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Mycopathologia (2017) 182:215–227 DOI 10.1007/s11046-016-0057-9

Pathogenesis of : Sensing the Host Tissue

Nilce M. Martinez-Rossi . Nalu T. A. Peres . Antonio Rossi

Received: 6 March 2016 / Accepted: 24 August 2016 / Published online: 2 September 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract The genera , , Keywords Á Molecular target Á and Epidermophyton include filamentous fungi that Cutaneous Á pH Á Heat shock proteins Á cause dermatophytosis, a superficial of the Fungal adhesion , stratum corneum, nail beds, and follicles. The ability of dermatophytes to adhere to these substrates and adapt to the host environment is essential for the Introduction establishment of infection. Several fungal enzymes and proteins participate in this adaptive response to the Fungal pathogenesis is dependent on both fungal and environment and to degradation. Transcription host factors that allow the establishment of the factors such as PacC and Hfs1, as well as heat shock in host tissue. This occurs concomitantly with proteins, are involved in sensing and adapting to the activation of the host , which aims acidic pH of the skin in the early stages of fungal–host to eliminate the . Several fungal virulence interaction. During growth, with keratin factors account for the establishment of infection and as the sole carbon source, the extracellular pH shifts contribute to tissue damage, including the degrada- from acidic to alkaline. This creates an environment in tion and use of host tissue as a nutrient source. which most of the known keratinolytic proteases However, the inflammatory response activated by the exhibit optimal activity. These events culminate in presence of the fungus or release of its metabolites the establishment and maintenance of the infection, and virulence factors also plays a significant role in which can be chronic or acute depending on the tissue damage during infection [1, 2]. Therefore, the dermatophyte species. This review focuses on these fungal–host interaction represents a dynamic and and other molecular aspects of the dermatophyte–host complex process that has been extensively studied interaction. over recent decades. This research has revealed certain features of several pathogenic and oppor- tunistic fungi that enable the establishment of & N. M. Martinez-Rossi ( ) Á A. Rossi mycoses. Deeper understanding of fungal pathogen- Department of Genetics, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Av Bandeirantes 3900, esis is crucial for the development of new therapeutic Ribeira˜o Preto, SP 14049-900, Brazil approaches and drugs. e-mail: [email protected] The fungal arsenal used to colonize host tissues comprises surface molecules responsible for attach- N. T. A. Peres Department of Morphology, Federal University of ment, secreted enzymes to convert host molecules into Sergipe, Aracaju, SE, Brazil nutrient sources, metabolic changes to metabolize 123 216 Mycopathologia (2017) 182:215–227 these components, thermotolerance, and dimorphism, who are in constant contact with them [10]. Dermato- whereby an individual fungal species converts phytes found in the soil degrade organic materials. between mycelia and forms depending on Individuals may contract dermatophytosis through environmental conditions [2]. The expression of contact with infected humans or or with fungal adhesins on the cell surface allows for rapid contaminated objects, primarily towels, manicure attachment to host tissue and the extracellular matrix, appliances, and hairbrushes [9]. Given the ubiquitous preventing elimination of the pathogen by the host nature of dermatophytes and the transmissibility defence mechanisms. Once enclosed in the host tissue, through direct and indirect contact, this group of the pathogen must scavenge nutrients to survive while pathogenic fungi is considered a public health prob- evading innate immune cells and molecules. Fungi lem, accounting for millions of dollars in healthcare secrete a broad spectrum of enzymes to degrade host expenses in the USA [11]. Circulatory and metabolic cells, such as proteases including collagenolytic and disorders, like diabetes mellitus, obesity, psoriasis, elastinolytic enzymes, lipases, nucleotidases, and hyperhidrosis, and immunosuppression, predispose mucolytic enzymes [3, 4]. Furthermore, several individuals to dermatophytosis, particularly tinea metabolic pathways are involved in fungal survival pedis (infection of the feet) and tinea unguium/ under stress conditions, such as nutrient shortages, (infection of the nails) [6]. Genetic oxidative and osmotic stresses, and exposure to factors are also associated with dermatophytosis, antifungal drugs. These pathways allow the fungus mainly innate immunity deficiencies [12]. to utilize different substrates for energy so that it may Based on conidia morphology, three genera of overcome the hostile environment of the host and, dermatophytes were recognized until now: Trichophy- thus, maintain the infection process [5]. ton, Microsporum, and Epidermophyton. Species are Fungi cause diseases that range from superficial classified, depending on whether their primary habitat cutaneous infections to profound and disseminated is, into anthropophilic, zoophilic, or geophilic [9]. cases reaching the , liver, spleen, kidneys, and Anthropophilic species are responsible for the major- the nervous system. Although cutaneous infections, or ity of human cases of dermatophytosis, followed by , are rarely life-threatening, they are zoophilic species [13]. In the above-mentioned study the most prevalent fungal infections worldwide, performed in France, anthropophilic species were the sometimes leading to a reduced quality of life in primary dermatophytes associated with human cases patients [6]. Etiologic agents of dermatomycosis (92.6 %), and was prevalent include dermatophytes, non-dermatophyte moulds, among onychomycoses (fingernails and toenails) [7]. and (like and Among dermatophytes, the most common are T. spp.), with dermatophytes being the most prevalent rubrum (tinea pedis, , tinea unguium, and [1]. A retrospective study surveying and ), Trichophyton interdigitale (tinea pedis nail infections from 2001 to 2011 in France showed and tinea cruris), (tinea capi- that dermatophytes were responsible for 67.9 % of tis), , and active cases of superficial infections, while non- ( and tinea corporis) [6, 13, 14]. dermatophytes accounted for the remaining 32.1 % The genomes of 24 dermatophyte strains are [7]. Dermatophytes cause superficial infections in both publicly available from the Broad Institute (http:// healthy and immunocompromised humans, with cases www.broadinstitute.org/annotation/genome/dermatop of deep infections associated with immunosuppression hyte_comparative/MultiHome.html). This massive [8]. Dermatophytic lesions on the skin are usually genome-sequencing project provided an analysis of round, erythematous, and itchy due to the inflamma- content and conservation across different spe- tory response triggered by the fungus and its metabo- cies. Comparative genome analyses revealed few lites [1]. In onychomycosis, nails become thicker and differences in genome organization and content separated from the nail bed; white spots and dystrophy among the species analysed, suggesting that differ- may also occur [9]. ences in gene regulation and post-transcriptional Animals such as dogs, cats, rabbits, and horses can mechanisms might be responsible for the niche- also become infected with dermatophytes. These specific adaptation of each strain [11]. An analysis of animals represent a source of infection for humans the expression of some coding for secreted 123 Mycopathologia (2017) 182:215–227 217 enzymes, such as proteases and lipases, showed that host, such as the acidic nature of the skin, the presence for some of the analysed genes, the expression levels of inhibitory molecules such as fatty acids and varied significantly between T. tonsurans and Tri- antimicrobial peptides, the action of phagocytic cells, chophyton equinum grown on keratin [15]. A com- and skin desquamation [9, 18, 19]. The attachment of parative proteomic analysis revealed differences in the dermatophytes to the host surface must occur rapidly amount and specificity of secreted proteins between T. to avoid fungal elimination. Adherence of dermato- rubrum and Trichophyton violaceum grown on soy phyte conidia to the stratum corneum occurs within medium [16]. Experimental models are required to 3–4 h [20], and the conidia germinate within 24 h. address questions about niche and host specificity of After 3 days, hyphae have spread through the skin different dermatophytes. Guinea pigs and mice have [21]. In the case of Trichophyton mentagrophytes, been used to elucidate immune responses triggered by fibril-like structures appear, which may help the zoophilic dermatophytes. However, for anthropophilic fungus adhere to the host tissue [22]. species, ex vivo and in vitro assays have been per- Adhesins are surface molecules expressed in the formed, providing insights into the pathogenic process fungal cell wall that allow rapid attachment to host and immune response triggered by these species tissues and the extracellular matrix. Some studies have (Fig. 1)[9, 17]. addressed the presence of adhesins in dermatophytes. In this review, we describe recent findings regard- Trichophyton rubrum and T. mentagrophytes express ing host–dermatophyte interactions, focusing on the surface glycoproteins that interact with galactose and immune responses triggered by fungal adhesion, mannose residues on the surface of Chinese hamster sensing, and adaptation to the host tissue. We partic- ovary (CHO) epithelial cells [23, 24]. In M. canis, the ularly highlight the role of skin pH sensing and the secreted protease of the subtilisin family Sub3 is mechanisms involved in the adaptive response of involved in adhesion to reconstructed feline epider- dermatophytes to ambient pH changes, including the mis, revealing that dermatophyte proteases play a role role of heat shock proteins. in attachment to host tissues [25]. SOWgp is an immunodominant antigen in the cell wall of Coccid- ioides immitis, a associated with Host–Dermatophyte Interaction and disseminated infection; the antigen is involved in virulence by helping the fungus attach to The establishment of dermatophytosis depends on the the extracellular matrix [26]. In T. rubrum, the sowgp fungus’s ability to overcome the natural barriers of the gene was upregulated in the early stages of in vitro nail

Fig. 1 Trichophyton rubrum ex vivo growth on nail and skin. Trichophyton rubrum conidia germinated on human nail (1 9 104 conidia/fragment) or skin (1 9 104 conidia/cm2) for 72 h. Human nail and skin are kindly provided by healthy donors [17]

123 218 Mycopathologia (2017) 182:215–227 and keratin growth, suggesting its involvement in activating the NLRP3 inflammasome [33–35]. dermatophyte virulence [17]. Although a fungal heat-sensitive compound was The first cells encountered by dermatophytes dur- responsible for this activation, the molecule triggering ing infection are keratinocytes, which play an impor- this pathway is still unknown [33, 35]. The inflam- tant role in host innate defence [9]. Infections caused masome is an intracellular protein complex that by zoophilic dermatophytes are acute and highly controls the activation of pro-inflammatory , inflammatory, while anthropophilic species cause recruiting inflammatory cells to control fungal infec- chronic disease with weak inflammatory response. tions [36]. In response to T. rubrum conidia, signalling Keratinocytes express a different profile in through IL-1R is important to control fungal growth, response to dermatophytes, which correlates with the given that IL-1R null allowed a faster inflammatory reaction triggered by various species. hyphal extension and a decreased release of IL-1b. Keratinocytes infected with the zoophilic dermato- Moreover, impairment of the IL-1R signalling also phyte Arthroderma benhamiae express pro-inflamma- decreased the IL17 response during mice infection tory genes and secrete cytokines to promote [35]. recruitment of inflammatory cells in the skin during Metabolic changes allow fungi to adapt to the host infection, tissue remodelling, and wound healing. environment and to use the molecules obtained to However, the anthropophilic species T. tonsurans acquire energy and disseminate through the tissue. The induces limited expression and secretion of cytokines glyoxylate cycle is important for fungal survival in the [27]. Skin from mice infected with A. benhamiae host, especially inside phagocytes [37]. In A. ben- exhibit infiltration of neutrophils, macrophages, and hamiae, the genes coding for the key enzymes of the dendritic cells and high levels of TGF-b, IL-1b, IL-6, glyoxylate cycle, malate synthase and isocitrate lyase, and IL-22 mRNA, suggesting a role for Th17 cells in were upregulated during infection of Guinea pigs [38]. the immune response against dermatophytes [28]. In the anthropophilic dermatophyte T. rubrum, three Interaction of A. benhamiae with keratinocytes genes from this pathway were upregulated during induced the expression of the hypA gene [29], a fungal growth in keratin (isocitrate lyase, malate synthase, hydrophobin that protects fungal cells from recogni- and citrate synthase), with malate and citrate synthase tion by the host immune cells. Neutrophils kill more also upregulated during ex vivo nail growth [17]. efficiently conidia from hypA null mutant and increase Once attached to the skin, nail or hair surface, the production of pro-inflammatory cytokines, sug- dermatophytes need to sense the host tissue and gesting its involvement in fungal escape from the host scavenge nutrients for survival. Fungi secrete several innate immunity [30]. hydrolytic enzymes to cleave macromolecules and Phagocytosis of T. rubrum conidia by macrophages allow them to be assimilated and used as an energy leads to upregulation of TNF-a and IL-10, and source. The most studied dermatophyte virulence downregulation of co-stimulatory and MHC II (Class factors are secreted proteolytic enzymes, mainly II molecules of the Major Histocompatibility Com- keratinolytic proteases [4]. During interaction with plex) molecules. Moreover, conidia can germinate the host as well as growth on keratinous substrates, forming hyphae inside the cell, causing the rupture of genes coding for proteases were upregulated in T. membrane, and subsequent death [31]. rubrum and A. benhamiae [38, 39]. However, dendritic cells kill T. rubrum conidia after Another important aspect of the interaction phagocytosis, which leads to the upregulation of IL- between dermatophytes and their hosts is the forma- 12, IL-10, and TNF-a. These cells were capable of tion of biofilms in nail infections, characterized by presenting antigens to autologous CD4? T cells, both thick biomasses embedded in an extracellular matrix derived from patients with dermatophytosis, promot- and containing dormant fungal elements [40–42]. ing proliferation and production of cytokines such as Biofilm formation could explain dermatophytomas, a IL-4, IL-10, and IFN-c [32]. form of onychomycosis refractory to standard anti- Recent studies showed that the dermatophytes fungal therapies where more than one Trichophyton schoenleinii, M. canis, and T. rubrum species may be present and living fungal elements are induced the release of the pro-inflammatory cytokine firmly adhered to the nail plate [42]. Therefore, the IL-1b in macrophages and dendritic cells, by ability of dermatophytes to form biofilms can be 123 Mycopathologia (2017) 182:215–227 219 considered a virulence factor, helping to protect the the ambient pH from acidic to alkaline, reaching fungi from ambient stress and providing metabolic values of 7.5–8.9 [9, 51]. In T. rubrum, transcription of cooperation and microbial communication. the gene encoding acetamidase is stimulated by an acidic environment. This enzyme produces acetate and ammonia. Acetate is then metabolized to acetyl-CoA Sensing Host pH by acetyl-CoA synthase, suggesting that ambient alkalinization correlates with the secretion of ammo- Initial contact with skin and nail plates occurs in an nia and the metabolism of acetate [52, 53]. During the acidic environment, with an average pH of 4.7 [43] due alkalinization process, dermatophytes respond by to a combination of molecules derived from glands, expressing enzymes that are functional at the current epidermal cells, and even resident flora. Among these ambient pH values; for instance, active acid and molecules are filaggrin–histidine–urocanic acid path- alkaline proteases are secreted at acid and alkaline pH way-related breakdown products, amino acids and values, respectively. This adaptive response is the alpha hydroxyl acids derived from sweat, acidic lipids essence of the pH regulatory system [54–56]. Further- such as cholesterol sulphate, and free fatty acids, more, growth on keratin leads to overexpression of which may derive from the hydrolysis of sebaceous genes encoding several proteases and membrane secretions by resident microflora [44–49]. Ammonia, transporter proteins, while these are only minimally carbon dioxide, and bicarbonate are also present in the expressed when the initial culture pH is 8.0 and skin and increase the skin pH. Thus, the maintenance glucose is the carbon source. Thus, the combination of of an acidic skin pH is the result of a complex the ambient pH shift and the presence of keratin as the biochemical process and the combination of various carbon source is necessary to induce the expression of chemical products. The acidic pH of the skin surface, these genes. This represents an efficient strategy for associated with specific fatty acids derived from skin the successful establishment, development, and main- lipids, represents a type of defence against the growth tenance of dermatophyte infection in the host. of . The invasive ability and virulence In the hard keratin present in the nail and hoof, the of dermatophytes correlate with their adherence peptide chains are firmly bonded together by disul- capacity. To establish a successful infection, arthro- phide bridges between cysteine residues, which are conidia must germinate rapidly and the hyphae must highly abundant in these proteins, forming a compact penetrate the body surface, or desquamation of the structure. The proteolytic digestion of hard is epithelium will eliminate them. Adherence of der- not possible without prior reduction in the disulphide matophytes to cell surfaces is due to the presence of bonds [57, 58], which depends on the production of glycoproteins containing mannan in the cell wall, sulphite by the dermatophytes. Cysteine dioxygenase possibly in a pH-dependent manner. Once established, (CDO) participates in the regulation of intracellular dermatophytes seek nutrients for growth and respond cysteine levels and contributes to the degradation of to the ambient pH by de-repressing genes encoding disulphide bonds in keratin proteins. The dermato- proteins and enzymes that have optimum activity at phyte cells begin producing sulphites and subse- acidic pH values, such as adhesins, lipases, phos- quently secrete them with the sulphite efflux pump phatases, DNAses, and keratinolytic proteases, among Ssu1 [59, 60]. Arthroderma benhamiae cdo1 and ssu1 many others. The acidic pH of the skin is optimal for knockout mutants were specifically growth defective these enzymes, allowing adherence and penetration of on hair and nails, revealing that these genes mediate the host tissue, uptake of nutrients, and survival against the growth of dermatophytes on these substrates [60]. host defence mechanisms. In T. rubrum, the ssu1 gene was upregulated during In vitro growth of T. rubrum and other is growth on keratin and ex vivo nail infection [17]. dependent on the initial culture pH, with an optimum Disruption of the pacC gene of T. rubrum, which pH of 4.0–5.0 in combination with an ambient pH shift codes for a transcription factor, correlates with from acidic to alkaline as keratin is utilized as the decreased growth on human nails, decreased secretion carbon source [39, 50]. The metabolism of some of keratinolytic proteases, and reduced conidiation (by amino acids—glycine, for instance—released from at least 3.7-fold) when grown on liquid medium skin proteins results in secretion of ammonia and shifts supplemented with keratin. These results indicate that 123 220 Mycopathologia (2017) 182:215–227

PacC somehow regulates the secretion of keratinolytic possibly with help from PalC and PalF [71]. In A. proteases, as well as growth, development, and nidulans, the PacC protein undergoes a two-step conidiation in T. rubrum [56]. Interestingly, disruption proteolytic process in response to a neutral/alkaline of the T. rubrum pacC gene did not affect either the pH [63]. First, PalA interacts with the YPXL/I motifs transcription patterns of the acetamidase and car- in PacC72 (full-length version of PacC), mediating a boxypeptidase genes or the progressive alkalinization protein–protein interaction that is required to convert of the culture medium during growth [61]. These PacC72 to PacC53. PalB, a calpain-like cysteine results indicate that the low activity level of the protease, likely mediates this proteolytic step. In the alkaline protease secreted by the pacC-1 mutant strain second, pH-independent step, PacC53 is proteolysed of T. rubrum is not related to the reduced alkaliniza- to PacC27. The PacC27 form limits transcription tion of the culture medium. under neutral/alkaline conditions of genes expressed preferentially at acidic pH levels. Thus, whatever the ambient pH, loss-of-function mutations in any of the The Functioning of PacC pal genes leads to a wild-type acidic growth phenotype [72]. That is, the full-length version of PacC would be Pathogenic fungi have the ability to sense and respond inactive in the absence of Pal signalling or under to the pH of the environment. This sensing is essential acidic growth conditions [63]. However, experimental for survival, growth, virulence, and dissemination in evidence has shown that the full-length version of different host niches. This adaptive response is depen- PacC in A. nidulans is also active at acidic pH levels dent on the highly conserved PacC/Pal signal transduc- [72]. Suppression subtractive hybridization experi- tion pathway, composed of at least seven genes—pacC, ments successfully identified novel genes upregulated palA, palB, palC, palF, palH,andpalI [62]—that in T. rubrum incubated at either acidic or alkaline pH mediate a myriad of metabolic events in filamentous, levels [61]. These genes participate in diverse cellular pathogenic, and model fungi, governing the response to processes, and genes upregulated at acidic or alkaline changes in the pH of the environment. The analysis of pH levels seem relevant to the initial stages of seven dermatophyte genomes revealed the presence of dermatophyte infection or its maintenance in the host, all genes in this PacC/Pal signal transduction pathway respectively. Interestingly, when incubated at either [19], suggesting that this signalling cascade is also acidic or alkaline pH levels, several genes display highly conserved among dermatophytes. diverse transcription profiles in a pacC- background. nidulans lacking the entire PacC-cod- This indicates that in T. rubrum, PacC has a diversity ing region mimics in neutral pH the acidic growth of metabolic functions in response to acidic and conditions of the wild-type strain. This mutant strain alkaline extracellular pH levels, as already reported grows very poorly, probably because the deletion for A. nidulans [73] and Neurospora crassa [74, 75]. prevents both the positive and negative actions of The identification of novel genes modulated by the PacC. Classical and molecular genetics analyses transcriptional regulator PacC provides new insights support the wide regulatory character of the into the pH sensing of T. rubrum. pacC gene, a Zn-finger regulator whose products directly mediate pH regulation. Like many acid- or alkaline-induced genes, the promoter regions of Secretion of Hydrolytic Enzymes alkaline-expressed genes such as ipnA, pacC, and the alkaline protease-encoding prtA contain a consensus Although it is well established that ambient pH affects sequence for binding of PacC (50-GCCARG-30) in the the growth, physiology, differentiation, and viability 1200 bp upstream of their initiation codes [63, 64]. of all , molecular responses to environmen- The six pal genes (palA, palB, palC, palF, palH, and tal pH changes still remain to be elucidated. In A. palI) are components of a signalling cascade that nidulans, loss-of-function mutations in the pal genes senses alkalinity and promotes the proteolytic cleav- reduced alkaline phosphatase activity but increased age of PacC [63, 65–70]. The ambient alkaline pH acid phosphatase activity, among other phenotypes signal is detected by PalI and PalH and transmitted mimicking growth at an acidic pH. Growth in alkaline downstream to the endosomal membrane complex, medium induces transcription of the pacC gene, but 123 Mycopathologia (2017) 182:215–227 221 the pal genes do not appear to be regulated by ambient on serine and threonine residues [90]. In proteins pH. Furthermore, the pal genes are involved in the glycosylated at both sites, it is not known which proteolytic cleavage of PacC. Thus, the molecular enzymatic step, N- or O-glycosylation, precedes the properties of the secreted acid phosphatases should be other [91]. Incomplete or altered glycosylation may identical in all pal mutants and wild-type strains, but affect the stability and half-life of proteins, thus based on the molecular mass, electrophoretic mobility, changing their activities or affinities towards sub- and chromatographic behaviour of the acid phos- strates [92, 93]. In T. rubrum, disruption of the pacC phatase secreted by the palB7 strain, this is not the case gene resulted, as already mentioned, in decreased [76]. In addition, the palB7 mutation alters the growth on human nails and decreased secretion of electrophoretic mobility of the constitutive acid phos- keratinolytic proteases in liquid medium when sup- phatase synthesized on medium with high inorganic plemented with keratin. Interestingly, PacC protein of phosphate (Pi). Indeed, the strains carrying the palB7 T. rubrum is possibly involved in the glycosylation of mutation and other pal- strains secreted a Pi-repress- these proteases through transcriptional modulation of ible acid phosphatase at pH 5.0 with low mannose and O- and N-linked mannosyltransferases [74]. Tran- N-acetylgalactosamine content compared to a control scriptional profiling of both the O-man and N-man strain [77, 78]. Incomplete post-translational manno- genes revealed a high level of complexity, because sylation of proteins could be the result of the palB7 transcription of these genes was affected by nutrients, mutation. Thus, the palB gene codes for a Ca2?- culture pH, and the functioning of the pacC gene. dependent protease with functions other than being Disruption of the pacC gene increased the expression directly involved in the proteolytic processing of of N-man at pH 5.0 in keratin cultures. Moreover, if PacC. Furthermore, mRNA differential display O-mannosylation precedes N-glycosylation in T. (DDRT-PCR) analyses identified two cDNAs in the rubrum, as demonstrated in yeast [91], this physio- wild-type strain not detected in the palB7 mutant strain logical effect is dependent on the function of the pacC that encode a mannosyltransferase and an NADH- gene at acidic pH. Interestingly, transcription of O- ubiquinone oxidoreductase (chain 4) [77]. man declined in the pacC- mutant in keratin at pH 5.0. In N. crassa, one of the metabolic responses to the Therefore, at acidic pH, PacC affects transcription of pH of the medium is the pH-dependent glycosylation the O-man and N-man genes positively and negatively, of secreted enzymes. For example, alkaline phos- respectively, in keratin cultures. The balance between phatase (Pho-2) synthesized at pH 7.8 differs from that N-man and O-man expression levels in cultures at synthesized at acidic pH only to the level of its acidic pH may be under the control of the PacC glycosylation [79–81]. Interestingly, almost the same transcription factor. Moreover, PacC affects nega- amount of this enzyme is secreted irrespective of tively the transcription of the N-man gene at pH 5.0 medium pH as measured by ELISA [80]. The lower and the O-man gene at pH 8.0, when the fungus is enzymatic activity for the protein secreted at pH 5.4 is cultured in keratin. Therefore, transcription of the N- probably due to the lower glycosylation level of this man and O-man genes might be required at different enzyme as compared to that of the protein secreted at culture pHs for the glycosylation of transported alkaline pH [80, 81]. Thus, the secretion of similar proteins, according to the stage of infection, which amounts of Pho-2 irrespective of medium pH in strains suggests a possible role in cell adhesion and activation grown in low-phosphate medium indicates that of signalling pathways regulating the production of expression of the pho-2 gene not only relies on enzymes that enable nutrient uptake for fungal devel- PacC-independent pH signalling mechanisms but also opment and maintenance in the host [94–96]. is not an alkaline-expressed gene [55, 82]. Protein secretion from a eukaryotic cell requires movement through the endoplasmic reticulum (ER) Heat Shock Genes and Interaction with pH and the Golgi apparatus, where in the course of Signalling in Fungal Pathogenesis trafficking, the secreted proteins undergo glycosyla- tion [83–89]. The glycosyl or mannosyl groups are Heat shock proteins (HSPs) are a conserved family of usually attached to either an amide group (N-glyco- molecular chaperones that participate in multiple sylation) or a hydroxyl group (O-glycosylation) found functions in cells. They aid in the stabilization and 123 222 Mycopathologia (2017) 182:215–227 correct folding of nascent polypeptides, the assem- another hsp70 gene was overexpressed after the bling of protein complexes, and the transport and culture medium shifted from acid to alkaline pH [61] sorting of proteins into their cellular compartments; and during interaction with human skin [113]. Indeed, they also participate in cell cycle control [97] and several genes from the Hsp70 family are overex- control programmed cell death [98]. Furthermore, pressed in A. benhamiae during experimental Guinea HSPs play a fundamental role in cellular recovery pig infection or in keratin culture. Interestingly, one of from several stress conditions and protection from these genes, hsp70 (S11394), is overexpressed during subsequent insults [97]. Two response elements reg- growth of A. benhamiae in keratin. This hsp70 gene is, ulate the expression of hsp genes in yeast: the heat however, downregulated in experimental Guinea pig shock element (HSE), which is bound by the tran- infection [38], reinforcing the conclusion of the scription factor Hsf1p [99, 100], and the stress authors that the set of genes overexpressed during response element (STRE), which is bound by the in vivo and in vitro experiments are not always the transcription factors Msn2/4p [101, 102]. These two same. binding sites are distributed differentially among the Thus, the relationship between fungal pathogenic- heat shock genes, suggesting that Hsf1p and Msn2p/4p ity and modulation of hsp genes is well documented, make distinct contributions to the expression of these as is the involvement of the pacC gene in the genes in response to stress [103, 104]. pathogenicity of T. rubrum [56], including the over- In yeast, the hsf gene is activated by multiple expression of this gene during fungal growth in keratin stresses, including heat, , glucose or human nails but not in human skin [17, 112]. starvation, pH, and salicylate [105]. Many targets for Recently, in an experiment involving a strain of T. HSF have been identified, including HSPs where HSF interdigitale with the pacC gene deleted (pacC-), it binds to the HSEs present in their promoters [106]. was revealed that pacC and hsf1 genes participate in The hsf gene is essential even in the absence of heat the same regulatory circuit to control hsp genes. This stress. report showed that pacC and hsf1 are overexpressed in During the invasion of host tissue by a pathogen, response to temperature shifts when T. rubrum or T. HSPs are released into the extracellular environment interdigitale are grown in keratin cultures. However, by the host cells in response to stressful circumstances, transcription of hsf1 is dramatically reduced in the probably as part of signalling to alert the rest of the pacC mutant, whereas the transcription levels of some of the potentially damaging situation hsp genes are upregulated independently of heat [97, 107]. Bacterial and fungal pathogens also increase induction [112]. These results suggest that PacC the expression of HSPs during infection of their controls the level of hsf1 expression and that the respective hosts [107, 108] revealing that both hosts association between PacC and Hsf1 determines the and pathogens depend on these proteins to protect amount of hsp transcripts present in the cell during a themselves from each other. Biofilm formation during stress response, including during the interaction with C. albicans infection depends on HSPs as well [109]. the host. HSPs are highly conserved among pathogens and play The Hsp90 protein is of particular importance in a crucial role in fungal pathogenicity and survival. fungi. It is highly abundant in cells constitutively and In dermatophytes, there are several examples of increases further under stress conditions. Some natural overexpression of HSP genes under conditions that compounds produced by microorganisms are used to simulate the interaction with the host tissue (Table 1). inhibit the Hsp90 ATPase activity, and these can be Three genes encoding putative Hsp70 proteins in A. used to study its functional role in the cell. An ex vivo benhamiae [38] and a gene encoding Hsp30 in T. nail interaction assay established the role of Hsp90 in T. rubrum [39, 52, 110] are overexpressed during keratin rubrum pathogenicity. Hsp90 inhibition by 17-ally- degradation. The hsp60, hsp70, and hsp78 genes lamino-17-demethoxygeldanamycin (17-AAG) attenu- exhibit increased expression when T. rubrum is grown ated fungal virulence on human nails in an inhibitor on human nails in vitro. However, there is no concentration-dependent manner, suggesting the difference in gene expression when the fungus is involvement of this HSP in pathogenicity [111]. The grown on human skin, suggesting a high specificity of involvement of the Hsp90 proteins of C. albicans and action of these proteins [111, 112]. Additionally, in pathogenicity has also been 123 Mycopathologia (2017) 182:215–227 223

Table 1 Representative list of hsps and related genes of dermatophytes upregulated upon exposure to host or to molecules of host tissue Host/tissue Organism/ID Gene/gene product References

Keratin Arthroderma benhamiae/S14252a Chaperone from the Hsp70 family [38] Arthroderma benhamiae/S11394a Hsp70 family protein [38] Arthroderma benhamiae/S03730a Hsp70 chaperone Hsp88 [38] T. rubrum/TERG_01659.3b hsp30 [39] T. rubrum/AAV33735c hsp30 [110] T. interdigitale DpacC/H101_01606.2b Hsp90 co-chaperone cdc37 [112] T. interdigitale DpacC/H101_05806.2b hsp ssc1, Hsp7-like protein [112] T. interdigitale DpacC/H101_02626.2b hsp70-like [112] T. rubrum/TERG_00838b pacC [17] Human nail T. rubrum/TERG_04141b hsp60 [111] T. rubrum/TERG_07949b hsp78 [111] T. rubrum/TERG_01883b hsp70 [112] T. rubrum/TERG_06398b Hsp90 co-chaperone cdc37 [112] T. rubrum/TERG_03206b hsp ssc1 [112] T. rubrum/TERG_00838b pacC [112] T. rubrum/TERG_04406b hsf1 [112] Human skin T. rubrum/TERG_06398b Hsp90 co-chaperone cdc37 [112] T. rubrum/TERG_03206b hsp ssc1 [112] T. rubrum/TERG_04406b hsf1 [112] T. rubrum/DW681773d hsp70 [113] T. rubrum/DW693707d hsp90 [113] Guinea pig infection Arthroderma benhamiae/S14167a heat shock protein 70 (hsp70)[38] Arthroderma benhamiae/S14252a chaperone from the Hsp70 family [38] a Gene Expression Omnibus (GEO) via accession number GSE15873 b Broad Institute of Harvard and MIT. http://www.broadinstitute.org/annotation/genome/dermatophyte_comparative/MultiHome. html c Accession number at GenBank (http://www.ncbi.nlm.nih.gov) d The GenBank/EMBL/DDBJ accession numbers for the EST sequences demonstrated [114, 115]. Blocking the activity of Successful establishment of infection is dependent on Hsp90 in T. rubrum leads to modulation of some hsp several fungal proteins and enzymes regulated by genes and the pacC gene, strengthening the relevance of multiple genes that are activated or repressed in Hsp90 to cellular physiology, independent of stress response to the ambient environment of the host. conditions. The concentration of pacC transcripts Dermatophyte genes and proteins that participate decreases during growth in keratin cultures containing significantly in this interaction are natural potential Hsp90 inhibitor, indicating an association between candidates for therapeutic targets. Candidate genes that pacC and hsp90 that may affect fungal virulence [111]. are present as a single, unique copy in the genome and are conserved among dermatophyte species have high potential as drug targets. The hsp90, hsf1,andpacC Conclusion and Perspectives genes fulfil these conditions and should therefore be further evaluated for their therapeutic potential. More- The interaction between dermatophytes and their over, chemical inhibition of Hsp90 results in increased specific hosts involves complex molecular mechanisms. susceptibility of T. rubrum to itraconazole and

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