M36 fungalysin identified in T. equinum and T. tonsurans was predicted to contain a GPI anchor unique among dermatophyte fungalysins.18 Analysis of the T. benhamiae secretome predicted that 22 proteases were likely to be GPI-anchored, including fungalysins, deuterolysins, subtilisins, and a range of peptidases.19 Analysis of the genome of T. rubrum var. raubitschekii IGIB-SBL-CI1 identified 16 enzymes belonging to the subfamilies S8A (12 genes; subtilisins), S8B (one gene; kexin) and S53 (three genes; sedolisin),23 indicating a need for further investigations of dermatophyte genomics. The dermatophyte proteases described in Table S1 are representative of published data and three examples from different species are given for each. In addition to the proteases described in Table S1, there remain many predicted/putative proteases that have not been characterised and hypothetical proteins with regions of homology to known protease families.17 For example, the T. benhamiae genome contains 9 predicted A1 aspartic proteases and 2 C40 peptidases. Of the 29 predicted metalloproteases (M10B, M12B, M14, M19, M20, M28, M35, M36, and M43 families) and 35 serine proteases (S1, S8, S9, S10, S28, S33, S41, and S53 families), only 13 and 20, respectively, have been confirmed by mass spectrometry,19 but not confirmed as proteases experimentally. It is possible that some of these genes are not expressed or are pseudogenes, such as the scpD homologue found in T. rubrum CHUV1673-05 which is interrupted by four stop codons.20 Preliminary sulfitolysis Although dermatophytes produce several proteases, most are not capable of degrading native keratin until the hydrolysis of cross-linking disulfide bonds by sulfitolysis has occurred (Fig.1).24–26 L-cysteine acts as the substrate for sulfite production by T. benhamiae27 and there is sufficient free cysteine in keratin to support sulfite formation.28 In T. benhamiae, cysteine is oxidised to cysteine sulfinic acid (cysteine sulfinate) by cysteine dioxygenase (Cdo1), leading, ultimately, to sulphite production.27 Cysteine dioxgenase was also identified in T. mentagrophytes and the recombinant enzyme produced cysteine sulfinic acid from cysteine.28 It is hypothesized that in dermatophytes, cysteine sulfinic acid undergoes transamination by glutamate-oxaloacetate transaminase (aspartate aminotransferase [AspAT]) to generate β-sulfinylpyruvate, which spontaneously decomposes to pyruvate and sulfite, as occurs in mice.29 Sulfite is secreted from the cell via the sulfite efflux pump Ssu1 (an integral membrane protein with 10 transmembrane domains), which belongs to the tellurite-resistance/dicarboxylate transporter (TDT) family.30 Extracellular sulfite could then reduce the disulfide bonds in keratin, facilitating proteolysis (Fig. 1 and Table S1).27 In an analogous system in T. mentagrophytes, cystine induced the expression and activation of Cdo1.31 The sulphite efflux pump Ssu1 is also produced by T. rubrum.30 Targeted gene knockouts of Cdo1 and Ssu1 in T. benhamiae resulted in a strain hypersensitive to cysteine and unable to grow on hair and nails, demonstrating a role for these enzymes in cysteine detoxification.27 In an alternative branch of the pathway, cysteine sulfinic acid can be metabolised to hypotaurine or taurine, via cysteine sulfinic acid decarboxylase and hypotaurine dehydrogenase, but this has no known role to play in keratin hydrolysis.15 Figure 1. View largeDownload slide Keratin sulfitolysis by dermatophytes, modified from24. Figure 1. View largeDownload slide Keratin sulfitolysis by dermatophytes, modified from24. Keratin hydrolysis: endoproteases The main groups of proteases produced by dermatophytes are shown in Figure 2 and described in Table S1. The major dermatophyte endoproteinases are subtilisins (S8A serine protease family) and fungalysins (M36 metallopeptidase family).17 These and other endoproteinases have undergone family expansions in the dermatophytes in comparison to most other pathogenic fungi.18,19 Figure 2. View largeDownload slide Keratin proteolysis by dermatophytes. Figure 2. View largeDownload slide Keratin proteolysis by dermatophytes. Growth of dermatophytes on keratin in vitro and in infection models typically results in overexpression of subtilisin genes, though the individual enzyme/s affected may vary according to the conditions and the fungal species involved. An enzyme later identified as Sub332 was isolated from M. canis grown in vitro and in/on M. canis hyphae (but not spores) in hairs of naturally infected cats33 and experimentally infected guinea pigs.34 The purified enzyme hydrolysed keratin azure, even in the absence of reducing agents, and a chymotrypsin/subtilisin test substrate, as did recombinant Sub3,35 and activity was inhibited by phenylmethylsulfonyl fluoride (PMSF).33,36 In M. canis, high keratinolytic activity was associated with increased severity of infections in guinea pigs, a correlation not seen for other hydrolytic enzymes.37 Subtilisins Sub1, Sub2, and Sub3 were cloned from M. canis IHEM15221, and mRNA of all three genes was detected by polymerase chain reaction (PCR) in the hairs of experimentally infected guinea pigs.32 All three genes contained the catalytic triad Asp/His/Ser characteristic of S8 subtilisins (IPR023828)38 as well as prepro- and signal peptide sequences (IPR010259) characteristic of secreted proteins.32 Also, sub1 and sub3, but not sub2, were transcribed in M. canis IHEM21239 arthroconidia, suggesting a role for the two proteases in the early stages of infection.39 The adherence of M. canis arthroconidia to reconstructed interfollicular feline epidermis was significantly reduced in the presence of serine protease inhibitors and by monoclonal antibodies to Sub3, suggesting that subtilisins, including Sub3, have a role in adherence,39 as postulated for the serine proteases of other fungi, including Candida spp.40 Experiments with an RNA-silenced-Sub3 strain of M. canis IHEM22957 also suggested that Sub3 had an important role in adherence, but was not necessary for the invasion of keratinized tissues.41 A role for Sub3 in adherence was also demonstrated with epidermis from humans and a range of animals, although the addition of recombinant Sub3 had no effect on adherence of this strain, indicating that Sub3 may be cell-associated.42 The propeptide of Sub3 acts as a noncompetitive inhibitor of Sub3 activity, but could not prevent adherence of M. canis to feline epidermis, probably being inactivated by cell surface-associated protease/s.43 For T. rubrum, serine protease activity was detected in crude culture supernatants44 and in isolated keratinases.45–47 Genes (sub1–7) encoding putative subtilisin serine proteases (S8 family) were identified in T. rubrum and phylogenetic analysis revealed sub2 as the likely ancestral gene.25 Extracellular Sub3 and Sub4 were produced when T. rubrum was grown on soy protein as the sole carbon and nitrogen source, and recombinant Sub3 and Sub4 could hydrolyse non-reduced and reduced keratin azure, but were less active against non-keratin proteins.25 Expressed sequence tags for sub1, sub5, and sub6, but not sub2, sub3, sub4 and sub7 were identified following growth of T. rubrum BMU01672 on YPG medium.48 Overexpression of subtilisin genes has been demonstrated in T rubrum strains grown on keratin for sub1, sub3, and sub 649 and separately for sub3 and sub550,51 and sub3 and sub4.58,59 In contrast, when T. rubrum ATCC52021 was grown on keratin, no such upregulation was detected.54 Real-time PCR analysis revealed high levels of sub7 expression following growth on human nail, whereas both sub7 and sub5 expression was elevated following growth on human stratum corneum.55 Elevated expression of sub1 was observed when T. rubrum CBS118892 was co-cultured with the HaCaT human keratinocyte cell line for 24 h.56 In a T. benhamiae LAU2354-2 guinea pig dermal infection model, upregulation of expression of sub6 by was detected, along with expression of sub1-3 and sub7, but not sub4, whereas growth in vitro on keratin was accompanied by strong upregulation of sub3 and sub4.57 In a later study, the genes for sub6, sub7, sub8, and sub10 were among the most highly expressed in vivo, whereas sub3 and sub4 were among the most highly expressed in vitro.19 Sub6, identified as the T. rubrum antigen/allergen Tri r 2,58,59 appears to have a role in dermatophyte virulence. Disruption of sub6 by site-directed mutagenesis in T. mentagrophytes ATCC28185 increased in vitro proteolytic activity and altered levels of expression of several protease genes. It also resulted in delayed onset of clinical symptoms, lower-grade lesions and reduced inflammation in infected guinea pigs. The reduced pathogenicity of the manipulated strain seemed to result in part from changes in the pattern of cytokine production.60 Of the enzymes secreted by T. rubrum during growth on keratin, Sub6 was the only subtilisin recovered from the nail beds of onychomycotic patients by Mehul and co-workers.61 Analysis of proteinase secretion by T. rubrum growing on human skin demonstrated that Sub3, Sub4, and Mep4 were probably the main proteinases responsible for the invasion of skin during infection.52 However, gene expression in cultures grown on excised human skin differed from that in cultures grown on media containing keratin, elastin, and collagen. The dermatophyte zinc-dependent metalloproteases (M36 family; fungalysins) were first identified in M. canis as three genes (mep1–3) homologous to the single Aspergillus fumigatus Mep gene.14,62,63 And mep2 and mep3 were expressed in M. canis infected guinea pigs, indicating a potential role in virulence/infection.63 Only one mep was identified in the geophile A. gypseum64, with significant homology to mep2 of T. rubrum and mep3 of T. tonsurans. This family was subsequently expanded to five genes (mep1–5) in T. rubrum, T. mentagrophytes and M. canis, all with high sequence homology and containing the characteristic HEXXE amino acid motif.65 When grown in vitro on soy protein, Meps accounted for 19–36% of total secreted protein and their activity was inhibited by the metalloprotease inhibitor o-phenanthroline.66 A comparison of the pathogenic potential of five metalloprotease genes from T. mentagrophytes led to the proposal that Mep4 and Mep5 were most likely to affect pathogenicity, determined in a guinea pig model and a keratin degradation test,67 whereas expression of only mep4 was significantly upregulated following growth in vitro on keratin, collagen, elastin or human skin sections.50,52 When T. rubrum was cultured with human keratinocytes (HaCaT), elevated expression of mep4 and subtilisin genes was observed,56 whereas in a feline M. canis skin infection model there was no evidence of mep expression during adhesion or early stages of invasion.68 The expression of protease genes, and production of the encoded proteases, is further complicated by the fact that keratin hydrolysis in vitro and infection of keratinous tissues are two distinct processes that require the production of different proteases and associated genes/proteins, a fact that has only recently become better understood. Keratin hydrolysis: exoproteases The action of endoproteases on keratin releases free peptides on which exoproteases may act.69,70 The extracellular aminopeptidases Lap1 and Lap2 (M28A family) and the dipeptidyl peptidases DppIV and DppV (S9 family) of T. rubrum grown on keratin were characterised by Monod and colleagues.71 DppV was originally identified as the T. tonsurans allergen Tri t 472, Tri r 4 of T. rubrum59 and Tri m 4 of T. mentagrophytes.73 Interestingly, when T. benhamiae was co-cultured with keratinocytes, expression of DppV was upregulated, but the expression of the other exoproteases described above was not.21 The leucine aminopeptidases Lap1 and Lap2 hydrolyse peptides from the N-terminus until they reach X-Pro or X-Ala sequences, which act as a stop.68 Transcriptional profiling of T. rubrum revealed that expression of lap1 and lap2 was upregulated during growth on medium containing keratin, but not elastin.49 Western blotting of culture supernatant of T. rubrum detected the presence of Lap1 and Lap2 and recombinant Lap1 and Lap2 hydrolysed leucine from the Leu-aminoacyl-4-methylcoumaryl-7-amide (AMC) substrate. Neither Lap hydrolysed the Gly-Pro-AMC DppIV substrate.71 Species/strain specific differences exist in Lap1 and Lap2 production between different strains of T. rubrum and T. violaceum.74 Secretome analysis of T. benhamiae proteins following keratin hydrolysis identified Lap1 and Lap2.21 An earlier study demonstrated that production of these enzymes was increased during proteolysis in vitro, but not in a guinea pig infection model.57 In a later study, the genes for Lap1 and Lap2 were among the most highly expressed by the same T. benhamiae strain during in vitro growth on keratin but not in an in vivo guinea pig infection model, whereas expression of lap2 alone was upregulated following growth on soy protein.19 Interestingly, when grown at neutral pH in soy protein medium, the major Lap produced by M. canis was Lap1, whereas Lap2 was the major Lap produced by T. benhamiae, T. rubrum, and T. violaceum.74,75 Dipeptidyl peptidase IV removes N-terminal dipeptides (Xaa-Yaa-|-Zaa) sequentially from polypeptides having unsubstituted N-termini preferentially when the penultimate residue (Yaa) is proline, and in some cases alanine, and when Zaa is neither proline nor hydroxyproline. Dipeptidyl peptidase V removes N-terminal dipeptides sequentially from polypeptides having unsubstituted N-termini preferentially when the penultimate residue (Yaa) is alanine. Recombinant DppIV from T. rubrum hydrolyzed Gly-Pro-AMC and Lys-Ala-AMC, but with a preference for X-Pro sequences. Recombinant DppV from T. rubrum CHUV 862-00 hydrolysed Lys-Ala-AMC, but not Gly-Pro-AMC; both enzymes were active between pH 6.5 and 10.5. Neither DppIV or DppV could hydrolyze tripeptides.71 The genes for DppIV and DppV were detected in four strains each of T. tonsurans and T. equinum and demonstrated 99.88 and 99.96% identity, respectively.76 Analysis of the secretome of T. benhamiae LAU2354 (CBS112371) following growth on keratin revealed the presence of DppIV and DppV,21 consistent with the data of Staib and colleagues.57 Expression of dppV was upregulated during co-culture of T. benhamiae with HaCaT keratinocytes, whereas lap1, lap2, and dppIV were not.21 A study of secreted proteins of two strains of T. rubrum and one of T. violaceum grown on soy protein detected DppIV from all three isolates, but DppV only from T. violaceum,74 indicating possible species/strain specificities in Dpp secretion. Transcription of the genes for DppIV and DppV were detectable in vitro in arthroconidia of three isolates of M. canis and dppIV was transcribed during adherence of these isolates and during invasion by two of the isolates in an ex vivo model of cat skin infection, indicating a possible role of these enzymes in infection,68 as observed for DppIV of Aspergillus fumigatus during colonization of collagen and elastin.77 Expression of the DppV gene was induced several-fold in a T. mentagrophytes clinical isolate following growth on medium containing, as the sole source of C and N, keratin, elastin, or blood plasma, but not human skin.73 It is hypothesized that the activity of DppIV and DppV provide access for the Laps to the next amino acids in peptides, potentially acting synergistically as demonstrated in Aspergillus oryzae and lactic acid bacteria.21,78,79 Carboxypeptidases hydrolyse C-terminal amino acids from peptides and this activity was detected in T. rubrum, T. benhamiae, and M. gypseum.69 Three serine carboxypeptidases (ScpA, ScpB, and ScpC) of the S10 family were produced by T. rubrum in vitro;53T. benhamiae expressed the genes for these carboxypeptidases in vitro and during a guinea pig infection.57 ScpA and ScpB of dermatophytes are unusual, in that, unlike their Aspergillus spp. homologues, they are not secreted, but are membrane-associated with GPI anchors, whereas ScpC does not have a GPI anchor, but may be vacuolar.20 All of the genomes of seven dermatophyte species examined (T. rubrum T. tonsurans, T. equinum, T. verrucosum, M. canis, M. gypseum, and T. benhamiae) possessed 11 S10 carboxypeptidase genes, except T. verrucosum, which had 12 carboxypeptidase genes.18 Trichophyton rubrum secreted two zinc-dependent metallocarboxypeptidases, McpA and McpB, (M14A family), when grown on protein. Recombinant McpA and ScpA of T. rubrum efficiently hydrolyzed N-(2-furanacryloyl)-L-phenylalanyl-L-phenylalanine (FAPP), a known carboxypeptidase A substrate.53 Expression of mcpA was increased when T. benhamiae CBS112371 was grown on keratin-soy medium and in a guinea pig infection model,57 whereas Tran et al. found in the same strain that mcpA was one of the most highly expressed during in vitro growth on keratin, but not in guinea pigs.19 Analysis of the same seven dermatophytes described above revealed the presence of four M14 metallocarboxypeptidase genes in the genome of all isolates, except T. benhamiae, which possessed five such genes.18 Analysis of the genome of T. rubrum var. raubitschekii isolated from a patient with onychomycosis, revealed 12 carboxypeptidase genes.23 Microenvironment pH and protease activity The pH of healthy skin and nails is mildly acidic;80 however, the metabolism of amino acids released during keratin breakdown results in a shift to an alkaline pH.14 The dermatophyte keratinases include some which exert optimal activity at the mildly acidic pH prevailing in the early stages of infection, and others with maximal activity at higher pH values found later during keratin breakdown (reviewed by Peres et al. 201051). Culture pH has been shown to affect gene expression and the pattern of protease production in T. rubrum.81 Both M. canis and T. benhamiae generated more endoprotease activity at neutral pH compared to pH 4.0, as was the case for most exoprotease activity of M. canis.75 The proteases secreted at acidic pH by T. benhamiae and M. canis include aspartic proteases and sedolisins/serine carboxypeptidases.75 Dipeptidyl peptidases may have pH-specific roles, as DppIV was one of the major proteases produced by T. benhamiae and M. canis at neutral pH, whereas DppV was more prevalent at acidic pH.75 T. rubrum genes involved in proteolysis and differentially expressed at acidic pH (5.0) included an S10 serine carboxypeptidase ScpB,20 dipeptidases, amino acid permeases, and a major facilitator superfamily di/tripeptide transporter, whereas genes differentially expressed at alkaline pH (8.0) included an amino acid permease and an arginine transporter.51,52 Peptide/Amino acid permeases Information on amino acid and peptide uptake by dermatophytes is relatively scarce, but the expression of 60 genes involved in transport functions were differentially regulated when T. rubrum was grown in the presence of human skin sections, albeit none specifically identified as amino acid/peptide transporters82 and 66 putative transporter genes were identified following growth on keratin, one of which demonstrated significant identity with the Aspergillus fumigatus LysP amino acid/peptide permease (acc. no. AAC98709).83 Additionally, dermatophyte genomes contain genes for ABC transporters that may be involved in amino acid/peptide uptake but have yet to be functionally characterized.84 The TruMDR2 gene, encoding an ABC transporter involved in antifungal resistance,85 in T. rubrum was disrupted, and this correlated with decreased ability of the mutant to grow on nail, indicating a possible role in keratin hydrolysis and uptake.83T. rubrum genes differentially expressed at acidic pH (5.0) included genes with homology to oligopeptide/amino acid permeases, for example, ptr2 and sec24, and a major facilitator superfamily di/tripeptide transporter, whereas genes differentially expressed at alkaline pH (8.0) included those with homology to an amino acid permease and an arginine transporter.51,52 Finally, multiple dermatophyte species also contain genes with homology to the amino acid permease gap1 of Saccharomyces cerevisiae and the proline permease prnB of Aspergillus nidulans that may be under control of Dnr1/AreA.86 Regulation of gene expression The regulation of gene expression in dermatophytes has received little attention. The PacC gene of T. rubrum ATCC MYA-3108 is homologous to the PacC/Rim101p family of pH-responsive transcription factors and expression of pacC is upregulated under alkaline conditions. Disruption of pacC decreased the ability of the mutant to grow on human nail, but not Sabouraud medium, and the secretion of keratinolytic proteases was decreased in a pacC mutant, indicating the probable importance of PacC in keratin hydrolysis and pathogenicity.87 Disruption of the T. rubrum PacC gene had no effect on transcription of the S10 carboxypeptidase ScpB gene.81 Interestingly, expression of pacC was downregulated when heat shock protein 90 (Hsp90) gene expression was chemically inhibited in T. rubrum CBS118892 (ATCC MYA-4607). Hsp90 inhibition also decreased growth of T. rubrum CBS118892 on human nail, but not skin, indicating a probable role of Hsp90 in keratin degradation and pathogenicity via PacC.88 Yamada and colleagues,86 isolated a global nitrogen regulatory gene, dnr1, from M. canis characteristic of the GATA family of transcription factors, and a homologue of the Aspergillus nidulans gene areA.89 These transcription factors activate the expression of genes, including those encoding proteases,90 and play a role in fungal virulence.91 When dnr1 was disrupted, the dnr1− mutants showed reduced growth on ammonia, but could use many amino acids for growth, suggesting a role in the regulation of nitrogen metabolism. The dnr1− mutants showed weak growth on keratin, suggesting that the dnr1 gene might have a role in the regulation of protease production and possibly amino acid uptake.19 Homologues of dnr1/areA have subsequently been identified in T. rubrum (82% identity; 664/808 aa), T. verrucosum (82% identity; 662/808 aa), T. benhamiae (80% identity; 655/820 aa), and T. mentagrophytes (83% identity; 666/805 aa).92 The transcriptional regulator StuA (an APSES transcription factor) has been partially characterized in T. benhamiae. APSES transcription factors have been associated with virulence in plant pathogenic fungi.93T. benhamiae stuA deletion mutants were unable to grow on hair and nail, unlike wild-type and stuA complemented strains, indicating a probable role in keratin degradation and pathogenicity.94 It is probable that other dermatophyte transcription factors remain to be discovered. Discussion The remarkable complexity of keratin biodegradation by dermatophytes begins with sulfitolysis, which involves the intracellular generation of sulfite from cysteine catabolism via the enzymes cysteine dioxygenase (Cdo1) It is hypothesized that in dermatophytes, cysteine sulfinic acid undergoes transamination by glutamate-oxaloacetate transaminase (AspAT; aspartate aminotransferase) to generate β-sulfinylpyruvate, which spontaneously decomposes to pyruvate and sulfite, as occurs in mice.25 The sulphite is exported via the sulphite efflux pump Ssu1 so that it can degrade the extensive disulphide bridges found in the keratin proteins (Fig. 1).27 However, sulfitolysis may not be essential in all cases as some secreted subtilisins can degrade keratin without prior reduction (Sub3 and Sub4 hydrolysis of keratin azure).21,32 The initial stages of keratin proteolysis are performed by endoproteases, of which dermatophytes have expanded families compared with other filamentous fungi, especially S8 family subtilisins and M36 family fungalysins (Fig. 2).18 The peptides generated by endoprotease activity are further hydrolysed by exoproteases, followed by uptake of tripeptides, dipeptides and amino acids for use in central metabolism. The expansion of protease families within the dermatophytes makes them well placed in nature to be able to utilise keratin and infect and invade keratinised tissues. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgments D.K.M. and C.S.S. contributed equally to the preparation of this manuscript. Declaration of interest Derry K Mercer is an employee of NovaBiotics Ltd. Colin Stewart is a former employee of NovaBiotics Ltd. References 1. Borman AM , Summerbell RC . Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses . In: Jorgensen JH , Pfaller MA , Carroll KC , Funke G , Landry ML , Richter SS , Warnock DW , eds. Manual Of Clinical Microbiology, vol 2 . Washington, DC : ASM Press , 2015 : 2128 – 2152 . Google Scholar CrossRef Search ADS 2. de Hoog GS , Dukik K , Monod M et al. Toward a novel multilocus phylogenetic taxonomy for the dermatophytes . Mycopathologia . 2017 ; 182 : 5 – 31 . Google Scholar CrossRef Search ADS PubMed 3. Borman AM , Campbell CK , Fraser M , Johnson EM . Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades . Med Mycol . 2007 ; 45 : 131 – 141 . Google Scholar CrossRef Search ADS PubMed 4. Nenoff P , Kruger C , Ginter-Hanselmayer G , Tietz HJ . Mycology, an update. Part 1: Dermatomycoses: causative agents, epidemiology and pathogenesis . J Dtsch Dermatol Ges . 2014 ; 12 : 188 – 209 . Google Scholar PubMed 5. Moll R , Divo M , Langbein L . The human keratins: biology and pathology . Histochem Cell Biol . 2008 ; 129 : 705 – 733 . Google Scholar CrossRef Search ADS PubMed 6. Bragulla HH , Homberger DG . Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia . J Anat . 2009 ; 214 : 516 – 559 . Google Scholar CrossRef Search ADS PubMed 7. Bruce Fraser RD , Parry DA . The role of disulfide bond formation in the structural transition observed in the intermediate filaments of developing hair . J Struct Biol . 2012 ; 180 : 117 – 124 . Google Scholar CrossRef Search ADS PubMed 8. Gong H , Zhou H , McKenzie GW et al. An updated nomenclature for keratin-associated proteins (KAPs) . Int J Biol Sci. 2012 ; 8 : 258 – 264 . Google Scholar CrossRef Search ADS PubMed 9. Rice RH , Xia Y , Alvarado RJ , Phinney BS . Proteomic analysis of human nail plate . J Proteome Res . 2010 ; 9 : 6752 – 6758 . Google Scholar CrossRef Search ADS PubMed 10. Woodley DT , O’Keefe EJ , Prunieras M . Cutaneous wound healing: a model for cell-matrix interactions . J Am Acad Dermatol . 1985 ; 12 : 420 – 433 . Google Scholar CrossRef Search ADS PubMed 11. McLean WH. Filaggrin failure: from ichthyosis vulgaris to atopic eczema and beyond . Br J Dermatol . 2016 ; 175 : 4 – 7 . Google Scholar CrossRef Search ADS PubMed 12. Eckert RL , Crish JF , Efimova T et al. Regulation of involucrin gene expression . J Invest Dermatol . 2004 ; 123 : 13 – 22 . Google Scholar CrossRef Search ADS PubMed 13. Nithya S , Radhika T , Jeddy N . Loricrin - an overview . J Oral Maxillofac Pathol . 2015 ; 19 : 64 – 68 . Google Scholar CrossRef Search ADS PubMed 14. Monod M. Secreted proteases from dermatophytes . Mycopathologia . 2008 ; 166 : 285 – 294 . Google Scholar CrossRef Search ADS PubMed 15. Kasperova A , Kunert J , Raska M . The possible role of dermatophyte cysteine dioxygenase in keratin degradation . Med Mycol . 2013 ; 51 : 449 – 454 . Google Scholar CrossRef Search ADS PubMed 16. Monod M , Mignon B , Staib S . Dermatophytes as saprophytes and pathogens . In: Sullivan DJ , Moran GP , eds. Human Pathogenic Fungi: Molecular Biology and Pathogenic Mechanisms . Norfolk, UK : Caister Academic Press , 2014 : 223 – 252 . 17. Rawlings ND , Barrett AJ , Finn R . Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors . Nucleic Acids Res . 2016 ; 44 : D343 – 350 . Google Scholar CrossRef Search ADS PubMed 18. Martinez DA , Oliver BG , Graser Y et al. Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection . MBio . 2012 ; 3 : e00259 – 12 . Google Scholar CrossRef Search ADS PubMed 19. Tran VD , De Coi N , Feuermann M et al. RNA Sequencing-based genome reannotation of the dermatophyte Arthroderma benhamiae and characterization of its secretome and whole gene expression profile during infection . mSystems . 2016 ; 1 : e00036 – 16 . Google Scholar CrossRef Search ADS PubMed 20. Zaugg C , Jousson O , Lechenne B , Staib P , Monod M . Trichophyton rubrum secreted and membrane-associated carboxypeptidases . Int J Med Microbiol . 2008 ; 298 : 669 – 682 . Google Scholar CrossRef Search ADS PubMed 21. Burmester A , Shelest E , Glockner G et al. Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi . Genome Biol . 2011 ; 12 : R7 . Google Scholar CrossRef Search ADS PubMed 22. Pierleoni A , Martelli PL , Casadio R . PredGPI: a GPI-anchor predictor . BMC Bioinformatics . 2008 ; 9 : 392 . Google Scholar CrossRef Search ADS PubMed 23. Latka C , Dey SS , Mahajan S et al. Genome sequence of a clinical isolate of dermatophyte, Trichophyton rubrum from India . FEMS Microbiol Lett . 2015 ; 362 : fnv039 . Google Scholar CrossRef Search ADS PubMed 24. Blyskal B. Fungi utilizing keratinous substrates . Int Biodeterior Biodegradation . 2009 ; 63 : 631 – 653 . Google Scholar CrossRef Search ADS 25. Jousson O , Lechenne B , Bontems O et al. Secreted subtilisin gene family in Trichophyton rubrum . Gene . 2004 ; 339 : 79 – 88 . Google Scholar CrossRef Search ADS PubMed 26. Kunert J. Keratin decomposition by dermatophytes: evidence of the sulphitolysis of the protein . Experientia . 1972 ; 28 : 1025 – 1026 . Google Scholar CrossRef Search ADS PubMed 27. Grumbt M , Monod M , Yamada T , Hertweck C , Kunert J , Staib P . Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump . J Invest Dermatol . 2013 ; 133 : 1550 – 1555 . Google Scholar CrossRef Search ADS PubMed 28. Kasperova A , Kunert J , Horynova M et al. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes . Mycoses . 2011 ; 54 : e456 – e462 . Google Scholar CrossRef Search ADS PubMed 29. Griffith OW. Cysteinesulfinate metabolism. altered partitioning between transamination and decarboxylation following administration of beta-methyleneaspartate . J Biol Chem . 1983 ; 258 : 1591 – 1598 . Google Scholar PubMed 30. Lechenne B , Reichard U , Zaugg C et al. Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes . Microbiology . 2007 ; 153 : 905 – 913 . Google Scholar CrossRef Search ADS PubMed 31. Kasperova A , Cahlikova R , Kunert J , Sebela M , Novak Z , Raska M . Exposition of dermatophyte Trichophyton mentagrophytes to L-cystine induces expression and activation of cysteine dioxygenase . Mycoses . 2014 ; 57 : 672 – 678 . Google Scholar CrossRef Search ADS PubMed 32. Descamps F , Brouta F , Monod M et al. Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo . J Invest Dermatol . 2002 ; 119 : 830 – 835 . Google Scholar CrossRef Search ADS PubMed 33. Mignon BR , Nikkels AF , Pierard GE , Losson BJ . The in vitro and in vivo production of a 31.5-kD keratinolytic subtilase from Microsporum canis and the clinical status in naturally infected cats . Dermatology . 1998 ; 196 : 438 – 441 . Google Scholar CrossRef Search ADS PubMed 34. Mignon BR , Leclipteux T , Focant C , Nikkels AJ , Pierard GE , Losson BJ . Humoral and cellular immune response to a crude exo-antigen and purified keratinase of Microsporum canis in experimentally infected guinea pigs . Med Mycol . 1999 ; 37 : 123 – 129 . Google Scholar CrossRef Search ADS PubMed 35. Descamps F , Brouta F , Vermout S , Monod M , Losson B , Mignon B . Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis . FEMS Immunol Med Microbiol . 2003 ; 38 : 29 – 34 . Google Scholar CrossRef Search ADS PubMed 36. Mignon B , Swinnen M , Bouchara JP et al. Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats . Med Mycol . 1998 ; 36 : 395 – 404 . Google Scholar CrossRef Search ADS PubMed 37. Viani FC , Dos Santos JI , Paula CR , Larson CE , Gambale W . Production of extracellular enzymes by Microsporum canis and their role in its virulence . Med Mycol . 2001 ; 39 : 463 – 468 . Google Scholar CrossRef Search ADS PubMed 38. Rawlings ND , Barrett AJ . Families of serine peptidases . Methods Enzymol . 1994 ; 244 : 19 – 61 . Google Scholar CrossRef Search ADS PubMed 39. Baldo A , Tabart J , Vermout S et al. Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes . J Med Microbiol . 2008 ; 57 : 1152 – 1156 . Google Scholar CrossRef Search ADS PubMed 40. Portela MB , Souza IP , Abreu CM et al. Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization . J Oral Pathol Med . 2010 ; 39 : 753 – 760 . Google Scholar CrossRef Search ADS PubMed 41. Baldo A , Mathy A , Tabart J et al. Secreted subtilisin Sub3 from Microsporum canis is required for adherence to but not for invasion of the epidermis . Br J Dermatol. 2010 ; 162 : 990 – 997 . Google Scholar CrossRef Search ADS PubMed 42. Bagut ET , Baldo A , Mathy A et al. Subtilisin Sub3 is involved in adherence of Microsporum canis to human and animal epidermis . Vet Microbiol . 2012 ; 160 : 413 – 419 . Google Scholar CrossRef Search ADS PubMed 43. Baldo A , Monod M , Mathy A et al. Mechanisms of skin adherence and invasion by dermatophytes . Mycoses . 2012 ; 55 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed 44. Meevootisom V , Niederpruem DJ . Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum . Sabouraudia . 1979 ; 17 : 91 – 106 . Google Scholar CrossRef Search ADS PubMed 45. Apodaca G , McKerrow JH . Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum . Infect Immun . 1989 ; 57 : 3072 – 3080 . Google Scholar PubMed 46. Asahi M , Lindquist R , Fukuyama K , Apodaca G , Epstein WL , McKerrow JH . Purification and characterization of major extracellular proteinases from Trichophyton rubrum . Biochem J . 1985 ; 232 : 139 – 144 . Google Scholar CrossRef Search ADS PubMed 47. Sanyal AK , Das SK , Banerjee AB . Purification and partial characterization of an exocellular proteinase from Trichophyton rubrum . Sabouraudia . 1985 ; 23 : 165 – 178 . Google Scholar CrossRef Search ADS PubMed 48. Wang L , Ma L , Leng W et al. Analysis of the dermatophyte Trichophyton rubrum expressed sequence tags . BMC Genomics . 2006 ; 7 : 255 . Google Scholar CrossRef Search ADS PubMed 49. Bitencourt TA , Macedo C , Franco ME et al. Transcription profile of Trichophyton rubrum conidia grown on keratin reveals the induction of an adhesin-like protein gene with a tandem repeat pattern . BMC Genomics . 2016 ; 17 : 249 . Google Scholar CrossRef Search ADS PubMed 50. Maranhao FC , Paiao FG , Martinez-Rossi NM . Isolation of transcripts over-expressed in human pathogen Trichophyton rubrum during growth in keratin . Microb Pathog . 2007 ; 43 : 166 – 172 . Google Scholar CrossRef Search ADS PubMed 51. Peres NT , Sanches PR , Falcao JP et al. Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum . BMC Microbiol . 2010 ; 10 : 39 . Google Scholar CrossRef Search ADS PubMed 52. Leng W , Liu T , Wang J , Li R , Jin Q . Expression dynamics of secreted protease genes in Trichophyton rubrum induced by key host's proteinaceous components . Med Mycol . 2009 ; 47 : 759 – 765 . Google Scholar CrossRef Search ADS PubMed 53. Zaugg C , Monod M , Weber J et al. 2009 . Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins . Eukaryot Cell . 2009 ; 8 : 241 – 250 . Google Scholar CrossRef Search ADS PubMed 54. Baeza LC , Bailao AM , Borges CL , Pereira M , Soares CM , Mendes Giannini MJ . cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin . Microbes Infect . 2007 ; 9 : 1415 – 1421 . Google Scholar CrossRef Search ADS PubMed 55. Chen J , Yi J , Liu L et al. Substrate adaptation of Trichophyton rubrum secreted endoproteases . Microb Pathog . 2010 ; 48 : 57 – 61 . Google Scholar CrossRef Search ADS PubMed 56. Komoto TT , Bitencourt TA , Silva G , Beleboni RO , Marins M , Fachin AL . Gene expression response of Trichophyton rubrum during coculture on keratinocytes exposed to antifungal agents . Evid Based Complement Alternat Med . 2015 ; 2015 : 180535 . Google Scholar CrossRef Search ADS PubMed 57. Staib P , Zaugg C , Mignon B et al. Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection . Microbiology . 2010 ; 156 : 884 – 895 . Google Scholar CrossRef Search ADS PubMed 58. Woodfolk JA , Sung SS , Benjamin DC , Lee JK , Platts-Mills TA . Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2 . J Immunol . 2000 ; 165 : 4379 – 4387 . Google Scholar CrossRef Search ADS PubMed 59. Woodfolk JA , Wheatley LM , Piyasena RV , Benjamin DC , Platts-Mills TA . Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity: sequence homology to two families of serine proteinases . J Biol Chem . 1998 ; 273 : 29489 – 29496 . Google Scholar CrossRef Search ADS PubMed 60. Shi Y , Niu Q , Yu X et al. 2016 . Assessment of the function of SUB6 in the pathogenic dermatophyte Trichophyton mentagrophytes . Med Mycol . 2016 ; 54 : 59 – 71 . Google Scholar PubMed 61. Mehul B , Gu Z , Jomard A , Laffet G , Feuilhade M , Monod M . Sub6 (Tri r 2), an onychomycosis marker revealed by proteomics analysis of Trichophyton rubrum secreted proteins in patient nail samples . J Invest Dermatol . 2016 ; 136 : 331 – 333 . Google Scholar CrossRef Search ADS PubMed 62. Brouta F , Descamps F , Fett T , Losson B , Gerday C , Mignon B . Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis . Med Mycol . 2001 ; 39 : 269 – 275 . Google Scholar CrossRef Search ADS PubMed 63. Brouta F , Descamps F , Monod M , Vermout S , Losson B , Mignon B . Secreted metalloprotease gene family of Microsporum canis . Infect Immun . 2002 ; 70 : 5676 – 5683 . Google Scholar CrossRef Search ADS PubMed 64. Kano R , Yamada T , Makimura K , Yamaguchi H , Watanabe S , Hasegawa A . Metalloprotease gene of Arthroderma gypseum . Jpn J Infect Dis . 2005 ; 58 : 214 – 217 . Google Scholar PubMed 65. Jongeneel CV , Bouvier J , Bairoch A . A unique signature identifies a family of zinc-dependent metallopeptidases . FEBS Lett . 1989 ; 242 : 211 – 214 . Google Scholar CrossRef Search ADS PubMed 66. Jousson O , Lechenne B , Bontems O et al. Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum . Microbiology . 2004 ; 150 : 301 – 310 . Google Scholar CrossRef Search ADS PubMed 67. Zhang X , Wang Y , Chi W et al. Metalloprotease genes of Trichophyton mentagrophytes are important for pathogenicity . Med Mycol . 2014 ; 52 : 36 – 45 . Google Scholar PubMed 68. Mathy A , Baldo A , Schoofs L ,et al. Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats . Vet Microbiol . 2010 ; 146 : 179 – 182 . Google Scholar CrossRef Search ADS PubMed 69. Danew P , Friedrich E , Mannsfeldt HG . Peptidase activity of skin-pathogenic fungi. I. Determination of leucineaminopeptidase, arylamidase, carboxypeptidase and acylase activity in Trichophyton rubrum and Microsporum gypseum . Dermatol Monatsschr . 1971 ; 157 : 232 – 238 [in German ]. Google Scholar PubMed 70. De Bersaques J , Dockx P . Proteolytic and leucylnaphthylamidase activity in some dermatophytes: preliminary results . Arch Belg Dermatol Syphiligr . 1973 ; 29 : 135 – 140 . Google Scholar PubMed 71. Monod M , Lechenne B , Jousson O et al. Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum . Microbiology . 2005 ; 151 : 145 – 155 . Google Scholar CrossRef Search ADS PubMed 72. Woodfolk JA , Slunt JB , Deuell B , Hayden ML , Platts-Mills TA . Definition of a Trichophyton protein associated with delayed hypersensitivity in humans: evidence for immediate (IgE and IgG4) and delayed hypersensitivity to a single protein . J Immunol . 1996 ; 156 : 1695 – 1701 . Google Scholar PubMed 73. Kaufman G , Berdicevsky I , Woodfolk JA , Horwitz BA . Markers for host-induced gene expression in Trichophyton dermatophytosis . Infect Immun . 2005 ; 73 : 6584 – 6590 . Google Scholar CrossRef Search ADS PubMed 74. Giddey K , Monod M , Barblan J et al. Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions . J Proteome Res . 2007 ; 6 : 3081 – 3092 . Google Scholar CrossRef Search ADS PubMed 75. Sriranganadane D , Waridel P , Salamin K et al. Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH . Proteomics . 2011 ; 11 : 4422 – 4433 . Google Scholar CrossRef Search ADS PubMed 76. Preuett BL , Schuenemann E , Brown JT , Kovac ME , Krishnan SK , Abdel-Rahman SM . Comparative analysis of secreted enzymes between the anthropophilic-zoophilic sister species Trichophyton tonsurans and Trichophyton equinum . Fungal Biol . 2010 ; 114 : 429 – 437 . Google Scholar CrossRef Search ADS PubMed 77. Rementeria A , Lopez-Molina N , Ludwig A et al. Genes and molecules involved in Aspergillus fumigatus virulence . Rev Iberoam Micol . 2005 ; 22 : 1 – 23 . Google Scholar CrossRef Search ADS PubMed 78. Byun T , Kofod L , Blinkovsky A . Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis . J Agric Food Chem . 2001 ; 49 : 2061 – 2063 . Google Scholar CrossRef Search ADS PubMed 79. O’Cuinn G , FitzGerald R , Bouchier P , McDonnell M . Generation of non-bitter casein hydrolysates by using combinations of a proteinase and aminopeptidases . Biochem Soc Trans . 1999 ; 27 : 730 – 734 . Google Scholar CrossRef Search ADS PubMed 80. Murdan S. The nail: Anatomy, physiology, diseases and treatment . In: Murthy SN , Maibach HI eds. Topical Nail Products and Ungual Drug Delivery . Boca Raton, FL, USA : CRC Press , 2013 : 1 – 35 . 81. Silveira HC , Gras DE , Cazzaniga RA , Sanches PR , Rossi A , Martinez-Rossi NM . Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling . Microb Pathog . 2010 ; 48 : 91 – 96 . Google Scholar CrossRef Search ADS PubMed 82. Liu T , Xu X , Leng W , Xue Y , Dong J , Jin Q . Analysis of gene expression changes in Trichophyton rubrum after skin interaction . J Med Microbiol . 2014 ; 63 : 642 – 648 . Google Scholar CrossRef Search ADS PubMed 83. Maranhao FC , Paiao FG , Fachin AL , Martinez-Rossi NM . Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis . J Med Microbiol . 2009 ; 58 : 163 – 168 . Google Scholar CrossRef Search ADS PubMed 84. Gadzalski M , Ciesielska A , Staczek P . Bioinformatic survey of ABC transporters in dermatophytes . Gene . 2016 ; 576 : 466 – 475 . Google Scholar CrossRef Search ADS PubMed 85. Fachin AL , Ferreira-Nozawa MS , Maccheroni W , Martinez-Rossi NM . Role of the ABC transporter TruMDR2 in terbinafine, 4-nitroquinoline N-oxide and ethidium bromide susceptibility in Trichophyton rubrum . J Med Microbiol . 2006 ; 55 : 1093 – 1099 . Google Scholar CrossRef Search ADS PubMed 86. Yamada T , Makimura K , Abe S . Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte , Microsporum canis. Med Mycol. 2006 ; 44 : 243 – 252 . Google Scholar CrossRef Search ADS 87. Ferreira-Nozawa MS , Silveira HC , Ono CJ , Fachin AL , Rossi A , Martinez-Rossi NM . The pH signaling transcription factor PacC mediates the growth of Trichophyton rubrum on human nail in vitro . Med Mycol . 2006 ; 44 : 641 – 645 . Google Scholar CrossRef Search ADS PubMed 88. Jacob TR , Peres NT , Martins MP et al. Heat shock protein 90 (Hsp90) as a molecular target for the development of novel drugs against the dermatophyte Trichophyton rubrum . Front Microbiol . 2015 ; 6 : 1241 . Google Scholar CrossRef Search ADS PubMed 89. Kudla B , Caddick MX , Langdon T et al. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans: mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger . EMBO J . 1990 ; 9 : 1355 – 1364 . Google Scholar PubMed 90. Marzluf GA. Genetic regulation of nitrogen metabolism in the fungi . Microbiol Mol Biol Rev . 1997 ; 61 : 17 – 32 . Google Scholar PubMed 91. Hensel M , Arst HN Jr. , Aufauvre-Brown A , Holden DW . The role of the Aspergillus fumigatus areA gene in invasive pulmonary aspergillosis . Mol Gen Genet . 1998 ; 258 : 553 – 557 . Google Scholar CrossRef Search ADS PubMed 92. Yamada T , Makimura K , Hisajima T , Ishihara Y , Umeda Y , Abe S . Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte , Trichophyton mentagrophytes. FEMS Microbiol Lett. 2009 ; 298 : 208 – 217 . Google Scholar CrossRef Search ADS 93. Zhao Y , Su H , Zhou J , Feng H , Zhang KQ , Yang J . The APSES family proteins in fungi: characterizations, evolution and functions . Fungal Genet Biol . 2015 ; 81 : 271 – 280 . Google Scholar CrossRef Search ADS PubMed 94. Krober A , Etzrodt S , Bach M ,. The transcriptional regulators SteA and StuA contribute to keratin degradation and sexual reproduction of the dermatophyte Arthroderma benhamiae . Curr Genet . 2017 ; 63 : 103 – 116 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Jan 17, 2018

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