1

Sporothrix schenckii complex biology: environment and fungal pathogenicity 1

Téllez MDa, Batista-Duharte Ab,c,d*, Portuondo Dc, Quinello Cc, Bonne-Hernández Rd, Carlos IZc* 2

a Faculty of Chemical Engineering. Oriente University. Ave Las Americas, Santiago de Cuba, Cuba, b 3

Immunotoxicology Laboratory, Toxicology and Biomedicine Center (TOXIMED), Medical Science 4

University, Autopista Nacional Km. 1 1/2 CP 90400, Santiago de Cuba, Cuba c Faculty of 5

Pharmaceutical Sciences. Universidade Estadual Paulista Julio Mesquita Filho, UNESP. Rua 6

Expedicionarios do Brasil 1621-CEP:14801-902, Araraquara, SP, Brazil, d Universidade Federal de 7

São Paulo, São Paulo-SP, Brasil, d CAPES/Brasil Grant. Foreigner Visiting Professor Program. 8

* Corresponding authors 9

Profa. Dra. Iracilda Zeppone Carlos: Faculty of Pharmaceutical Sciences of Araraquara. Universidade 10

Estadual Paulista Julio Mesquita Filho, UNESP. Araraquara, São Paulo. Brazil. CEP:14801-902 11

Phone 55 16 3301-5712. E-mail: carlosiz@fcfar.unesp.br 12

Prof Dr. Alexander Batista-Duharte: Phone 55 16 3301-5713. E-mail: batistaduhartea@gmail.com 13

Abstract 14

Sporothrix schenckii is a complex of various species of fungus found in soils, plants, decaying 15

vegetables and other outdoor environment. It is the etiological agent of sporotrichosis in humans and 16

several animals. Humans and animals can acquire the disease through traumatic inoculation of the 17

fungus into subcutaneous tissue. Despite the importance of sporotrichosis as a disease, which is 18

currently regarded as an emergent disease in several countries, the factors driving its increasing 19

medical importance are still largely unknown. There are only a few studies addressing the influence of 20

the environment on the virulence of these pathogens. However, some recent discoveries made in 21

studies of the biology of S. schenckii are demonstrating that adverse conditions in its natural habitats 22

can trigger the expression of different virulence factors that confer survival advantages in both animal 23

hosts and in the environment. In this review, we provide updates on the important advances in the 24

understanding of the biology of S. schenckii and the modification of their virulence linked to 25

demonstrated or putative environmental factors. 26

Keywords: Sporotrichosis, Sporothrix schenckii complex, virulence, environmental, ‘dual use’ 27

Word count of summary: 170 28

Word count of main text: 6689 29

Total number of tables: 1 Total number of figures: 1 30

Microbiology Papers in Press. Published August 18, 2014 as doi:10.1099/mic.0.081794-0

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1. Introduction 31

Sporotrichosis is an acute or chronic granulomatous mycosis of humans and mammals that has a 32

worldwide distribution. Initially the causal agent of this disease was thought to be a unique thermally 33

dimorphic fungus Sporothrix schenckii; however, it has been recently proposed, based on physiologic 34

and molecular aspects, that S. schenckii is a complex of various species (Lopes-Bezerra, 2006; 35

Marimon et al., 2007; Marimon et al., 2008; Evangelista et al., 2014). Sporotrichosis was originally 36

described in 1898 by Schenck at the Johns Hopkins Hospital in Baltimore, who recognized that the 37

infection was most likely due to a previously undescribed fungal pathogen (Schenck, 1898). Two years 38

later, Hektoen and Perkins confirmed these observations in a report of a second case and a detailed 39

morphological description of the pathogen, which included studies involving laboratory animals 40

(Hektoen and Perkins 1900). Other cases in the United States and western Europe were recognized, 41

especially in France, where large numbers of cases were reported in the early 20th century (de 42

Beurmann, 1912). 43

Recently, S. schenckii has received particular attention due to the increased number of infections 44

caused worldwide. Currently, the presence the S. schenckii complex and cases of sporotrichosis have 45

been reported from all of the continents (Madrid et al., 2009; Barros et al., 2010) and in different 46

geographic regions, such as tropical and subtropical regions. Some areas have declared sporotrichosis 47

an emerging health problem, with a growing interest for sanitary authorities (Feeney et al., 2007; Hay 48

and Morris-Jones, 2008; López-Romero et al., 2011; Messias, 2013). Sporotrichosis is now the most 49

common subcutaneous mycosis in the Americas (especially Brazil, Mexico, Peru, Colombia and 50

Uruguay) and also in Japan, India and South Africa (Carrada-Bravo et al., 2013). A particular 51

geographical area called Abancay, in the south central Peruvian highlands, is considered a 52

hyperendemic area, with an estimated incidence of approximately 50–60 cases per 100,000 inhabitants 53

per year (Bustamante and Campos, 2001). Meanwhile in Brazil there are increasing reports of 54

sporotrichosis in areas where the disease was rare decades ago, with an intriguing zoonotic 55

transmission by cats (Borges et al., 2013; Rodrígues et al., 2013a,b). In Europe, sporotrichosis is less 56

common, although it was also a common disease in France in 1900 but declined after two decades. 57

Currently, sporotrichosis is intermittently reported in several others countries, such as Italy, Spain, 58

Portugal, United Kingdom and Turkey (Criseo et al., 2008; Ojeda et al., 2011; Dias et al., 2011; 59

Gürcan et al., 2007). Other interesting observation is that from the beginning of the AIDS (Acquired 60

Immunodeficiency Syndrome) era, new clinical forms of disseminated sporothrichosis have been 61

reported, including cerebral, endocardial and ocular involvement (Galhardo et al., 2010; Ramos-e-62

Silva et al., 2012; Silva-Vergara et al., 2012). 63

3

A presence or even an abundance of the fungus in nature is not enough to explain the development of 64

the disease. Although the fungus is frequently isolated in environmental and commercial samples, it is 65

unclear why sporotrichosis has a low incidence. However, the different local emerging outbreaks 66

reaching sometimes epidemic proportions lead to questions regarding the relevance of the host in the 67

development of the disease (López-Romero et al., 2011), the virulence associated with genetic 68

polymorphisms in the S. schenckii complex (Sasaki et al., 2014), and other environmentally associated 69

factors not sufficiently studied yet. 70

The aim of this work is to provide an update on the recent advances in the understanding of Sporothrix 71

schenckii complex biology, addressing different ways through which environmental factors may 72

modify the virulence of the fungus, as a possible contribution to the sporotrichosis outbreaks. 73

2- The Sporothrix schenkii complex and sporotrichosis 74

The genus Sporothrix is a species-rich taxon of Ascomycetes, which varies according to the ecological 75

niche, frequency, distribution and virulence. Some of these fungi have the potential to survive in 76

mammalian hosts and are able to cause damage in multiple species of animals, including humans (de 77

Hoo, 1974; Fernandes et al., 2013). S. schenckii is recognized as a cryptic species complex including 78

Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, Sporothrix luriei, Sporothrix pallida 79

(formerly Sporothrix albicans) and S. schenckii sensu strict (Marques et al 2014). With the exception 80

of S. pallida, the other Sporothrix species have been reported to cause sporotrichosis in humans and 81

animals (Lopes-Bezerra, 2006; Marimon et al., 2007, Marimon et al., 2008, Romeo and Criseo, 2013; 82

Evangelista et al., 2014). 83

The fungi belonging to the S. schenckii complex are ascomycetous dimorphic organisms (Ascomycota, 84

Pyrenomycetes, Ophiostomatales, Ophiostomataceae), naturally found in substrates such as 85

living and decayed vegetation, animal excreta, and soils plentiful in cellulose, with a pH range of 86

3.5 to 9.4, a mean temperature of 31°C, and a relative humidity above 92%. This fungus is 87

phenotypically characterized by the ability to produce conidias in its filamentous form, and 88

cigar-shaped yeast-like cells when cultured at 35–37°C or as the infectious form in animals and 89

humans (Lopes-Bezerra, 2006). 90

Sporotrichosis affects humans and other mammals, such as cats, dogs, rats, armadillos, and horses. As 91

the fungus is abundant in soil, wood and moss, most infections occur following minor skin trauma in 92

people with outdoor occupations or hobbies, such as gardening, farming, hunting or other activities 93

involving close contact with vegetation and soil. The zoonotic transition from ill or carrier animals 94

(mainly cats) to man is gaining a growing importance. Similarly, an inoculation may also occur after 95

motor vehicle accidents and in laboratory personnel handling of Sporothrix-infected specimens (Barros 96

et al., 2007; Hay et al., 2008; Romeo and Criseo; 2013). 97

4

Human disease has different clinical manifestations and can be classified into fixed cutaneous, 98

lymphocutaneous, disseminated cutaneous, and extracutaneous or systemic sporotrichosis. The fixed 99

cutaneous sporotrichosis is located in the skin and is restricted to the injury site without lymphatic 100

involvement. The lymphocutaneous sporotrichosis is the most common manifestation and is 101

characterized by papular and nodular, superficial ulcers and/or vegetative plaque lesions in the skin 102

and along the trajectory of the draining lymph vessels. The disseminated cutaneous form consists of 103

multiple and distant lesions in the skin due to hematogenous dissemination or multiple inoculations of 104

the fungus. In the extracutaneous or systemic sporotrichosis, multiple cutaneous and visceral lesions 105

involving the liver, joints, spleen, bones, marrow bones, lungs, eyes, testis and central nervous system 106

associated with cellular immunodeficiency have been described (Edwards et al., 2000; Rocha et al., 107

2001; Ramos-e-Silva et al., 2012). Primary pulmonary sporotrichosis is another clinical type of this 108

infection caused by the inhalation of infectious conidia in contaminated environments. (Aung et al., 109

2013). 110

3-The response of the Sporothrix schenkii complex to changing environmental conditions 111

112

The origin and maintenance of virulence in dimorphic fungi is enigmatic because an interaction with a 113

mammalian host is not a requisite for fungal survival and virulence (Steenbergen et al., 2004). This 114

phenomenon is called by Casadewall ‘ready-made’ virulence (Casadewall et al., 2003). The influence 115

of different environmental contaminants, on pathogenic or non-pathogenic fungi has been studied 116

(Zafar et al., 2007; Gadd, 1993; Valix, 2001, 2003; Anahid et al., 2011). S. schenkii complexes are 117

present in nature in diverse environmental conditions, including polluted environments (Cooke and 118

Foter 1958; Dixon et al., 1991; Ulfig, 1994; Ulfig et al., 1996; Kacprzak, 2005; Pečiulytė 2010; Chao 119

et al., 2012; Yazdanparast et al., 2013), in a wide pH range from 2.2 to 12.5 (Tapia et al., 1993; 120

Ferreira et al., 2009) the floor of swimming pools (Staib 1983), desiccated mushrooms (Kazanas, 121

1987), fleas, ants and horse hair (Carrada-Bravo, 2013). Moreover, specific indoor environments also 122

select for certain stress-tolerant fungi and can drive their evolution towards acquiring medically 123

important traits (Gostinčar et al., 2011). 124

3.1 Physical factors: S. schenkii is able to resist extreme conditions, such as very low temperatures for 125

several years (Pasarell and Mcginnis, 1992; Mendoza et al., 2005), extreme osmotic pressure 126

(Castellani, 1967; Borba, 1992; De Capriles, 1993; Mendoza et al., 2005; Ferreira et al., 2009 ) and. 127

Some studies have revealed that different levels of ultraviolet light (UV) exposure by S. schenckii 128

strains resulted in a conserved viability, although with a high frequency of morphological variants, 129

depending on the strain and UV dose. The main morphological variants were smaller colonies or 130

changes in shape. Stable and non-stable morphological variants were found in the population, and the 131

5

reversion of the mutant phenotype was always to the wild-type phenotype (Torres-Guerrero 132

and Arenas-López; 1998). In another study, the gamma radiation effects on the yeast cells of S. 133

schenckii were analyzed, showing that yeast cells remained viable up to 9.0 kGy; however, synthetic 134

protein metabolism was strongly affected, while a dose of 7.0 kGy retained viability, metabolic 135

activity, and morphology but abolished the capacity to produce infection (de Souza et al., 2011). 136

3.2 Metals: Microorganisms, including fungi, have been shown to possess an ability to survive by 137

adapting or mutating at high concentrations of toxic heavy metals. In general, two mechanisms have 138

been proposed for heavy metal tolerance in fungi: 1) extracellular sequestration with chelation and 139

cell-wall binding, mainly employed in the avoidance of metal entry and 2) intracellular physical 140

sequestration of metal by binding to metallothioneins (MT) sequestered as insoluble phosphates or 141

removed from the cell via transporters such as CadA, ZntA or PbrA, preventing the damage caused by 142

metals to sensitive cellular targets and reducing the metal burden in the cytosol. (Anahid et al., 2011; 143

Jarosławiecka and Piotrowska-Seget, 2014). Most fungi synthesize siderophores that chelate iron, 144

which is ultimately taken up as a siderophore-iron complex (Kosman et al., 2003). The capacity to 145

accumulate iron is critical for the survival of fungal pathogens in different conditions (Schaible et al., 146

2004). Unlike other fungi, such as S. cerevisiae (Kaplan et al., 2006), S. schenckii is capable of 147

producing its own siderophores in response to low iron availability (Pérez-Sánchez et al., 2010). This 148

mechanism can be involved in the pathogenicity and survival under conditions of environmental stress 149

or inside the host. 150

3.3 Chemical contaminants: Environmental fungi can biodegrade aromatic hydrocarbons in their own 151

habitat (Cerniglia 1997). An interesting report revealed that several fungi, including S. schenckii, were 152

isolated from air biofilters exposed to hydrocarbon-polluted gas streams and assimilated volatile 153

aromatic hydrocarbons as the sole source of carbon and energy. The data in this report show that many 154

volatile-hydrocarbon-degrading strains are closely related to, or in some cases clearly conspecific with, 155

the very restricted number of human-pathogenic fungal species causing severe mycoses, especially 156

neurological infections, in immunocompetent individuals (Prenafeta-Boldu et al., 2006). In addition, 157

the effect of fungicides against several environmental pathogenic fungi was evaluated, and S. schenkii 158

had the greatest resistance to these products compared to the other fungi (Morehart and Larsh, 1967). 159

3.4 Other microorganisms: A phenomenon insufficiently studied is the interaction of the pathogenic 160

fungi with other microorganisms in the neighborhood. Chaturvedi et al. evaluated the in vitro 161

interactions between colonies of Blastomyces dermatitidis and six other zoopathogenic fungi. The 162

interactions were found to range from neutral with H. capsulatum and C. albicans to strongly 163

antagonistic with Microsporum gypseum, Pseudallescheria boydii, and Sporothrix schenckii and 164

included lysis by Cryptococcus neoformans (Chaturvedi et al., 1988). In another study, Cryptococcus 165

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neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, 166

suggesting that fungal pathogenic strategies may arise from environmental interactions with 167

phagocytic microorganisms (Steenbergen et al., 2003). In another interesting report, Steenbergen et al., 168

examined the interactions of three dimorphic fungi, Blastomyces dermatitidis, Histoplasma 169

capsulatum, and S. schenckii, with the soil amoeba, Acanthameobae castellanii. The ingestion of the 170

yeast by this amoeba resulted in amoeba death and fungal growth. For each fungal species, the 171

exposure of yeast cells to amoebae resulted in an increase in hyphal cells (Steenbergen et al., 2004). 172

The biochemical events during phagocytosis by either A. castellanii or immune phagocytes appear 173

similar, suggesting that the ‘respiratory burst’ enzyme(s) responsible for oxyradical generation in these 174

two cell types is structurally related (Davis et al., 1991). Thus, soil amoebae may contribute to the 175

selection and maintenance of pathogenic dimorphic fungi in the environment, conferring these 176

microbes with the capacity for virulence in mammals. 177

Despite that are necessary more studies evaluating the influence of different environmental factors on 178

the physiology and pathogenicity of the S. schenkii complex, all the available data suggest that the 179

strategies that pathogenic fungi acquire to survive this environmental competition may, in turn, 180

provide the ability to infect animals and may further allow the emergence of opportunistic pathogens 181

from these microenvironments (Baumgardner, 2012) (Figure 1). 182

4. Fungal elements involved in environmental resistance and pathogenicity 183

Although is known that many external influences can affect the pathogenicity of the S. schenkii 184

complex as environmental pathogenic fungi, these influences and mechanisms have not been 185

sufficiently studied in this complex. However, the presence of the same molecules interacting with 186

environmental toxins described in other fungus (Casadewall et al., 2003) is a good reason to 187

hypothesize that similar mechanism may be acting to face these extreme conditions. As Casadewall 188

reported for C. neoformans (Casadewall et al., 2003), several virulence factors in S. schenckii also 189

appear to have ‘dual use’ capabilities for the survival in both animal hosts and in the environment 190

(Table 1). 191

4.1 Dimorphism: The dimorphic fungi comprise a group of important human pathogens and represent 192

a family of seven phylogenetically related ascomycetes that include Blastomyces dermatitidis, 193

Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis, 194

Penicillium marneffei and Sporothrix schenckii. These fungi possess the unique ability to switch 195

between mold and yeast in response to thermal stimuli and other environmental conditions. In the 196

environment, they grow as mold that produces conidia or infectious spores, which, when transmitted to 197

humans or other susceptible mammalian hosts, are capable of converting to pathogenic yeasts that 198

cause serious infection (Klein and Tebbets 2007). 199

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The dimorphism in the S. schenckii complex has also been associated with the ability to adapt to 200

environmental changes and to yield an increased virulence (Nemecek et al., 2006; Gauthier et al., 201

2008). This fungus exhibits mycelium morphology in its saprophytic phase at 25°C in laboratory 202

conditions and a yeast morphology in host tissues at 35-37°C. On the other hand, in the environment 203

with different grades of temperatures they are able to keep the mycelial form. The formation of yeast 204

cells was thought to be a requisite for the pathogenicity of S. schenckii; however, the mechanisms that 205

regulate the dimorphic switch remain unclear. Some recent findings have begun to clarify these 206

mechanisms. 207

The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, and 208

they are involved in fungal dimorphism and pathogenicity. Valentín-Berríos et al. described a new G 209

protein α subunit gene in S. schenckii: ssg-2, and they demonstrated that the SSG-2 Gα subunit of 210

40.90 kDa interacts with the cytosolic phospholipase A2 (cPLA2), participating in the control of the 211

dimorphism in this fungus (Valentín-Berríos et al., 2009). 212

The yeast-to-mycelium transition is dependent on calcium uptake (Serrano et al., 1990; Rivera-213

Rodríguez et al., 1992). In S. schenckii, a Ca2+/Calmodulin-dependent protein kinase (CaMK) 214

encoded by the calcium/calmodulin kinase I (sscmk1) gene (GenBank accession no. AY823266) was 215

described (Valle-Aviles et al., 2007). Experiments using different inhibitors of the CaMK pathway 216

showed that they inhibited the transition from yeast cells to hyphae, which suggests a 217

calcium/calmodulin pathway is involved in the regulation of the dimorphism in S. schenckii (Valle-218

Aviles et al., 2007). Similarly, RNAi technology was used to silence the expression of the sscmk 1 219

gene. The RNAi transformants were unable to grow as yeast cells at 35°C and showed a decreased 220

tolerance to this temperature (Rodriguez-Caban et al., 2011). 221

The mitogen-activated protein kinase (MAPK) cascade and cyclic AMP (cAMP) signaling pathways 222

are known to be involved in fungal morphogenesis and pathogenic development. The MAPK and 223

cAMP pathways are both activated by an upstream branch, two-component histidine protein kinase 224

(HPK) phospho-relay system (Hou et al., 2013). Nemecek et al. reported that a long-sought regulator 225

controls the switch from a non-pathogenic mold form to a pathogenic yeast form in dimorphic fungi. 226

They found that DRK1, a hybrid dimorphism-regulating histidine kinase, functions as a global 227

regulator of the dimorphism and virulence in B. dermatitidis and H. capsulatum and is required for the 228

phase transition from mold to yeast, expression of virulence genes, and pathogenicity in vivo. 229

(Nemecek et al., 2006). More recently, Hou et al. developed a molecular cloning, characterization and 230

differential expression of DRK1 in S. schenckii. In this report, quantitative real-time RT-PCR revealed 231

that SsDRK1 was more highly expressed in the yeast stage compared with the mycelial stage, which 232

indicated that SsDRK1 may be involved in the dimorphic switch in S. schenckii (Hou et al., 2013). 233

8

Zhang et al. reported other proteins involved in the dimorphism process. The Ste20-related kinases are 234

involved in signaling through the mitogen-activated protein kinase pathways and in morphogenesis 235

through the regulation of cytokinesis and actin-dependent polarized growth (Zhang et al., 2013). The 236

expression of other proteins that may affect biosynthetic and metabolic processes were found by 237

Zhang’s group, such as Cullin-3, CdcH and 4-coumarate-CoA ligase. The expression of these genes 238

was detected predominantly in the yeast form and due to the yeast phase, which is the form of S. 239

schenckii found in the host; the expression of these genes could reflect the ability of the yeast phase to 240

adapt to growth-limiting environments. They also demonstrated an upregulation of the Hsp70 241

chaperone Hsp88 in the development of the yeast phase of S. schenckii at 37°C; however, the precise 242

role of this gene remains unclear (Zhang et al., 2012). 243

Lipids are thought to play important roles in the regulation of the dimorphism and virulence in 244

pathogenic fungi. Generally, the ratio of phospholipid/ergosterol is less than 1 in the yeast form and 2-245

20 in the mycelial form cells in C. albicans and S. schenckii. During the transition from the yeast to 246

mycelial forms, phosphatidylinositol and phosphatidylserine are reduced, 247

whereas phosphatidylcholine increases. Phospho-lipase D is activated during this transition (Kitajma, 248

2000). 249

Phorbol-12-myristate-13-acetate (PMA), a tumor promoting agent and PKC activator, was found to 250

stimulate germ tube formation and germ tube growth by yeast cells induced to undergo a transition to 251

the mycelium form in a concentration-dependent manner. PMA also had a stimulatory effect on DNA 252

and RNA synthesis in cells induced to undergo the yeast to mycelium transition and was found to 253

inhibit cell duplication and bud formation in yeast cells induced to re-enter the budding cycle. In 254

contrast, Polymyxin B, an inhibitor of PKC, inhibited germ tube formation by yeast cells. This 255

inhibition could be overcome if PMA or calcium were added to the medium, suggesting that the 256

inhibition obtained in the presence of this antibiotic was due to the inhibition of PKC. These results 257

support the involvement of PKC in the control of the dimorphic expression in S. schenckii (Colon-258

Colon and Rodriguez-del Valle, 1993). 259

4.2 Genetic polymorphism: Recently, there has been an increasing interest in studying the biology of 260

the S. schenckii complex, and particular attention has been focused on its molecular phylogeny, which 261

has improved our knowledge of the taxonomy, pathogenic characteristics and the epidemiology of this 262

pathogenic fungus. Recent studies have shown important differences in the virulence and drug 263

resistance profiles among different species in the S. schenckii complex. S. brasiliensis is the most 264

virulent species in comparison with S. globosa and S. mexicana, which show little or no virulence in 265

murine models (Fernández-Silva et al., 2012; Fernandes et al., 2013). In addition, the test for 266

susceptibility to antifungal drugs showed that S. schenckii s. str. and S. brasiliensis were highly 267

9

susceptible to most of the antifungals tested in vitro in comparison with the more resistant, S. globosa 268

and S. mexicana species (Marimon et al., 2008). These and other observations suggest that possible 269

genetic differences may be involved. 270

The genomic organization and chromosome number in different species in the S. schenckii complex 271

remain unknown. However, sequencing studies in the calmodulin-encoding gene in the different 272

members of the S. schenkii complex have offered valuable information for elucidating the relationships 273

and differences among these species and their role in pathogenic manifestations (Romeo et al., 2011). 274

A recent study revealed a high diversity in the calmodulin gene (regulated by Ca+2) of S. schenckii. In 275

contrast, S. brasiliensis and S. globosa appeared to be more homogenous, with a low genetic diversity. 276

The electrophoretic karyotype profiles of S. brasiliensis isolates showed less variability than those 277

observed in S. schenckii sensu strictus isolates (Sasaki et al., 2014). These results were consistent with 278

the phylogenetic data, where the variability among isolates within the species was less frequent in S. 279

brasiliensis than in S. schenckii sensu strictus (Marimon et al., 2006; 2007; Rodrigues et al., 2013). 280

Future studies are necessary to determine the role of different genes from the species of the S. 281

schenckii complex in the virulence, drug resistance and environmental resistance. Changes in the 282

expression of such genes might be beneficial to S. schenckii survival from the natural environment to 283

within a host during infection. Comparative genomics and transcriptomics analysis between highly 284

pathogenic Sporothrix species and species with reduced or absent pathogenicity certainly will 285

accelerate the discovery of new proteins for diagnostics, drug targets and vaccines (Romeo and Criseo, 286

2013). 287

4.3 Cell wall: The fungal wall protects the cell from drastic changes in the external environment; it is 288

the first point of contact with the host (Mora-Montes et al., 2009). The S. schenckii cell wall is 289

composed of glucans, galactomannans, rhamnomannans, chitin, glycoproteins, glycolipids and melanin 290

(Travassos et al., 1977; Travassos and Lloyd 1980; Lopes-Bezerra et al., 2006; López-Romero et al., 291

2011). Despite are necessary more studies to know their detailed structure, it seems to be that a proper 292

wall cell composition is required for supporting external stress and for virulence (Madrid et al., 2010; 293

López-Esparza et al., 2013). 294

4.4 Melanin: An interesting mechanism to resist environmental adverse conditions is the production of 295

melanin, also implicated in the pathogenesis of several important human fungal pathogens. Several 296

types of melanin have been described in the fungal kingdom but the majority is derived from 1.8-297

dihydroxynaphthalene (DHN) and is known as DHN-melanin (Helene et al., 2012). Melanin has been 298

referred to as 'fungal armor,' due to the ability of the polymer to protect microorganisms against a 299

broad range of toxic insults, warranting the survival of fungi in the environment and during infection 300

(Gómez and Nosanchuk, 2003). Melanization reduces the susceptibility to enzymatic degradation, the 301

10

toxicity from heavy metals, ultraviolet and nuclear radiation, extremes of temperature, oxygen and 302

nitrogen free radicals, which may afford the fungus protection against similar insults in 303

the environment (Wang and Casadevall 1994 a, b; Rosas and Casadewall 1997, 2001, Taborda et al., 304

2008, Gessler et al., 2014). Melanin also reduces the susceptibilities of pathogenic fungi to different 305

antifungal drugs (van Duin et al., 2003; Nosanchuk and Casadevall 2006) and to different immune 306

mechanisms in the host, especially phagocytosis (Steenbergen et al., 2004). 307

S. schenckii produces melanin or melanin-like compounds in vitro. While melanin is an important 308

virulence factor in other pathogenic fungi, this pigment also has a similar role in the pathogenesis of 309

sporotrichosis (Morris-Jones et al., 2003). Melanized cells of the wild-type S. schenckii and the albino 310

grown on scytalone-amended medium were less susceptible to killing by chemically generated 311

oxygen- and nitrogen-derived radicals and by UV light than were the conidia of mutant strains. Wild 312

type melanized conidia and the scytalone-treated albino were also more resistant to phagocytosis and 313

killing by human monocytes and murine macrophages than were the unmelanized conidia of two 314

mutants (Romero-Martinez et al., 2000). Similar to C. neoformans and P. brasiliensis, S. schenckii can 315

utilize phenolic compounds to augment melanin production, which may be associated with a 316

concomitant increase in the protection against unfavorable conditions in both the environment and 317

during infection (Almeida-Paes et al., 2009). 318

4.5 The expression of adhesins: The adhesion ability of a fungus is important in the colonization of 319

different environmental extracts and in the host is essential for colonization and the establishment of 320

infection. Specific microbial adhesins mediate adherence to host tissues by participating in 321

sophisticated interactions with some of the host proteins that compose the extracellular matrix 322

(Teixeira et al., 2013). The adhesion-encoding genes are not constitutively expressed. Instead, 323

adhesion is under tight transcriptional control by several interacting regulatory pathways. The switch 324

from non-adherence to adherence may allow yeasts to adapt to stress (Verstrepen and Klis, 2006). The 325

adhesion genes are activated by diverse environmental triggers such as carbon and/or nitrogen 326

starvation, changes in pH or ethanol levels (Verstrepen et al., 2003; Sampermans et al., 2005). Fungi 327

need to adhere to the appropriate host tissues to establish an infection site. Apart from being a stress-328

defense mechanism, adhesion is also crucial for fungal pathogenesis: (Verstrepen and Klis, 2006). 329

The outermost layer of the cell wall of S. schenckii has molecules involved in the adhesion of the 330

fungus to host tissues and has a central role in host-pathogen interactions, mediating several 331

interactions associated with the pathogenesis of this microorganism processes. It has been reported that 332

S. schenckii adhered to fibronectin and laminin in soluble and immobilized forms (Lima et al., 1999, 333

2001, 2004) and that this is a key step for the transposition of the endothelial barrier (Figueiredo et al., 334

2007). Independently, Ruiz-Baca et al. (2008) and Nascimento characterized a 70 kDa glycoprotein 335

11

(gp70) on the cell wall of S. schenckii that mediates the adhesion of the fungus to the dermal and 336

subendothelial matrix. This gp70 and another protein with a slightly lower molecular mass (67 kDa) 337

were detected from different isolates. This variation might be related to differences in glycosylation 338

(Teixeira et al., 2009). Interestingly, sera obtained from patients with sporotrichosis reacted mainly 339

with the 40 and 70 kDa antigens (Scott and Muchmore, 1989; Lopes-Alves et al., 1994). In addition, 340

hyperimmune sera of mice infected with S. schenckii reacted with gp 70 (Birth and Adams, 2005), and 341

monoclonal antibodies specific for gp70 were protective against experimental infection (Nascimento et 342

al., 2008). Moreover, recently the Lopes-Becerra´s group reported that reduced level of gp70 343

expression was found in virulent S. brasiliensis and S schenckii strains, while a high expression of this 344

molecule is associated with a lower virulence profile of the strains. This would be other evidence this 345

antigen induces a protective host response (Castro et al., 2013). 346

In other pathogenic microorganism, Staphylococcus aureus, many molecules involved in adhesion are 347

also involved in different immune evasion mechanisms, not related to the specific process of 348

adhesion (Zecconi and Scali, 2013). However, in the case of the S. schenckii complex, the role of their 349

adhesins as immune evasion factors is unknown. 350

4.6 Enzyme production: The Pires de Camargo group studied the different enzymatic activities 351

related to fungal virulence of 151 Brazilian S. schenckii isolates from 5 different geographic regions of 352

Brazil. All (100%) of the S. schenckii isolates presented urease and DNase activities. Only three 353

(15.78%) isolates (one from the North and two from the Southeast region) showed gelatinase activity, 354

and five (26.31%) isolates (one from the North, three from the Northeast, and one from the Southeast 355

region) showed proteinase activities of 0.68, 0.88, 0.85, 0.55, and 0.78, respectively. Additionally, 356

only four (21.05%) isolates (one from the North, one from the Central West, and two from the 357

Southeast region) showed caseinase activities of 0.75, 0.87, 0.89, and 0.87, respectively (Ferreira et al., 358

2009). Another report of isolates from Venezuela confirmed the urease activity (Mendoza et al., 2005), 359

while another study in India reported that all of the mycelial forms of S. schenckii could split urea 360

(Ghosh et al., 2002). 361

However, Fernandes et al. from the same group of Pires de Camargo, also found that while the “highly 362

virulent” S. schenckii isolates show a profile of secreted enzymes (proteinase, caseinase, gelatinase, 363

DNase and urease), most of these enzymes were not observed in the hypervirulent species S. 364

brasiliensis (Fernandes et al., 2009). This observation indicates that the mechanisms promoting 365

pathogenesis are much more complex, perhaps not conserved among closely related Sporothrix species 366

and most likely involve different virulence factors to evade the host immune system (Romeo and 367

Criseo, 2013). 368

12

An interesting enzyme present in S. schenkii complex is the nitroreductase, a member of a group of 369

enzymes that reduces the wide range of nitroaromatic compounds and has potential industrial 370

applications (Stopiglia et al., 2013). Nitroreductase activity has been detected in a diverse range of 371

bacteria and in yeast (Lee et al., 2008; Xu et al., 2007; Aviv et al., 2014). A recent study in an 372

emergent Salmonella enterica serovar Infantis strain, demonstrated that the fixation of adaptive 373

mutations in the DNA gyrase (gyrA) and nitroreductase (nfsA) genes, conferred resistance to 374

quinolones and nitrofurans and contributes to stress tolerance and pathogenicity of this bacteria (Aviv 375

et al., 2014). The possible role of the nitroreductase in the S. schenckii tolerance to environmental 376

adverse conditions and in the host, the resistance to oxidative stress, to antifungal drug and other 377

aspects involved in the pathogeny are matters needing to be studied. 378

Additionally, the recent finding in S. schenkii of intracellular molecules and signals associated with the 379

heterotrimeric G protein alpha subunit SSG-1, involved in the response to different external adverse 380

conditions, help to explain how this fungus is able to survive under external stress (environmental and 381

inside the host) (Valentín-Berríos et al., 2009; Pérez-Sánchez et al., 2010). 382

5. Infections of an alternative host 383

Sporothrichosis is a human mycosis; however, ultimately there is an alarming increase in the 384

frequency of infections in domestic animals, principally in cats, which has led to an increase in the 385

relevant importance from an epidemiological perspective (Smilack; Reed et al., 1993; De Lima et al., 386

2001; Barros et al., 2008; Lloret et al., 2013). Its ease of transmission to cohabitant humans and other 387

animals, especially by bites and scratches, makes it a significant zoonosis (Read et al., 1982; Nusbaum 388

et al., 1983; Dunstan et al., 1986). Cats develop disseminated skin ulcers, with often fatal infection 389

(Lloret et al., 2013). The fungus is spread from an ulcer or from bites and scratches. The propensity of 390

cats to transmit the infection to humans may be attributable to the large number of organisms 391

associated with the lesions in most infected cats (Yegneswaran et al., 2009). 392

However, there are reports of the presence of S. schenckii in samples from the nails of apparently 393

healthy domestic cats (Schubach et al., 2001, 2002). The cat habit of digging holes and covering their 394

excrements with soil and sand, and sharpening its nails on trees, woods or trunks, are most likely 395

responsible for the carriage of the fungus on its nails and claws, even as healthy carriers (Carrada-396

Bravo and Olvera Macías 2013). These healthy carriers can be important in the dissemination of the 397

fungus and the occurrence of sporothrichosis cases when the conditions are favorable. Human 398

infections have also been associated with insect stings, fish handling, and the bites or scratches from 399

birds, dogs, squirrels, horses, reptiles, parrots and rodents, even though clinical symptoms may not be 400

present in these animals (Werner and Werner, 1994; Kauffman, 1999; Liu and Lin, 2001; Ranjana et 401

al., 2001; Haddad et al., 2002; Barros et al., 2011; Carrada-Bravo and Olvera-Macías, 2013). 402

13

403

6. The ability to escape from host immune mechanisms 404

Members of the S. schenckii complex are able to produce localized infections in immunocompetent 405

hosts. They have developed different mechanisms permitting the escape from host immunological 406

mechanisms. 407

6.1 Phagocytosis inhibition: It has long been reported that phagocytosis is an important defense 408

mechanisms against S. schenckii (Cunningham et al., 1979; Oda et al., 1982). The recognition of the 409

fungus is mediated by molecules such as Toll-like receptors (TLRs): TLR2 (Negrini et al., 2013; 410

2014), TLR4 (Sassa et al., 2012) and carbohydrate receptors (Guzman-Beltran et al., 2012). Sialic acid 411

residues are expressed at the cell surface of S. schenckii (Alviano et al., 1982). These residues protect 412

unopsonized fungal cells from phagocytosis by resident mouse peritoneal macrophages. The enzymatic 413

removal of sialic acids from the external layers of S. schenckii envelopes renders yeast cells more 414

susceptible to phagocytosis (Oda et al., 1982; Alviano et al., 1999). Other in vitro studies have shown 415

that galactomanan, ramnomanan and a lipid extract purified from the fungal cell wall are able to inhibit 416

the phagocytosis of S. schenckii yeast by peritoneal macrophages (Oda et al., 1983; Carlos et al., 417

2003). 418

Several processes associated with phagocytosis, such as the cytotoxicity mediated by reactive oxygen 419

and nitrogen species (ROS and NO), are involved in the destruction of invading organisms, and their 420

virulence may be related to a differential susceptibility to these mediators (Fernandez et al., 2000; 421

Carlos et al., 2009; Sassa et al., 2012; Xiao-Hui et al., 2008). A study demonstrated that the S. 422

schenckii catalase is involved in the defense against the ROS induced by environmental changes in 423

vitro, and they proposed that catalase, as a main component of antioxidant defense, may contribute to 424

the survival of S. schenckii during infection. Moreover, it is known that ergosterol is the major sterol 425

observed in fungal membranes (Weete, 1989), while ergosterol peroxide, the putative product of the 426

H2O2 dependent enzymatic oxidation of ergosterol (Bates et al., 1976), is a constituent of the 427

membrane of S. schenckii, which could inactivate or scavenge toxic oxygen products that are formed 428

in phagocytic cells (Basaga, 1990; Sgarbi et al., 1997). It is conceivable that in S. schenckii, ergosterol 429

peroxide is formed as a protective mechanism to evade reactive oxygen species during phagocytosis 430

(Sgarbi et al., 1997). 431

Carlos et al. studied the effect of three different components of the S. schenckii wall cell (a lipid 432

extract, exoantigens and the alkali-insoluble fraction) and observed a drastic inhibition of the 433

phagocytosis of yeast in murine peritoneal macrophages previously treated with a lipid extract. This 434

inhibitory effect was associated with a high production of Tumor Necrosis Factor alpha (TNF-α) and 435

Nitric oxide (NO) (Carlos et al., 2003). Studies performed in our laboratory demonstrated a high 436

14

fungal burden in a murine model of S. schenckii infection during the 2nd and 4th after a previous high 437

production of NO (unpublished data). Similarly, Niedbala et al. reported that NO induces a population 438

of CD4(+)CD25(+)Foxp3(-) regulatory T cells (NO-Tregs) that suppress the functions of 439

CD4(+)CD25(-) effector T cells in vitro and in vivo (Niedbala et al., 2007; 2011). The helper 17 440

(Th17) cells are important in antifungal mechanisms (Hernández-Santos and Gaffen, 2012). 441

Interestingly, recently it was discovered that NO-Tregs suppressed Th17 but not Th1 cell 442

differentiation and function, suggesting a differential suppressive function between NO-Tregs and 443

natural Tregs (nTregs) (Niedbala et al., 2013). In addition, severe infection promotes the release of 444

proinflammatory mediators, including TNF-α and NO, with the subsequent induction of molecules, 445

such as IL-10, Fas L and CTLA-4, which lead to the suppression of the T cell response (Fernandes et 446

al., 2008). 447

6.2 Production of proteases: Many species of human pathogenic fungi secrete proteases. Fungal 448

proteases have been intensively investigated as potential virulence factors. It is evident that secreted 449

proteases are important for the virulence of dermatophytes because these fungi grow exclusively in the 450

stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources (Monod et al., 451

2002). The proteases secreted by C. albicans are involved in the adherence process and penetration of 452

tissues and in interactions with the immune system, thereby stimulating inflammatory process in the 453

infected host. (Wu 2013, Pietrella, 2013). S. schenckii produces proteases that are able to cleave 454

different subclasses of human IgG, suggesting a sequential production of antigens and molecules that 455

could interact and interfere with the immune response of the host (da Rosa et al., 2009). Other effects 456

of proteases secreted by S schenckii are being investigated. 457

6.3 Transitory early immunosuppression: Different reports derived from mouse models of 458

infections with S. schenkii reveal a transitory early immunosuppression, favoring a fungal burden. 459

Studies performed by Carlos et al. demonstrate the production of interleukin-1 (IL-1) and TNF by 460

adherent peritoneal cells from BALB/c mice, which was measured at week 2, 4, 6, 8 and 10 after 461

intravenous inoculation with 106 S. schenckii yeasts. Compared with age-matched controls, IL-1 and 462

TNF production by adherent peritoneal cells from S. schenckii-infected mice was reduced severely at 463

week 4 and 6 of infection and was greater than normal at weeks 8 and 10. Moreover, between week 4 464

and 6 of infection, there was a depression of the delayed type hypersensitivity response to a specific 465

whole soluble antigen and an increase in fungal multiplication in the livers and spleens of the infected 466

mice. Thus, the deficits of cell-mediated immunity in the mice with systemic S. schenckii infection 467

may derive, in part, from an impaired amplification of the immune response due to the abnormal 468

generation of IL-1 and TNF (Carlos et al., 1994). Other reports have demonstrated that, although nitric 469

oxide NO is an essential mediator in the in vitro killing of S. schenckii by macrophages, the activation 470

15

of the NO system in vivo contributes to the immunosuppression and cytokine balance during the early 471

phases of infection with S. schenckii (Fernandes et al., 2008). 472

6.4 The liberation of exo-antigens: A peptide-polysaccharide released from the fungal cell wall, 473

called exoantigen (ExoAg), has been identified as an important virulence factor for sporotrichosis 474

(Nascimento et al., 2008; Teixeira et al., 2009). The main immunogenic proteins of the cell wall (gp 70 475

and gp 40) are present in the ExoAg, and it is thought that the secretion of this outer component can 476

distract several immune mechanisms and serve as a fungal evasion mechanism (Carlos et al., 2009). 477

This hypothesis needs to be confirmed. 478

6.5 Opportunistic infections in immunodeficient hosts: Infection with S. schenckii causes a 479

localized lymphocutaneous disease in the immunocompetent host, while it frequently results in 480

disseminated disease in the immunocompromised patient. Disseminated sporotrichosis occurs in 481

individuals with impaired cellular immunity, such as in cases of neoplasia, transplantation, diabetes, 482

and especially acquired immunodeficiency syndrome (AIDS) (Wroblewska et al., 2005; Silva-Vergara 483

et al., 2012; Freitas et al., 2012). The extracutaneous forms of sporotrichosis without skin 484

manifestations and no previous history of traumatic injuries have been described in renal transplant 485

recipients not treated with antifungal prophylaxis (Gewehr et al., 2013). Additionally, this disease has 486

been described in a patient with X-linked chronic granulomatous disease (CGD) (Trotter et al., 2014). 487

Interestingly, more than 20 cases of sporotrichosis meningitis have been reported, several of these 488

were associated with immunosuppression (Silva-Vergara et al., 2005; Vilela et al., 2007; Galhardo et 489

al., 2010). 490

4. Concluding remarks and future perspectives 491

The vast majority of human pathogenic fungi are environmental fungi, which normally live outside the 492

human body and can cause infections in certain susceptible hosts. These fungi have most likely gained 493

their pathogenic potential in environmental niches, which resemble certain aspects of the host body. In 494

these environmental niches, fungi acquire the capacity to adhere to surfaces, form biofilms, compete 495

with bacteria, acquire all necessary nutrients and deal with changes in temperature, UV radiations, pH, 496

osmolarity, environmental chemical contaminants, meteorological factors and other physical, chemical 497

and biological stress factors. In addition, these fungi may be challenged by amoebae, which share 498

many characteristics with human phagocytes (Davis et al., 1991). Thus, in nature or in animal hosts, 499

fungal cells must respond efficiently to changing environmental conditions in order to survive (Pérez-500

Sánchez et al., 2010). 501

The S. schenkii complex uses signal transduction pathways to sense the environment and to adapt 502

quickly to changing conditions (Rodriguez-Caban et al., 2011). Despite these findings, little is known 503

about the genetic mechanisms of virulence and drug resistance in the S. schenckii complex. The 504

16

ecological niches occupied by the S. schenckii complex are extraordinarily complex and variable and 505

most likely include biologic elements such as other fungi, viruses, bacteria, animals, protists, algae and 506

plants; together with physical (e.g., temperature, humidity, radiations) and chemical components (pH, 507

metals, hydrocarbons, pesticides, etc.). 508

The chronic effect of some of these components including heavy metals, UV radiations and pesticides 509

with immunotoxic effects in potential hosts, such as cats and other animals and outdoor workers such 510

as farmers and gardeners, can contribute to the occurrence of fungal infection. The understanding of 511

the interactions between fungi and their potential hosts in the environment is in its infancy; however, 512

initial observations suggest that this will be an extremely rich area of investigation for exploring the 513

fundamental questions of fungal pathogenesis (Casadewall et al., 2003; Prenafeta-Boldu et al., 2006). 514

This knowledge will contribute to the design of new strategies for the control of sporothrichosis. 515

516

Competing Interests: The authors declare that no competing interests exist. 517

518

519

520

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170. Xu J, Yang Q, Qian X, Samuelsson J, Janson JC. 2007. Assessment of 4-nitro-1,8-naphthalic 931

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171. Yazdanparast SA; Dargahi H; Shahrokhi S; Horabad RF. 2013. Isolation and Investigation of 934

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29

172. Yegneswaran PP; Sripath H; Bairy I; Lonikar V; Rao R; Prabhu S. 2009. Zoonotic 937

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report of a first case in southern Karnataka, India. Int J Dermatol, 48, 1198–1200. 939

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Med. 31(6):1343-8. 948

949

Table 1. Some attributes of S. schenckii complex with demonstrated or putative effects in the protection 950

against environmental stressors and as virulence factor in the host (dual use) 951

952

953

954

955

956

957

958

959

960

961

962

963

964

965

966

967

968

969

970

971

972

973

974

975

976

Attribute

Function

In the environment In the host Selected references

Cell wall Protects the cell from drastic changes in

the external environment

Protects the cell from

aggressive conditions in the

host tissue

Oda et al., ., 1983; Carlos et

al., 2003; Madrid et al., .

2010; López-Esparza et al.,

2013

Melanin Ultraviolet shielding, extreme

temperature protection, reduced

susceptibility to enzymatic degradation

Resistant to phagocytosis and

oxidative killing by phagocytic

cells. Antifungal drug

resistance

Almeida-Paes et al., 2009

Romero-Martinez et al.,

2000

Morris-Jones et al., 2003

Ergosterol Protection against oxidative killing by

soil amoebas?

Protection against oxidative

killing by phagocytic cells

Sgarbi et al., 1997

Dimorphism Mycelium morphology in its saprophytic

phase

Yeast morphology in host

tissues at 35-37°C

Nemecek et al., 2006;

Gauthier et al., 2008

Genetic polymorphism Generation of strain diversity to survive

to environmental stress?

Antifungal drug resistance.

Different virulence profile

Romeo and Criseo 2013

Adhesins In other fungi, adhesion genes are

activated by diverse environmental

triggers like carbon and/or nitrogen

starvation, changes in pH or ethanol

levels.

Adhesion to the dermal and

subendothelial matrix,

transposition of the

endothelial barrier,

Immunomodulators

Verstrepen et al., 2003;

Sampermans et al., 2005;

Figueiredo et al., .,2007;

Lima et al., . ,1999, 2001,

2004; Ruiz-Baca et al., .

2008; Nascimento et al., . ,

2008; Teixeira et al., 2013

Proteinase Nutritional function Tissue damage; degradation of

antibodies

De Rosa et al., 2009; Monod

et al., 2002

Catalase Protection against ROS of soil amoebas? Protection against ROS of host

phagocytes

Davis et al., 1991; Xiao-Hui

et al., 2008;

Superoxide dismutase Protection against oxygen derived

oxidants?

Intracellular growth Pérez-Sánchez et al., 2010

Nitroreductase Tolerance to environmental

contaminants?

Resistance to NO in

phagocytes?

Stopiglia et al., 2013

Aviv et al., 2014

Siderophores Iron uptake in the environmental Iron uptake inside the host Pérez-Sánchez et al., 2010

SSG-1 Survival under conditions of stress and nutrient limitation inside the

human host or the environment .

Pérez-Sánchez et al., 2010

30

977

978

979

980

981

982

983

Fig 1. Environmental stressor promotes Sporothrix schenckii virulence.The origin of virulence in S. 984

schenckii should be related to interactions of the pathogen with different environmental challengers 985

present in their natural habitat. These adaptive changes permit to acquire survival capacity tending to a 986

higher virulence. * Dimorphism, Genetic polymorphism, Cell wall, Melanin, Expression of adhesins, 987

Enzyme production. 988

Extreme temperatures, pH, osmotic pressure and humidity. radiations, chemicals.

soil amoebas.

Environment Host tissue

Adhesion, resistant to phagocytosis and killing by phagocytic cells, tissue damage, degradation of antibodies, iron uptake, immunosupresion, antifungal drug resistance

Sporothrix schenckii complex (Constitutive and inducible

virulence factors)

Melanin Cell wall Ergosterol

Dimorphism

Genetic polymorphism

Adhesins

Proteinases

Catalase

Superoxide

dismutase

Siderophore

s

SSG-1