R e vie w

Future Microbiology

Review

Sporothrix schencki complex and sporotrichosis, an emerging health problem Everardo López Romero1, María del Rocío Reyes-Montes2, Armando PérezTorres2, Estela Ruiz Baca3, Julio C Villagómez-Castro1, Héctor M Mora-Montes1, Arturo Flores-Carreón1 & Conchita Toriello†2 Universidad de Guanajuato, Guanajuato, México; 2Universidad Nacional Autónoma de México, Mexico City, México; 3Universidad Juárez del Estado de Durango, Durango, México † Author for correspondence: Tel.: +52 555 623 2461 n [email protected] 1

Sporothrix, a human pathogen with complex biology & ecology

rP

ro

of

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix Mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers such as a 70 -kDa glycoprotein are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O -linked glycan structure known in S. schenckii. It contains an a-mannobiose core followed by one a-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzyme’s catalytic domain in S. schenckii.

10.2217/FMB.10.157 © 2011 Future Medicine

Future Microbiol. (2011) 6(1), xxx–xxx

A

ut

ho

Sporothrix is a genus of ubiquitous fungi including not only common soil saprobes but also human, insect and plant parasites. This anamorphic (asexual state) genus is found in an identical biotope as the ascomycete Ophiostoma (formerly Ceratocystis), which shows similar morphological and physiological traits, and both fungi are widely distributed in nature. Because of the indistinguishable conidial morphology of some Ophiostoma species with Sporothrix schenckii Hektoen and Perkins [1] , as well as the synthesis of rhamnomannans as the main surface antigens of both fungi, Mariat [2] proposed Ophiostoma stenoceras (formerly Ceratocystis stenoceras) as the sexual (teleomorph state) of S. schenckii. However, analysis of mDNA restriction profiles [3] , macro-restriction patterns with two enzymes [4] and DNA sequences of internal transcribed spacer (ITS) regions of the ribosomal RNA operon [5] confirmed the separation of the two fungi. At present, a teleomorphic (sexual state) S schenckii has not yet been described; however, an overview of the systematics of the Sordariomycetes based on a four-nuclear-locus (nSSU and nLSU rDNA, TEF and RPB2)

phylogeny [6] suggests that S. schenckii is an anamorphic Ophiostoma (Ophiostomataceae, Oph iostomat a le s, Sord a riomyc et id ae, Sordariomycetes, Ascomycota, Fungi [201] ). Significant genetic variability has been shown in S. schenckii by molecular research [4–9] , suggesting that this fungus is not a single species but a complex of numerous cryptic species. Molecular analysis showed that Sporothrix albicans, Sporothrix inflata and Sporothrix luriei are different from S. schenckii [10,11] . Phenetic and genetic approaches proposed three new species, Sporothrix brasiliensis, Sporothrix globosa and Sporothrix mexicana [10] . All these species within the S. schenckii complex are of medical interest. The Sporothrix spp. names mentioned in the original publications will be used in the following sections. Members of the S. schenckii species complex are dimorphic fungi. The mycelial morphotype in nature penetrates the human host through skin abrasions produced by fungal-contaminated plants or animals (or, more rarely, is inhaled) and converts to the yeast morphotype. Mycelia show hyaline, regularly septated, thin hyphae, 1–2 µm in diameter, and oval, pyriform, or elongated conidia, 1.5–3 to 3–6 µm, which are

Keywords n adhesin n emerging mycosis n glucosamine-6-P synthase n protein glycosylation n Sporothrix schenckii n sporotrichosis

part of

ISSN 1746-0913

1

Review

López Romero, del Rocío Reyes-Montes, Pérez-Torres et al.

production is variable among different strains of the fungus and melanized S. schenckii isolates appear to cause infection more easily than those producing low amounts of this pigment [19–21] . The cell wall composition and polysaccharides of S. schenckii have been widely reviewed previously [22–25] , showing mannose, glucose and rhamnose constituents; this last sugar is very rare in other human pathogens. Rhamnomannans with monorhamnosyl side chains predominate in conidia and yeast cells, whereas dirhamnosyl side chains predominate in hyphae [22] . Peptidorhamnomannans isolated from different culture media and by diverse methods are used in various immunological diagnostic tests and epidemiological studies. In particular, sporotrichin, a culture filtrate-derived peptide polysaccharide [26,27] is especially efficient for detecting delayedtype hypersensitivity and is widely used in epidemiological research. Glycosylation of these molecules is discussed below.

A

ut

ho

rP

ro

of

born singly or in groups produced sympodially on denticulate conidiogenous cells (sympodial conidia). Another type of conidia produced are thick-walled, dark brown and are usually borne individually on short denticles along the sides of hyphae [12] . These conidia are regarded as sessile conidia and they show different shapes among Sporothrix species of clinical relevance [10] . The yeast morphotype is readily obtained on different culture media at 37 or 25°C on a basal medium with glucose, neutral pH (7.2) and aeration [13,14] . Yeast cells are fusiform and ovoid of 2.5–5 to 3.5–6.5 µm, with single, double or multiple budding. Yeast forms may originate from the sides and tips of hyphae near septae. Although rarely observed in tissue, yeast cells form ‘asteroid bodies’ that were described 100 years ago as a central yeast cell surrounded by an extracellular eosinophilic material forming spicules [15] . Yeast cells are also observed in human and experimental host tissues as elongated cells of different forms, generally described as ‘cigar bodies’. This fungus is able to grow over a wide pH range, the mycelial morphotype from 3.0 to 12.5 and the yeast morphotype from 2.4 to 9.5. Urease activity was only observed in the mycelial morphotype [16] . In general, the mycelium morphotype of Sporothrix spp. grows well at 25–28°C on mycological media but fails to grow at 40°C. The key phenotypic features for recognizing the species of Sporothrix spp. are the morphology of the sessile pigmented conidia, growth at 30, 35 and 37°C, and the assimilation of sucrose, raffinose and ribitol [10] . Melanin production of S. schenckii has been described in conidial [17] and yeast cells [18] . The fungus’ ability to produce melanin over a broad pH range is advantageous for its survival in environmental and pathogenic conditions [19] . S. schenckii produces melanin via the 1,8-dihydroxynaphthalene (DHN) pentaketide pathway in conidia, but not in hyphae [17] . However, yeast cells can also produce melanin in vitro and during mammalian infection [18] . In recent work, Almeida-Paes et al. showed melanin particles derived from hyphae grown in l-DOPA medium, providing evidence in support of l-DOPA melanin synthesis distinct from the DHN process described in this fungus [19] . Furthermore, in another study, S. schenckii yeast cells cultivated in a high brain and heart infusion culture medium were highly virulent, revealing a high expression of melanin on the fungal cell wall, thus supporting the pathway of l-DOPA melanin biosynthesis that involves the use of phenolic compounds from rich brain medium as melanin substrate [20] . Melanin

2

Future Microbiol. (2011) 6(1)

Sporotrichosis, an emerging health problem

S. schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis [28,29] , which is an acute or chronic subcutaneous mycosis of humans and other mammals. Sporotrichosis can be acquired by a traumatic contact with live or decayed vegetation, any traumatism with a fungus-contaminated object or organic material, through insect bites (rarely), and recently, even through cat contact or scratches [30] . On rare occasions it might be acquired by spore inhalation. In recent times, sporotrichosis may develop as a very serious disseminated disease with visceral and osteoarticular involvement in immunocompromised individuals, particularly people with AIDS [31] . Sporotrichosis has been considered to be occupational due to the fact that infected patients are generally workers closely related to vegetation exposure where the causal fungi have their ecological niche [29,32,33] . One of the most famous sporotrichosis epidemics occurred in 3000 gold miners from South Africa infected by timbers on which the fungus was growing [28] . Recently, the largest epidemic of this mycosis due to zoonotic transmission was described in Rio de Janeiro, Brazil [30,34–37] . Between 1998 and 2004, 1503 cats, 64 dogs and 759 humans were diagnosed by isolation of S. schenckii in culture [30,35,36] . As a rule, feline disease preceded human and canine diseases, and the individuals most frequently affected included housewives taking care of future science group

Sporothrix schencki complex & sporotrichosis, an emerging health problem

A

ro

of

order to clearly define the S. schenckii complex and the specific ecology and epidemiology of this complicated fungal taxa. Described for the first time by Schenck in the USA in 1898, sporotrichosis is well known throughout the world, although it is more frequent in tropical and subtropical climates of the American continent, Asian countries and Australia, while it is rare in Europe. Sporotrichosis is characterized by a wide range of cutaneous and systemic clinical manifestations. Localized cutaneous (Figure 1A) and subcutaneous are the most common forms of sporotrichosis, and it is characterized by ulcerative lesions, associated regional lymphangitis and lymphadenopathy. A distinctive clinical finding is the presence of hard, spherical nodules along lymphatic vessels (Figure 1B) . Extracutaneous sporotrichosis can involve the lung, joints, bones and other organs. The systemic form of sporotrichosis is usually associated with immunocompromised patients [22] . Interestingly, in these

ho

rP

cats with sporotrichosis [30] . Epidemiologically, the isolation of the fungus from nails and oral cavity of cats indicates that transmission can occur through a scratch or a bite, whereas isolation from the nasal fossae and cutaneous lesions suggests the possibility of infection through secretions [34,36,37] . The zoonotic epidemic in Rio de Janeiro arose as an emerging zoonosis based on the unusual human disease and was probably secondary to a traumatic exposure to cats infected by S. schenckii [38] . More recently, a small cluster of human sporotrichosis cases occurred in the southwest of western Australia, where sporadic cases had been identified. More than 50% of these cases occurred in the Busselton-Margaret River region, where no cases had previously been recorded. The majority of the cases were linked to hay exposure, while a few involved camping trips or domestic gardening [39] . Although sporotrichosis has been rare in Europe, two new cases were reported in southern Italy in 2007 and 2008 [40,41] . Even when the fungus is isolated from environmental samples, the incidence of the mycosis is low, which leads to questions regarding the relevance of the host in the development of the disease as well as the virulence and species of the different fungal strains found in nature. In previous investigations, S. schenckii strains always showed a great difference in virulence [42,43] remaining unclear whether these differences were attributable to different species of the complex or to different isolates of the same species [44] . Early studies with the newly described species of Sporothrix in an immunocompetent murine model showed that S. brasiliensis was the most virulent species, followed by S. schenckii and then S. globosa, while S. mexicana and S. albicans showed low or no virulence at all in this model [44] . However, although S. globosa was demonstrated to be a low virulent species in mice, a recent report describes a human case of lymphocutaneous sporotrichosis caused by S. globosa in Brazil, this being the first description of this species in that country [45] . The species was identified by microscopic morphology and assimilation of different carbon sources and confirmation was made by sequencing the nuclear calmodulin gene. Interestingly, this isolate grew at 37°C, that is, different from S. globosa when this species was first described [10] and that appears unable to grow at 37°C. It is now imperative to correctly identify Sporothrix species by phenotypic and molecular (sequencing) studies in

Review

A

ut

B

future science group

Figure 1. Clinical aspects of the most common cutaneous infections by Sporothrix schenckii located in upper extremities. Typically, an ulcer nodule of fixed cutaneous sporotrichosis (A) is confined exclusively to the skin. The lymphangitic spread of infection results in lymphocutaneous disease (B) with ‘ascending’ nodular (ulcerated or not) lesions developed along lymphatic vessels (courtesy of A Bonifaz). This type of cutaneous sporotrichosis is associated with lymphadenopathy.

www.futuremedicine.com

3

Review


However, the authors mention ITS-RFLP as a method with a greater discriminatory power than mtDNA-RFLP. In a further study, Ishizaki et al. added other isolates from India, Thailand, Mexico, Guatemala, Colombia and Brazil to the mtDNA-RFLP and ITS-RFLP analyses and found another two new mtDNA types (Type 31 and Type 32) for all S. schenckii isolates [61] . Electrophoretic karyotype analysis of eight S. schenckii isolates from Japan divided them into three types with six to eight chromosomes [62] . Single strand conformation polymorphism (SSCP) with oligonucleotides designed from the region of a putative gene that codes for a membrane transporting protein was used to study 20 S. schenckii isolates previously classified by mtDNA-RFLP [63] . The isolates were divided into three types corresponding to those previously obtained in a karyotype analysis [62] . With another molecular technique, random amplified polymorphic DNA (RAPD-PCR), Lee et al. analyzed ten S. schenckii clinical isolates in conjunction with other fungi [64] . The results demonstrated that each random primer amplified characteristic band patterns from the DNA of eight of the ten isolates, but different band patterns were obtained with the other two isolates, suggesting a different Sporothrix anamorph from Korea. Mesa-Arango et al. analyzed the phenotypic characteristics (conidia size and thermaltolerance) and genotypes (using RAPD-PCR) of isolates from Mexico, Guatemala and Colombia [42] . The isolates from Colombia showed larger conidia and were thermal-sensitive relative to the isolates from Mexico and Guatemala. Great variability was observed among the S. schenckii isolates, which formed four groups according to their geographical origin. No relationship was found among the polymorphic patterns and the clinical forms of the disease, although the isolates from Mexico were separated into two groups: clinical and environmental. Reis et al. tried out a study using RAPD-PCR to identify the genetic relationship between S. schenckii strains from patients and from cats involved in an outbreak of sporotrichosis in Brazil [65] . The polymorphic patterns obtained from the isolates from patients were very similar to patterns obtained from the isolates of cats studied, suggesting a common source and mechanism of infection. With another technique, amplified fragment length polymorphism analysis, Neyra et al. studied the genetic diversity of S. schenckii isolates from Peru and reference strains from other countries [66] . The isolates from Peru were divided into two separate homogeneous groups

ho

rP

ro

of

cases primary skin lesions are scattered or often absent. Reports of sporotrichosis in HIV-positive patients have been increasing in different parts of the world [46–48] , and although sporotrichosis is not the most common fungal infection in immunocompromised patients, it is difficult to treat [47] . Although sporotrichosis as a human disease has a lingering feature, this mycosis can appear in a population for the first time or increase its incidence or geographic range, as was reported in western Australia and Rio de Janeiro, Brazil. Moreover, the sporotrichosis outbreak in Brazil showed expansions in host and vector ranges with potential serious human health impacts that defined this sporotrichosis event as an emerging infectious disease dominated by zoonosis. Several socio–economic (human and animal demography), environmental (climate changes) and ecological (pathogen changes, changes in farming practices and new urban developments) factors could be relevant to the occurrence of more sporotrichosis foci (‘hotspots’) as emerging human and zoonotic diseases, albeit mostly nonlife-threatening localized infections. However, the increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, will define sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular epidemiology & detection

A

ut

Several molecular techniques have been used to check the genetic variability of the S. schenckii complex species. However, only a limited number of these approaches were found to be useful. Among them, restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) and rDNA and gene sequencing have been powerful tools for epidemiological analysis and for intra-specific clustering or typing of S. schenckii isolates (Table 1) . A general overview of the genetic variability of the Sporothrix schenckii complex is provided here. Isolates of S. schenckii from different sources and geographic origins were analyzed by mtDNA RFLP and clustered into two large phylogenetic groups: group A with strains predominantly from South America and Africa, and group B with strains predominantly from Australia and Asia, with 30 mtDNA types altogether [49–59] . Watanabe et al. used rDNA ITS1-RFLP to study the phylogeny among the existing S. schenckii types and then grouped S. schenckii isolates into four rDNA types (rDNA types I–IV), which supported the results of mtDNA typing [60] .

4




Review

Table 1. Summary of the principal methods of molecular typing and detection of Sporothrix spp. Molecular method

Comments

Ref.

RFLP of mtDNA

Sporothrix schenckii isolates from all the world as: 32 mtDNA types with two groups A = South Africa, North, Central and South America B = Japan, Australia, China, Korea S. schenckii not a single species but a complex of numerous cryptic species

[7]

[10,11,67]

[71–75]

ro

sequences of calmodulin, revealing for the first time that in addition to S. schenckii sensu stricto, S. globosa is found in Mexico, Central and South America [67] . DNA fingerprinting with M13 primer sequencing of the ITS-rDNA region was performed in Sporothrix isolates from a sporotrichosis outbreak transmitted by cats in Rio de Janeiro, Brazil [68] . Although nine subtypes were found, they were not associated with specific clinical forms, and ITS sequence analysis suggested a common origin for the outbreak. In the same study, the minimal inhibitory concentration (MIC) values to amphotericin B, itraconazole, posaconazole, ravuconazole and terbinafine were low for the isolates, and no differences were found among isolates from different clinical forms of the disease. These last results differ from another antifungal susceptibility study of five species of Sporothrix [69] , where significant differences were found among these species, with S. brasiliensis showing the best response to antifungals, and S. mexicana the worst response. Further studies concerning antifungal susceptibility for this genus are greatly needed.Sequencing and phylogenetic analysis of the D1–D2 region of the 28S rRNA gene of Sporothrix isolates from garden soil samples and commercial fertilizers, including S. schenckii ATCC 10268 and two clinical strains from Italy, revealed enough differences to justify the separation of the examined isolates into two principal groups (environmental and clinical) [70] . This separation between clinical and environmental isolates correlates with the RAPD-PCR analysis of Sporothrix strains from Mexico [42] and with the sequence analysis of S. schenckii ITS rDNA [5] .

A

ut

ho

rP

with a high level of variability among them and without a correlation with the clinical forms of sporotrichosis. Recently, based on an analysis of the ITS rDNA region, de Beer et al. suggested that more than one species may exist in S. schenckii [5] . Furthermore, using phylogenetic analyses comparing LSU rDNA data at the genus level and using ITS and partial b-tubulin sequences at the species level of isolates from soil and wood together with those of clinical isolates of Sporothrix species in the O. stenoceras–S. schenckii complex, de Meyer et al. demonstrated that the human pathogenic strains form an aggregate of several cryptic species [9] . On the other hand, sequence analysis of three protein-coding loci (chitin synthase, b-tubulin and calmodulin) of S. schenckii isolates from different geographical locations was used to clarify whether the variability of this fungus was due to species divergence or intra-specific diversity. Sequence analysis proposed at least six putative phylogenetic species prevalent in different geographical regions [7] . Another study of 127 S. schenckii isolates with the nuclear calmodulin gene and other phenotypic features differentiated S. albicans, S. inflata and S. schenckii var. luriei from S. schenckii and proposed three new species: S. brasiliensis, S globosa and S. mexicana [10] . A later communication proposed S. schenckii var. luriei as the species S. luriei based on its phenotypic characteristics and a multilocus sequence analysis, which are different from other species in the S. schenckii complex [11] . Furthermore, Madrid et al. studied another 32 clinical and environmental isolates identified morphologically as S. schenckii from Mexico, Guatemala and Colombia through partial gene

[5,9]

of

DNA sequence of ITS rRNA; LSUrDNA, partial calmodulin sequences Sequence analysis of protein coding Six distinct putative phylogenetic species among S. schenckii species complex loci for chitin synthase, b-tubulin, calmodulin Calmodulin sequences Sporothrix species of medical relevance: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix Mexicana and S. schenckii Nested PCR By different DNA probes: 28S rDNA, chitin synthase 1 gene, 18S rRNA, DNA topoisomerase II gene

[49–61]



5

Review


All recent molecular tools have had a direct application on phylogenetic studies showing the former S. schenckii species as a complex of different species. The delineation of new phylogenetic groups and their identification is critical and will have a strong impact on several applied biological areas, such as epidemiology, prevention (vaccine development) and the diagnosis of the disease. Molecular detection

A

ut

ho

rP

ro

of

The definite diagnosis of sporotrichosis is achieved through the isolation of S. schenckii from clinical samples, and in some cases, the diagnosis is supported with immunological procedures. However, both isolation and immunodiagnosis pose limitations, such as cultures of the biopsy specimens frequently yielding negative results and immune cross-reactions with other fungal species causing similar diseases. Among the different methods, PCR is now a viable option for disease diagnosis, especially for immunocompromised patients. A nested PCR assay has been designed for the detection of S. schenckii in clinical samples, which uses an 18S rRNA gene sequence as a blank [71] . The nested PCR showed a sensitivity of 40 fg of S. schenckii DNA, detecting one colony forming unit (CFU) of S. schenckii in tissue samples. Validation of the nested PCR assay in clinical isolates and infected experimental mice showed its high sensitivity and specificity, suggesting that this is a quick diagnostic assay with sufficient precision to be clinically useful for patients with sporotrichosis. This nested PCR identified S. schenckii with all the described types of mtDNA and in isolates of the fungus from different parts of the world [72] . Another probe based on the 28S rRNA gene was for detecting any pathogenic or saprobic fungi during the first PCR, whereas specific S. schenckii oligonucleotides were used for the second PCR to detect the fungus in clinical samples. The probe showed a high level of specificity when tested with related pathogenic fungi and clinical samples [73] . Another specific S. schenckii oligonucleotide was designed based on the chitin synthase 1 gene for detecting S. schenckii in tissue samples, another useful molecular tool for the diagnosis of human and animal sporotrichosis [74] . In recent work, Kanbea et al. developed specific primers for three species based on the nucleotide sequences of the DNA topoisomerase II genes of S. schenckii, S. schenckii var. luriei and O. stenoceras [75] . Three gene-specific primers (SSHF31 and SSHR97 for S. schenckii,

and SSLF64 for S. schenckii var. luriei) were designed and evaluated for their specificities in PCR amplifications. PCR targeting the DNA topoisomerase II gene (SSHF31/SSHR97) specifically and rapidly distinguished S. schenckii from its related species. Although several molecular methods have been described for the detection and identification of S. schenckii, they have not been probed with other recently described clinical species of the S. schenckii complex and, therefore, they are not yet assessed for species differentiation. Other molecular probes for detecting Sporothrix spp. in environmental and clinical samples will probably evolve from future gene sequencing approaches and lead to efficient and quick sporotrichosis diagnosis and correct identification of the new fungal species. Furthermore, the discrimination among clinically relevant species of Sporothrix would be probably important in those cases of disseminated sporotrichosis in immunocompromised patients, because of significant differences in susceptibility and resistance to antifungals. For instance, S. brasiliensis was the species that showed the best response whereas S. mexicana showed the worst response to terbinafine, which was the most active drug, followed by ketoconazole and posaconazole [69] . However, in cases of fixed and lymphocutaneous sporotrichosis, species identification would probably not result in any difference in the therapeutic treatment, although it should nonetheless be useful for epidemiology purposes.

6


Host–pathogen interactions & the role of adhesins in pathogenesis

The cell wall (CW), the outermost part of fungi and other organisms, mediates all host– pathogen interactions and can be a modulating factor affecting the host’s immune response [76] . The most external layer of the CW has an amorphous microfibrillar material involved in adherence to host tissues [77] . Virulence has been defined as the relative capacity of a microbe to cause damage in a host [78] . The central point of the concept is that the relevant outcome of host–microbe interactions is host damage. Disease only becomes apparent when host damage reaches a certain threshold [79] . Defining microbial pathogenesis in the context of host response or host damage permits the inclusion of many variables that affect the host–pathogen interaction. Thus, virulence is a relative property of the pathogen that is modulated by host susceptibility and resistance [78] . future science group


ro

of

inhibit FN binding but have no significant effect on LM binding. These results suggest the presence of adhesins in the S. schenckii cell surface. Since the interaction of S. schenckii yeasts with soluble ECM proteins can facilitate their dissemination to extracutaneous sites, adherence to ECM components has been postulated to be a virulence factor [101,102] . Moreover, yeast cells of S. schenckii efficiently adhere to and invade human umbilical vein endothelial cells both in vitro and in vivo, in a process modulated by IL-1b, TGF-b and divalent cations that could play a role in the dissemination of the fungus from the primary site of cutaneous infection to other organs. Two endothelial cell surface proteins of 90 and 135 kDa could be involved in the interaction with S. schenckii [77] . A 70-kDa glycoprotein (Gp70) from the CW of S. schenckii yeast cells that is regularly distributed along the fungal wall mediates adhesion to ECM components of mouse tail dermis, and it appears to be specific for S. schenckii because it was not immunodetected in other pathogenic or non-pathogenic fungi [103] . Nascimento et al. isolated a glycoprotein of the same molecular weight from S. schenckii with adhesin-like activity for FN and LM and demonstrated that it is involved in adhesion of the fungus to the subendothelial matrix [104] . Sequence analysis of this gp70 molecule showed significant homology with an 89-kDa surface glycoprotein that has been reported to be involved in epithelial cell junctions [105] . Other pathogenic fungi express CW glycoproteins with molecular weights close to 70 kDa that also exhibit adhesin properties. For instance, the CWs of A. fumigatus conidia and C. albicans germ tubes present 72-kDa and 68-kDa proteins (respectively) that show adhesin activity for LM and FN [106,107] . Teixeira et al. demonstrated the presence of the so-called gp70 component in the surface of different isolates of S. schenckii [108] . A slight decrease in the molecular weight (from 70 to 67 kDa) owing to a difference in the glycosylation state of the protein was observed in the less virulent isolates. The most virulent strains showed higher adhesion capabilities and expressed at least four FN binding proteins in addition to gp70, whereas the least virulent strains showed only the 67-kDa protein. In our laboratory, treatment of purified Gp70 with endoglycosidase H reduced its molecular weight to 66 kDa, indicating that approximately 5.7% of the Gp70 mass consists of N-linked oligosaccharides. b-elimination, on the other hand, had no effect on molecular weight [103] . Altogether,

A

ut

ho

rP

The S. schenckii CW consists of alkali-soluble and -insoluble glucans, which are found in yeast and conidial morphotypes [80] . Very few glycoproteins (such as peptidorhamnomannan and peptidorhamnogalactan) have been extracted from yeast-like cells [81,82] . The best characterized glycoprotein is peptidorhamnomannan, which exhibits 14.2% protein, 33.5% rhamnose and 57% mannose [83] . The serum of sporotrichosis patients and the mannose-recognizing lectin Concanavalin A (Con A) reacts with peptidorhamnomannan [83,84] , whose main chains are formed by a-1,6-linked mannosyl units [85] containing a-d-glucuronic acid residues [86] . Furthermore, a-d-glucuronic acid residues mono- and di-substituted by terminal nonreducing rhamnose have been revealed as significant antigenic determinants [84] . It is notable that treatment of the CW rhamnomannan protein complex with mild alkali (b-elimination), in addition to the loss of O-linked oligosaccharides, released a main antigenic peptide of 70 kDa [87] . Results derived from immunoblot analysis have revealed proteins with molecular weights in the range of 22 to 70 kDa, and serum obtained from sporotrichosis patients reacts mainly with the 40- and 70-kDa antigens [84,88] . Moreover, hyperimmune serum from rats infected with S. schenckii reacts with a 70-kDa antigen [89] . Pathogen adherence is crucial for colonization and establishment of infection. The adhesion process has been extensively studied in a great variety of pathogenic fungi such as C. albicans, Aspergillus fumigatus, Histoplasma capsulatum and Paracoccidiodes brasiliensis [90–95] but very little is known of this process in S. schenckii. Entactin, fibronectin (FN), laminin (LM) and types II and IV collagen are some of the extracellular matrix (ECM) targets that recognize adhesins from some pathogenic fungi [96,97] using a variety of mechanisms, such as protein– protein and lectin-like interactions [98,99] . Several CW components have been described that are involved in the adhesion process of pathogenic fungi, such as mannoproteins and integrin-like proteins [98] . Because adhesion to cell surfaces or ECM molecules is the first step to infection and dissemination of pathogens in the host, it is relevant that yeast cells and conidia of S. schenckii can bind to type II collagen, FN and LM; additionally, yeast cells can also bind to fibrinogen, whereas conidia can bind neither fibrinogen nor thrombospondin [100,101] . Some CW carbohydrate-containing fractions from yeast-like cells

Review



7

Review


N-linked glycosylation

Upon translocation into the ER lumen, nascent proteins are recognised by the oligosaccharyl transferase complex, which transfers preassembled Glc3Man9GlcNAc2 oligosaccharides from dolichol-phosphate precursors to asparagine residues contained within the motif Asn-X-Ser/ Thr (where X may be any amino acid except proline) [120] . Soon after protein glycosylation, the N-linked glycan core undergoes processing by ER a-glycosidases: the three glucose units are trimmed by ER a-glucosidases I and II, and one mannose residue is removed by the ER a1,2mannosidase, generating the oligosaccharide Man8GlcNAc2 isomer B [121] . The glycoproteins are then transported to the Golgi complex, and the N-linked glycans are further modified by a set of glycosyl transferases and hydrolases. In lower eukaryotes, such as Saccharomyces cerevisiae and C. albicans, the modifications are exclusively carried out by mannosyl transferases, generating high mannose N-linked glycans, commonly named mannans [119,122,123] . In higher eukaryotes like filamentous fungi, the N-linked glycan core is further processed by Golgi mannosidases and different glycosyl transferases, generating complex hybrid N-glycans, which may contain mannose, fucose, galactose, xylose, glucoronic acid and others [120] . As with other fungi, S. schenckii probably has similar mechanisms for the elaboration of N-linked glycans, yet little is known about this biosynthetic pathway. So far, there is neither biochemical nor genetic information about generation of the N-linked glycan core, but we can infer that it is synthesized by this fungus because ER a-glycosidases with the ability to trim the N-linked glycan core have been described [124] . In addition, our group has isolated SsALG11, the ortholog of S. cerevisiae ALG11 [Mora-Montes H and Flores-Carreón A, Unpublished Data] , which encodes an a1,2-mannosyltransferase involved in Glc3Man9GlcNAc2 synthesis [125,126] , as well as SsCWH41 and SsROT2, the genes encoding ER a1,2-glucosidases I and II, respectively [Mora-Montes H and Flores-Carreón A, Unpublished Data] . The ER a1,2-mannosidase has been purified and characterized from both filamentous and yeast forms of S. schenckii [124] . This 75-kDa membrane-bound enzyme belongs to glycosyl hydrolases family 47. It processes the N-linked glycan core Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 isomer B, and if the incubation reactions are conducted for more than 12 h, further trimming occurs to generate Man7GlcNAc2 isomer B and Man6GlcNAc2 [124] . This N-linked

rP

ro

of

these data indicate that the glycoprotein isolated by Teixeira et al. [108] and by us most likely corresponds to the same adhesin. Although adhesins from the fungal CW have potential pathogenic attributes, they are also highly immunogenic and antibodies to these molecules are produced during fungal infection. Thus, the fungal CW has a dual status: it has properties of both a Trojan horse and an Achilles heel. An example of this assumption is the recent evidence of antibody-mediated passive protection in a murine model of sporotrichosis. This protection was obtained with a monoclonal antibody specific for the 70-kDa S. schenckii cell wall glycoprotein injected before, during, and after experimental infection. Passive protection was evidenced as a significant reduction in the number of CFUs in the organs of infected mice [104] . These findings point to the relevance of the host response to the pathogenic outcome of fungus–host interactions. To gain deeper insight into the mechanisms of fungal adhesion, host responses and pathogenesis, more work will be required to identify additional cell surface antigenic components that can also be used for the diagnosis and treatment of sporotrichosis. Protein glycosylation in Sporothrix

A

ut

ho

Glycosylation of proteins is a posttranslational modification found in virtually all eukaryotic cells. Upon translocation of nascent proteins into the lumen of the endoplasmic reticulum (ER), specialized enzymes catalyze the addition of the C-terminus of polypeptides (GPI anchors) and sugar moieties to asparagine (N-linked glycosylation), serine or threonine residues (O-linked glycosylation). These glycoproteins are then modified further in the Golgi complex and finally transported to different compartments within the cell, where they play essential roles in cell physiology [109] . As mentioned previously, the S. schenckii cell wall is composed of sugar polymers and glycoproteins, which represent approximately 80 and 20% of cell wall dry weight, respectively [80] . The latter play important roles in the ability of S. schenckii to hydrolyze host proteins [110] and in adhesion to host cells and extracellular components [101–103,108] . In addition, glycoproteins are essential for fungal virulence, cell wall integrity and host immune recognition [76,111–119] . Thus, these observations highlight the importance of glycosylation pathways for the fungal cell, and they suggest that studies of these biosynthetic pathways might unveil new targets for antifungal strategies.

8




O -linked glycosylation

A

ut

of

ho

rP

As with N-linked glycosylation, the O-linked glycosylation pathway begins in the ER, where the protein-O-mannosyltransferase (PMT) transfers a mannose unit from the glycolipid dolichol-phosphate-mannose to serine or threonine residues present in nascent proteins [109] . In contrast to N-linked glycosylation, there is no sequence motif for the addition of O-linked sugars. The PMT activity controls the elaboration of O-linked glycans, and it is encoded by a gene family that may contain three to seven members, depending on the organism [117,131] . In pathogenic fungi, such as C. albicans and Cryptococcus neoformans, PMT2 is essential for viability, and disruption of PMT1 or PMT4 led to defects in cell wall assembly, cell morphology and virulence [117,132,133] . The activities of dolichol-phosphate-mannose synthase (the enzyme responsible for the elaboration of dolichol-phosphate-mannose) and PMT have both been characterized in membrane fractions from S. schenckii [134] , but the size of the PMT gene family and its relevance for S. schenckii virulence remain to be established. Upon O-linked mannosylation in the ER, glycoproteins are transported to the Golgi complex, and the O-linked glycans are further elongated by glycosyl transferases. In S. cerevisiae and C. albicans, this action is performed by a-mannosyltransferases encoded by members of the MNT1/KTR1 and MNN1 gene families [122,123] . These enzymes have a significant degree

of similarity among members of the same family, resulting in redundant and promiscuous enzyme activities that participate in more than one glycosylation pathway at the same time [119,122,123] . We are currently characterizing the MNT1/ KTR1 gene family in S. schenckii, and thus far, the ortholog of S. cerevisiae KTR1 (MNT1 in C. albicans) has been isolated [Mora-Montes H &Flores-Carreón A, Unpublished Data] . Because the encoded enzyme participates in both N-linked and O-linked glycosylation in S. cerevisiae [135] , but only in O-linked glycan elaboration in C. albicans [123] , it is difficult to predict a putative role in S. schenckii glycosylation pathways. Heterologous expression and biochemical characterisation of S. schenckii MNT1 is in progress to elucidate its role in the biosynthesis of glycoproteins. Filamentous fungi can elongate O-linked glycans exclusively with mannose residues as do lower eukaryotes; alternatively, they can synthesise complex oligosaccharides containing, among others, galactose, glucose, rhamnose and sulfate groups [136,137] . So far, the main glycan present in peptidorhamnomannan is the only O-linked glycan structure described in S. schenckii [83] . This oligosaccharide contains up to five monosaccharide units, and it is composed of an a-mannobiose core followed by one a-glucuronic acid unit, which may be mono- or di-rhamnosylated [83] . However, the identities of the enzymes involved in the elaboration of this O-linked glycan remain unknown.

ro

glycan core-processing profile is similar to that described for other ER a1,2-mannosidases [127– 129] , and is likely to happen in vivo, where slow processing controls the sorting of glycoproteins undergoing ER-associated degradation [130] . Thus far, we have been unable to demonstrate Golgi a-mannosidase activity in S. schenckii, suggesting that the N-linked glycosylation pathway in this organism is similar to that present in lower eukaryotes, which generates high mannose N-linked glycans. Early works have characterized some of the glycan structures present in rhamnomanan and cell wall [22–24] . However, these studies were performed using glycans extracted with 2% KOH at 100ºC. This hard treatment cleaves both N- and O-glycosidic linkages and, therefore, it is difficult to sort any resolved glycan structure as N-linked or O-linked glycan. Thus, further work has to be done to assign the already solved glycan structures as N-linked or O-linked oligosaccharides.

Review


Glucosamine 6-P-synthase, a potential target for antifungal drugs

Fungal infections are a public health problem that have stimulated changes in modern chemotherapy, due mainly to a significant increase in the number of patients infected with HIV and the emergence of strains exhibiting multi-drug resistance (MDR), which has limited the clinical use of antifungal agents [138] . The presence of Sporothrix strains with different susceptibility to a number of drugs has been described [69,139] , and this seems to correlate with the geographical source of the isolates [140] . These observations have increased the interest in developing new antifungal compounds directed to more specific targets in this and other human-pathogenic fungiAnalysis of the hexosamine biosynthetic pathway (HBP) has become very important in the study of pathogenesis of fungi and protozoa in the mammalian host [141–143] . HBP, which is involved in the mechanisms of eukaryotic tissue protection against damage caused by diabetes, ischemia and www.futuremedicine.com

9

Review


fungi [148] . Notably, despite being an oligomeric enzyme catalyzing a complex two-step reaction, GlcN-6-P synthase activity exhibited typical hyperbolic kinetics with both substrates. Apart from the difference in the oligomeric structure, it is well-documented that the eukaryotic but not the prokaryotic GlcN-6-P synthase is down-regulated by the final product of the Leloir pathway [148] . As expected, the enzyme from S. schenckii is inhibited by UDP-GlcNAc with an IC50 value of 0.12 mM [156] , which is lower than that estimated for the enzymes from C. albicans and S. cerevisiae [150,157] but higher than that obtained in rat [158] and human liver [159] cells. In rat cells, inhibition by UDP-GlcNAc is competitive with respect to Fru-6-P, whereas in fungi it is non-competitive for both substrates [155–158] . It has been described that in Blastocladiella emersonii and other fungi such as Aspergillus nidulans, phosphorylation and dephosphorylation of GlcN-6-P is a major modulator of enzyme sensitivity to UDP-GlcNAc [160,161] . By contrast, phosphorylation–dephosphorylation of GlcN-6-P synthase in C. albicans [157] and S. schenckii [156] does not affect the enzyme’s sensitivity to UDP-GlcNAc. All enzymes involved in the biosynthesis of fungal and bacterial cell walls have been considered as targets for antibiotics because inhibition of their activity or suppression of their respective genes may be lethal [148] . GlcN-6-P synthase from fungi is a potential target for antimycotic treatment because the enzyme exhibits differences with respect to mammalian cells in terms of biochemical characteristics and physiological responses to inhibition [162] . Ultrastructural studies of GlcN-6-P synthase from C. albicans have shown that it consists of two catalytic domains, one at the N-terminus (amino acids 1–345) and another at the C-terminus (amino acids 346–712), and these have functional GAH (glutamine amide hydrolyzing) and ISOM (hexose phosphate-isomerising) activities, respectively. Although the GAH domain is monomeric, the native ISOM domain is tetrameric and contains the binding site for UDP–Glc–NAc. Residues 709–712 function as an intramolecular channel connecting both domains, and it has a molecular gate at the Trp97 residue that functions to open and close the channel [149,163] . Information on the oligomeric structure of GlcN-6-P synthase has allowed significant progress in the development of drugs specifically targeted against catalytic domains of the enzyme.

A

ut

ho

rP

ro

of

trauma [144–146] , can be regarded as a branch of glycolysis where fructose-6-phosphate (Fru-6-P) is first converted into glucosamine-6-phosphate (GlcN-6-P), which is finally transformed into uridine 5-diphospho N-acetylglucosamine (UDP-GlcNAc) by the so-called Leloir route. UDP-GlcNAc, the end product of the pathway, is the activated donor of N-acetylglucosamine for the synthesis of a plethora of macromolecules, such as peptidoglycan; lipopolysaccharides and teichoic acids in bacteria; chitin in fungi, insects and crustaceans; and glycoproteins, glycosaminoglycans and mucopolysaccharides in mammals (Figure 2) [147] . The first and rate-limiting step in the HBP is catalyzed by glucosamine6-phosphate synthase (l-glutamine:d-fructose6-phosphate amidotransferase; EC 2.6.1.16; GlcN-6-P synthase), which irreversibly converts Fru-6-P into GlcN-6-P using glutamine as the ammonia donor. Because of its functional position in hexosamine metabolism and its biochemical characteristics, this enzyme has been proposed as a strategic target in antifungal chemotherapy [148,149] . GlcN-6-P synthase has been extensively studied in C. albicans [148] , S. cerevisiae [150] and Escherichia coli [151] . In prokaryotic cells, the enzyme is a dimer of two identical subunits and has a molecular weight of 130–150 kDa, whereas its eukaryotic counterpart exhibits a tetrameric structure and a molecular mass of 320–340 kDa [148] . In E. coli, the enzyme has been purified and biochemically characterized [152] . The genes coding for the enzyme in bacteria, eukaryotic organisms and mammals are known as glmS, GFA and GFAT, respectively. In bacteria and fungi, the enzyme is essential for cell wall synthesis, and suppression of the respective genes is lethal. However, bacteria may reverse the forward reaction of GlcN-6-P deaminase to carry out the work of GlcN-6-P synthase. In mammals, two genes (GFA1 and GFA2) have been reported, whereas only one (GFA1) exists in fungi [148] . GlcN-6-P synthase from humans [153] , C. albicans [154] and S. cerevisiae [155] has been purified by overexpression of the respective genes in E. coli. Recently, we purified to homogeneity and biochemically characterised the native form of the enzyme from S. schenckii [156] . The protein exhibited a homotetrameric structure consisting of four subunits of 79 kDa, a molecular mass of 350 kDa and a pI of 6.2. The estimated Km values of 1.12 and 2.2 mM for Fru-6-P and l-glutamine, respectively, are well within the corresponding ranges of 0.2– 1.56 mM and 0.4–3.8 mM obtained in other

10





HO

OH

CH2OH

O

OH OH NH2

O

D-Glucosamine-6phosphate

L-Glutamine

OH

NH2

HO

CH2OP

OH OH NH-CO-CH3

O

OP NH-CO-CH3

O

OH

O–P–O–P

URIDINE

NH-CO-CH3

O

UDP-N-acetylD-glucosamine

HO

CH2OH

of

ro N-acteylglucosamine pyrophosphorylase

N-acetyl-D-glucosamine1-phosphate

OH

CH2OH

HO

Acetylglucosaminephosphate mutase

N-acetyl-D-glucosamine6-phosphate

HO

CH2OP

Glucosamine-6-phosphate acetyltransferase

rP

Fungi, insects and crustaceans Chitin

Eukaryotic cells Glycoproteins Glycosaminoglycans Mucopolysaccharides

Bacteria Peptidoglycan Lipopolysaccharides Teichoic acids

Figure 2. Scheme showing the synthesis of UDP-N-acetylglucosamine from D-fructose-6-phosphate and L-glutamine, and the committed step catalyzed by glucosamine-6-phosphate synthase in the so-called Leloir pathway. Also illustrated is the role of the final product as a precursor for a plethora of amino-sugar-containing bio-macromolecules in a vast diversity of organisms. For simplicity, secondary reactants and products are not indicated.

NH2

O

Glucosamine-6phosphate synthase

O

+

OH

CH2OP

D-Fructose-6-phosphate

ho

ut

A


Review

11

Review


better inhibitors of fungal growth than ADGP, maybe because of their greater lipophilicity and increased uptake by fungal cells [149,162,170] . We investigated the effects of ER and ADMP on GlcN-6-P synthase activity and S. schenckii growth. In contrast to previous reports in other organisms, ER was a stronger inhibitor than ADMP, suggesting possible conformational or molecular differences between the enzyme of S. schenckii and that from other fungi. On the other hand, ER and the ER-containing oligopeptides Nva-ER and Nva-Lys-ER weakly affected fungal growth (11–39%), whereas ADMP and its lipophylic derivatives N-butanoil-ADGP and N-hexanoil-ADGP inhibited growth to values over 90% [156] . The ability of specific inhibitors of GlcN-6-P synthase to retard the growth of S. schenckii strengthens the role of this enzyme as a strategic target for antifungal therapy, and it should stimulate future research on new more potent and selective inhibitors.

A

ut

ho

rP

ro

of

Bacilysin has been used as a potent inhibitor of mycelial growth and viability of C. albicans [164] . This drug is transported into C. albicans through a specific dipeptide permease or by an oligopeptide transport system in the yeast or mycelial forms, respectively [164,165] . Once inside the cell, an epoxy-dipeptidase produces a C-terminal amino acid anticapsin that acts as an analogue of glutamine and causes irreversible inhibition [165] . Other glutamine and transition state intermediate analogs, such as N3- (4-methoxyfumaroyl)-L-2,3diaminopropanoic acid (ER) and 2-amino2-deoxy-d-glucitol-6-phosphate (ADGP), respectively, are strong inhibitors of the fungal enzyme, although their antifungal activity is poor due to their slow transport through the cytoplasmic membrane. Kanosamine (3-amino-3-deoxy-glucopyranose) and azaserine [O-(2-diazoacetyl)-L-serine] are analogs of Fru-6-P and L-glutamine, respectively, and they function as competitive inhibitors of the enzyme. However, they have not been used widely due to the adverse effect of azaserine (under investigation in the treatment of Type 2 diabetes) on the viability of human pancreatic islets in vitro [166,167] and the poor effect of kanosamine on the growth of C. albicans [168] . Although glutamine is the source of nitrogen for a large number of enzymes, ER and its derivatives are specific inhibitors of GlcN-6-P synthase because of the active site geometry of this enzyme. ER requires an active transport mechanism to gain entrance into the cell and exert its inhibitory effect. Thus, different hydrophobic derivatives, such as the acetoxylmethyl ester of ER or l-lysyl-lnorvalyl-ER, have been designed to facilitate their penetration into the cell. As does ER, its derivatives inhibit enzyme activity competitively acting as active site-directed inactivators and blocking the N-terminal glutamine-binding domain of the enzyme. These compounds have the advantage of being good inhibitors of the growth of both C. albicans [169] and H. capsulatum [141] . Another group of GlcN-6-P synthase inhibitors have a structure that mimics the transition state of the reaction taking place in the C-terminal sugar isomerizing domain. These include ADGP, ADMP (-D-manitol-6–2amino-2-deoxy phosphate) and hydrophobic derivatives of ADGP (N-acyl-, N-alkyl- or N,N-dialkyl-). ADGP derivatives show an inhibitory effect on activity in vitro similar to those of ADGP and ADMP, but they are

12


Future perspectives & conclusions

All recent molecular research on Sporothrix species support the notion that S. schenckii is no longer the sole species causing sporotrichosis. This complex of species that dwell in soil and may become human and animal pathogens urgently needs to be studied to understand their mechanism of acquisition of pathogenicity, the different species that may attack humans and their susceptibility to antifungals. Future research should focus on basic fungal cellular development, particularly the identification of cell surface antigens, as these may give rise to novel diagnostic tests, as well as strategic targets for antifungal chemotherapy such as essential enzymes or otherwise metabolic events that are crucial for tissue recognition, adhesion and fungal dissemination. These efforts will undoubtedly result in new and more efficient alternatives to prevent and/or control sporotrichosis. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary Sporothrix, a human pathogen with complex biology & ecology Sporothrix schenkii should no longer be considered a single species; rather, it is a complex of phylogenetically distinct species. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix Mexicana and S. schenckii.

n n

Sporotrichosis: an emerging health problem? Sporotrichosis is an acute or chronic subcutaneous mycosis of mammals acquired by traumatic contact with live or decayed vegetation or any traumatism with objects or organic material contaminated by Sporothrix species. n Sporotrichosis is an occupational disease but may be also acquired by zoonotic transmission, with some features of an emerging infectious disease with regional and global relevance to public health. n

Molecular epidemiology & detection To date, there are 32 mitochondrial (mtDNA) types of S. schenckii strains divided in two groups: group A (South Africa and America) and group B (Asia and Australia). n A nested PCR assay identifies S. schenckii of all the mtDNA types and in isolates recovered from different regions of the world; PCR assays allow rapid diagnosis of sporotrichosis especially in immunocompromised patients. n Future molecular and biochemical work must ascertain the Sporothrix species under study.

of

n

Host–pathogen interactions & the role of adhesins in pathogenesis

Several components of the S. schenckii cell wall act, as in other fungi, as adhesins and immunogenic inducers. Components of the extracellular matrix and endothelial cell surface proteins are targets of S. schenckii adhesins. n A 70-kDa glycoprotein (Gp70), apparently specific to S. schenckii but similar in molecular weight to other fungal glycoproteins, mediates fungal adhesion and virulence. n

ro

n

Protein glycosylation in Sporothrix

Glycoproteins are essential for fungal virulence, cell wall integrity and host immune recognition. There is little information about the biosynthesis of glycoproteins in S. schenckii. Thus far, ER a1,2-mannosidase, dolichol-phosphatemannose synthase and protein-O-mannosyltransferase are the only enzyme activities characterised in this organism. n The main glycan peptidorhamnomannan is the only O-linked glycan structure known in S. schenckii. It contains an a-mannobiose core followed by one a-glucuronic acid unit, which may be mono- or di-rhamnosylated. n

rP

n

Glucosamine 6-P-synthase, a potential target for antifungal drugs

Glucosamine-6-P synthase plays a limiting role in hexosamine biosynthesis and has been proposed as a strategic target for antifungal chemotherapy. n Knowledge of the oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzyme’s catalytic domain. n The native enzyme from S. schenckii was recently purified and its sensitivity to ER, ADMP and derivatives thereof was studied. n ER was a stronger enzyme inhibitor than ADMP, but the latter inhibited fungal growth with a much higher efficiency.

ut

ho

n

Bibliography

1.

2.

3.

4.

5.

A

Papers of special note have been highlighted as: n of interest nn of considerable interest

n

Hektoen L, Perkins CF: Refractory subcutaneous abscesses caused by Sporothrix schenckii. J. Exp. Med. 5, 77–89 (1900).

6.

Mariat F: Adaptation de Ceratocystis a la vie parasitaire chez l’animal. Etude de l’aquisition d’un pouvoir pathogéne comparable a celui de Sporothrix schenckii. Sabouraudia 9, 191–205 (1971).

7.

Suzuki K, Kawasaki M, Ishizaki H: Analysis of restriction profiles of mitochondrial DNA from Sporothrix schenckii and related fungi. Mycopathologia 103, 147–151 (1988). O’Reilly LC, Altman SA: Macrorestriction analysis of clinical and environmental isolates of Sporothrix schenckii. J. Clin. Microbiol. 44, 2547–2552 (2006). de Beer ZW, Harrington TC, Vismer HF,


nn

8.

Wingfield BD, Wingfield MJ: Phylogeny of the Ophiostoma stenoceras–Sporothrix schenckii complex. Mycologia 95, 434–441 (2003).

9.

Sporothrix schenckii appears to be a complex of species. Zhang N, Castlebury LA, Miller AN et al.: An overview of the systematics of the Sordariomycetes based on a four-gene phylogeny. Mycologia 98, 1076–1087 (2006). Marimon R, Gené J, Cano J, Trilles L, Dos Santos Lazera M, Guarro J: Molecular phylogeny of Sporothrix schenckii. J. Clin. Microbiol. 44, 3251–3256 (2006). Demonstrates that S. schenckii is a complex of at least six putative phylogenetic species. Watanabe S, Kawasaki M, Mochizuki T, Ishizaki H: RFLP analysis of the internal transcribed spacer regions of Sporothrix schenckii. Jpn. J. Med. Mycol. 45, 165–175 (2004).


de Meyer EM, de Beer W, Summerbell RC et al.: Taxonomy and phylogeny of new wood- and soil-inhabiting Sporothrix species in the Ophiostoma stenoceras–Sporothrix schenckii complex. Mycologia 100, 647–661 (2008).

10. Marimon R, Cano J, Gené J, Sutton D,

Kawasaki M, Guarro J: Sporothrix brasiliensis, S. globosa, and S. mexicana. Three new Sporothrix species of clinical interest. J. Clin. Microbiol. 45, 3198–3206 (2007). n

Shows a clear correlation between molecular data and phenotypic features, and confirms CAL gene as a good marker for the recognition of three new Sporothrix species.

11. Marimon R, Gené J, Cano J, Guarro J:

Sporothrix luriei: a rare fungus from clinical origin. Med. Mycol. 46, 621–625 (2008a). 12. Nicot J, Mariat F: Caracteres morphologiques

et position systématique de Sporothrix schenckii agent de la sporotrichose humaine.

13


14. Resto S, Rodríguez del Valle N: Yeast cell

cycle of Sporothrix schenckii. J. Med. Vet. Mycol. 26, 13–24 (1988). 15. Hiruma M, Kawada A, Ishibashi A:

Ultrastructure of asteroid bodies in sporotrichosis. Mycoses 34, 103–107 (1991). 16. Ghosh A, Maity PK, Hemashettar BM,

Sharma VK, Chakrabarti A: Physiological characters of Sporothrix schenckii isolates. Mycoses 45, 449–454 (2002). 17. Romero-Martínez R, Wheeler M, Guerrero-

Plata A, Rico G, Torres-Guerrero H: Biosynthesis and function of melanin in Sporothrix schenckii. Infect. Immun. 68, 3696–3703 (2000). 18. Morris-Jones R, Youngchim S, Gomez BL

et al.: Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection. Infect. Immun. 71, 4026–4033 (2003). 19. Almeida-Paes R, Frases S, Fialho Monteiro

de antígenos miceliales-levaduriformes de diferentes fases de crecimiento de Sporothrix schenckii. Rev. Mex. Mic. 2, 131–138 (1986). 28. Rippon JW: Tratado de Micología Médica.

Interamericana McGraw-Hill, México DF, MX 351–380 (1990). 29. Bonifaz A: Micología Médica Básica.

McGraw-Hill, Mexico City, Mexico, 167–184 (2010). 30. Barros MBL, Schubach A, Francesconi-Do-

Valle AC et al.: Cat-transmitted sporotrichosis epidemic in Rio de Janeiro, Brazil: description of a series of cases. Clin. Infect. Dis. 38, 529–535 (2004). 31. Durden FM, Elewski B: Fungal infections in

HIV patients. Semin. Cutan. Med. Surg. 16, 200–207 (1997). 32. Dixon DM, Salkin IF, Duncan RA et al.:

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis. J. Clin. Microbiol. 29, 1106–1113 (1991).

33. Gonzalez-Ochoa A: Contribuciones recientes

al conocimiento de la esporotricosis. Gac. Med. Mex. 95, 463–474 (1965).

34. Barros MB, Schubach TM, Gutierrez-

GalhardoMC et al.: Sporotrichosis: an emergent zoonosis in Rio de Janeiro. Mem. Inst. Oswaldo Cruz 96, 777–779 (2001).

ho

PC, Gutierrez-Galhardo MC, ZancopéOliveira RM, Nosanchuk JD: Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates. Microbes Infect. 11, 554–562 (2009).

27. Arenas G, Toriello C: Actividad inmunológica

20. Teixeira PAC, Alves de Castro R, Rodrigues

35. Schubach TMP, Schubach A, Okamoto T

et al.: Evaluation of an epidemic of sporotrichosis in cats: 347 cases (1998–2001). J. Am. Vet. Med. Assoc. 224, 1623–1629 (2004).

ut

Lanzana FF et al.: l-DOPA accessibility in culture medium increases melanin expression and virulence of Sporothrix schenckii yeast cells. Med. Mycol. 48, 687–695 (2010). 21. Cooper CR, Dixon DM, Salkin IF:

Laboratory-acquired sporotrichosis. J. Med. Vet. Mycol. 30, 169–171 (1992).

A

22. Travassos LR, Lloyd KO: Sporothrix schenckii

and related species of Ceratocystis. Microbiol. Rev. 44, 683–721 (1980). 23. Lloyd KO, Bitoon MA: Isolation and

purification of a peptido-rhamnomannan from the yeast form of Sporothrix schenckii. Structural and immunochemical studies. J. Immunol. 107, 663–671 (1971). 24. Mendonca L, Gorin PAJ, Lloyd KO, Travassos

LR: Polymorphism of Sporothrix schenckii surface polysaccharides as a function of morphological differentiation. Biochemistry 15, 2423–2431 (1976). 25. Lopes-Bezerra LM, Schubach A, Costa OR:

Sporothrix schenckii and sporotrichosis. An. Acad. Bras. Cienc. 78, 293–308 (2006). 26. Gonzalez-Ochoa A, Figueroa ES:

Polisacáridos del Sporotrichum schenckii, Datos immunológicos. Intra-dermoreacción

14

41. Criseo G, Malara G, Romeo O, Puglisi

Guerra A: Lymphocutaneous sporotrichosis in an immunocompetent patient: a case report from extreme southern Italy. Mycopathologia 166, 159–162 (2008). 42. Mesa-Arango AC, Reyes-Montes MR,

Pérez-Mejía A et al: Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of sporotrichosis. J. Clin. Microbiol. 40, 3004–3011 (2002). 43. Dixon DM, Duncan RA, Hurd NJ: Use of a

mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest US epidemic of sporotrichosis. J. Clin. Microbiol. 30, 951–954 (1992). 44. Arrillaga-Moncrieff I, Capilla J, Mayayo E

et al.: Different virulence levels of the species of Sporothrix in a murine model. Clin. Microbiol. Infect. 15, 651–655 (2009).

of

Blasini G: Effects of pH, temperature, aeration and carbon source on the development of the mycelia or yeast forms of Sporothrix schenckii from conidia. Mycopathologia 82, 83–88 (1983).

en el diagnóstico de la esporotricosis, Rev. Inst. Salubr. Enferm. Trop. Mexico City 8, 143–153 (1947).

45. Oliveira MME, Almeida-Paes R, Medeiros

Muniz M, Lima Barros MB, Gutierrez Galhardo MC, Zancopé-Oliveira RM: Sporotrichosis caused by Sporothrix globosa in Rio de Janeiro, Brazil: Case report. Mycopathologia 169, 359–363 (2010).

ro

Mycopathol. Mycol. Appl. 49, 53–65 (1973). 13. Rodríguez del Valle N, Rosario M, Torres-

rP

Review

36. Schubach TMP, Schubach A, Okamoto T

et al.: Canine sporotrichosis in Rio de Janeiro, Brazil, clinical presentation, laboratory diagnosis and therapeutic response in 44 cases (1998–2003). Med. Mycol. 44, 87–92 (2006).

37. Schubach A, Barros MB, Wanke B: Epidemic

sporotrichosis. Curr. Opin. Infect. Dis. 21, 129–133 (2008). 38. Schubach A, de Lima Barros MB, Schubach

TM et al.: Primary conjunctival sporotrichosis: two cases from a zoonotic epidemic in Rio de Janeiro, Brazil. Cornea 24, 491–493 (.2005). 39. Feeney KT, Arthur IH, Whittle AJ, Altman

SA, Speers DJ: Outbreak of sporotrichosis, Western Australia. Emerg. Infect. Dis. 13, 1228–1231 (2007). 40. Baroni A, Palla M, Novene MR et al.:

Sporotrichosis: success of itraconazole treatment. Skin Med. 6, 41–44 (2007).


46. Rocha MM, Dassin T, Lira R, Lima EL,

Severo LC, Londero AT: Sporotrichosis in patient with AIDS: report of a case and review. Rev. Iberoam. Micol. 18, 133–136 (2001). 47. Hardmann S, Stephenson I, Jenkins DR,

Wiselka MJ, Johnson EM: Disseminated Sporothrix schenckii in a patient with AIDS. J. Infect. 51, e73–e77 (2005). 48. Callens SF, Kitetele F, Lukun P et al.:

Pulmonary Sporothrix schenckii infection in a HIV positive child. J. Trop. Pediatr. 52, 144–146 (2006). 49. Suzuki K, Kawasaki M, Ishizaki H: Analysis

of restriction profiles of mitochondrial DNA from Sporothrix schenckii and related fungi. Mycopathologia 103, 147–151 (1988). 50. Takeda Y, Kawasaki M, Ishizaki H:

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan. Mycopathologia 116, 9–14 (1991). 51. Ishizaki H, Kawasaki M, Aoki M, Miyaji M,

Nishimura K, Garcia Fernandez JA: Mitocondrial DNA analysis of Sporothrix schenckii in Costa Rica. J. Med. Vet. Mycol. 34, 71–73 (1996). 52. Ishizaki H, Kawasaki M, Aoki M, Matsumoto

T, Padhye AA, Mendoza M, Negroni R: Mitochondrial DNA analysis of Sporothrix schenckii in North and South America. Mycopathologia 142, 115–118 (1998). 53. Ishizaki H, Kawasaki M, Aoki M, Vismer H,

Muir D: Mitochondrial DNA analysis of Sporothrix schenckii in South Africa and



Australia. Med. Mycol. 38, 433–436 (2000).

67. Madrid H, Cano J, Gené J, Bonifaz A,

Toriello C, Guarro J: Sporothrix globosa, a pathogenic fungus with widespread geographical distribution, Rev. Iberoam. Micol. 26, 218–222 (2009).

54. Ishizaki H, Kawasaki M, Mochizuki T, Jin

XZ, Kagawa S: Environmental isolates of in China Sporothrix schenckii. Jpn. J. Med. Mycol. 43, 257–260 (2002). 55. Ishizaki H, Kawasaki M, Aoki M et al.:

RM, Francesconi Do Valle AC et al.: Molecular epidemiology and antifungal susceptibility patterns of Sporothrix schenckii isolates from a cat-transmitted epidemic of sporotrichosis in Rio de Janeiro, Brazil, Med. Mycol. 46, 141–151 (2008).

56. Lin J, Kawasaki M, Aoki M, Ishizaki H, You

G, Li R: Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China. Mycopathologia 148, 69–72 (1999).

Guarro J: In vitro antifungal susceptibilities of five species of Sporothrix. Antimicrob. Agents Chemother. 52, 732–734 (2008).

61. Ishizaki H, Kawasaki M, Anzawa K et al.:

63. Sugita Y: Molecular analysis of DNA

polymorphism of Sporothrix schenckii. Jpn. J. Med. Mycol. 41, 11–15 (2000). 64. Lee JB, Lee SC, Won HY: Analysis of random

amplified polymorphic DNA (RAPD) for Sporothrix schenckii and related fungi. Korean J. Med. Mycol. 5, 113- (2000). 65. Reis RS, Almeida-Paes R, Muniz Mde M,

Tavares PM et al.: Molecular characterization of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil. Mem. Inst. Oswaldo Cruz 104, 769–74 (2009). 66. Neyra E, Fonteyne PA, Swinne D, Fauche F,

Bustamante B, Nolard N: Epidemiology of human sporotrichosis investigated by amplified fragment length polymorphism. J. Clin. Microbiol. 43, 1348–1352 (2005).


biologically active peptide-rhamnogalactan from Sporothrix schenckii. J. Dermatol. 3, 25–29 (1976).

83. Lopes-Alves LM, Mendonca-Previato L,

73. Sandhu GS, Kline BC, Stockman L, Roberts

GD: Molecular probes for diagnosis of fungal infections. J. Clin. Microbiol. 33, 2913–2919 (1995).

74. Kano R, Nakamura Y, Watanabe S,

Tsujimoto H, Hasegawa A: Identification of Sporothrix schenckii based on sequences of the chitin synthase 1 gene. Mycoses 44, 261–265 (2001).

n

This is the first report demonstrating the use of a nested PCR assay to detect S. schenckii.

75. Kanbe T, Natsume L, Goto I et al.: Rapid and

specific identification of Sporothrix schenckii by PCR targeting the DNA topoisomerase II gene. J. Dermatol. Sci. 38, 99–106 (2005). 76. Bates S, Hughes HB, Munro CA et al.: Outer

chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J. Biol. Chem. 281, 90–98 (2006). 77. Figueiredo CC, Lima OC, Carvalho L et al.:

The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules. Microb. Pathog. 36, 177–188 (2004). 78. Casadevall A, Pirofski LA: Host–pathogen

interactions: redefining the basic concepts of virulences and pathogenicity. Infect. Immun. 67, 3703–3713 (1999). 79. Pirofski LA, Casadevall A: The meaning of


Fournet B, Degrand P, Previato J: O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii. Glycoconj. J. 9, 75–81 (1992).

84. Lopes-Alves LM, Travassos LR, Previato JO,

Chen HD: Identification of Sporothix schenckii of various mtDNA types by nested PCR assay. Med. Mycol. 22, 1–5 (2009).

ut

A

Yamaguchi H. Karyotiping by PFGE of clinical isolates of Sporothrix schenckii. FEMS Immunol. Med. Mic. 13, 147–154 (1996).

82. Nakamura Y: Purification and isolation of a

72. Xu TH, Lin JP, Gao XH, Wei H, Liao W,

Mitochondrial DNA analysis of Sporothrix schenckii in India, Thailand, Brazil, Colombia, Guatemala and Mexico. Jpn. J. Med. Mycol. 50, 19–26 (2009). 62. Tateishi T, Murayama SY, Otsuka F,

purification of a peptide-rhamnomannan from the yeast form of Sporothrix schenckii: structural and immunochemical studies. J. Immunol. 107, 663–671 (1971).

ro

of Sporothrix schenckii in clinical samples by a nested PCR assay. J. Clin. Microbiol. 41, 1414–1418 (2003).

ho

Ishizaki H: RFLP analysis of de internal transcribed spacer regions of Sporothrix schenckii. Jpn. J. Med. Mycol. 45, 165–175 (2004).

Describes the first analysis of S. schenckii cell wall composition.

81. Lloyd KO, Bitoon MA: Isolation and

71. Hu S, Chung WH, Hung SI et al.: Detection

rP

60. Watanabe S, Kawasaki M, Mochizuki T,

n

of

sequencing and phylogenetic analysis of environmental Sporothrix schenckii strains: comparison with clinical isolates. Mycopathologia 169, 351–358 (2010).

59. Arenas R, Miller D, Campos-Macias P:

Epidemiological data and molecular characterization (mtDNA) of Sporothrix schenckii in 13 cases from Mexico. Int. J. Dermatol. 46, 177–179 (2007).

Travassos LR: Soluble and insoluble glucans from different cell types of Sporothrix schenckii. Exp. Mycol. 3, 92– 105 (1979).

70. Criseo G, Romeo O: Ribosomal DNA

58. Mora-Cabrera M, Alonso RA, Ulloa-Arvizu

R, Torres-Guerrero H: Analysis of restriction profiles of mitochondrial DNA from Sporothrix schenckii. Med. Mycol. 39, 439–344 (2001).

80. Previato JO, Gorin PAJ, Haskin RH,

69. Marimon R, Serena C, Gené J, Cano J,

57. Kawasaki M et al. Molecular epidemiology of

Sporothrix schenckii in Mexico, Brazil and Spain. Presented at: 4th Congress of the International Society for Human and Animal Mycology. Buenos Aires, Argentina, 2000.

microbial exposure, infection colonisation, and disease in clinical practice. Lancet Infect. Dis. 2, 628–632 (2002).

68. Gutierrez-Galhardo MC, Zancopé-Oliveira

Mitochondrial DNA analysis of Sporothrix schenckii from China, Korea and Spain. Jpn. J. Med. Mycol. 45, 23–25 (2004).

Review

nn

Mendonca-Previato L: Novel antigenic determinant from peptidorhamnomann of Sporothrix schenckii. Glycobiology 4, 281–284 (1994). Describes the structure of the O-linked glycan from peptidorhamnomannan, the first glycan structure resolved in S. schenckii.

85. Travassos LR, Gorin PA, Lloyd KO:

Discrimination between Sporothrix schenckii and Ceratocystis stenoceras rhamnomannans by proton and carbon-13 magnetic resonance spectroscopy. Infect. Immun. 9, 674–680 (1974). 86. Gorin PA, Haskins RH, Travassos LR,

Mendoca-Previato L: Further studies on the rhamnomannans and acidic rhamnomannans of Sporothrix schenckii and Ceratocystis stenoceras. Carbohydr. Res. 55, 21–33 (1977). 87. Lima OC, Lopes Bezerra LM: Identification

of a Concanavalin A binding antigen of the cell surface of Sporothrix schenckii. J. Med. Vet. Mycol. 35, 167–172 (1997). 88. Scott EN, Muchmore HG: Immunoblot

analysis of antibody response to Sporothrix schenckii. J. Clin. Microbiol. 27, 300–304 (1989). 89. Nascimento RC, Almeida SR: Humoral

immune response against soluble and fractionate antigens in experimental sporotrichosis. Immun. Med. Microb. 43, 241–247 (2005). 90. Mendes-Giannini JS, Taylor ML, Bouchara

JB et al.: Pathogenesis II. Fungal responses to host responses: interaction of host cells with fungi. Med. Mycol. 38(Suppl. 1), 113–123

15

Review


104. Nascimento RC, Espíndola NM, Castro RA,

et al.: Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis. Eur. J. Immunol. 38, 3080–3089 (2008).

Zenteno E, Toriello C: Histoplasma capsulatum yeast-cells attach and agglutinate human erythrocytes. Med. Mycol. 42, 287–292 (2004). 93. González A, Gómez BL, Diez S, et al.:

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins. Infect. Immun. 73, 2486–2495 (2005).

Pneumocystis carinii infection in human. Clin. Diagn. Lab. Immunol. 6, 149–155 (1999). 106. Tronchin G, Bouchara JP, Annaix V et al.:

Fungal cell adhesion molecules in Candida albicans. Eur. J. Epidemiol. 7, 23–33 (1991). Expression and identification of a lamininbinding protein in Aspergillus fumigatus conidia. Infect. Immun. 65, 9–15 (1997). et al.: Cell surface expression of adhesins for fibronectin correlates with virulence in Sporothrix schenckii. Microbiology 155, 3730–3738 (2009). n

rP

110. Da Rosa D, Gezuele E, Calegari L, Goñi F:

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG. Rev. Inst. Med. Trop. Sao Paulo 51, 1–7 (2009).

ho

adherence of Candida albicans to extracellular matrix proteins. Microbiology 141, 2681–2684 (1995). 98. Hostetter MK : Adherence molecules in

Endoplasmic reticulum a-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host–fungus interaction. Eukaryot. Cell 6, 2184–93 (2007).

ut

99. Hostetter MK: Integrin-like proteins in

JR: Histoplasma capsulatum yeast phasespecific protein Yps3p induces Toll-like receptor 2 signaling. J. Neuroinflamm. 5, 30 (2008).

A

et al.: Dendritic cell interaction with Candida albicans critically depends on N-linked mannan. J. Biol. Chem. 283, 20590–20599 (2008). 114. Netea MG, Brown GD, Kullberg BJ, Gow

NA: An integrated model of the recognition of Candida albicans by the innate immune system. Nat. Rev. Microbiol. 6, 67–78 (2008).

102. Lima OC, Figueiredo CC, Previato JO et al.:

Involvement of fungal cell wall components the adhesion of Sporothrix schenckii. Microb. Pathog. 69, 6874–80 (2001). 103. Ruiz-Baca E, Toriello C, Pérez A, et al.:

Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall of Sporothrix schenckii involved in fungal adherence to dermal extracellular matrix. Med. Mycol. 47, 185–196 (2009).

16

119. Mora-Montes HM, Bates S, Netea MG et al.:

A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host–fungus interactions. J. Biol. Chem. 285, 12087– 12095 (2010a).

120. Kornfeld R, Kornfeld S: Assembly of

asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631–664 (1985).

121. Herscovics A: Importance of glycosidases in

mammalian glycoprotein biosynthesis. Biochim. Biophys. Acta 1473, 96–107 (1999). 122. Lussier M, Sdicu AM, Bussey H: The KTR

and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1426, 323–334 (1999). 123. Munro CA, Bates S, Buurman ET et al.:

Mnt1p and Mnt2p of Candida albicans are partially redundant a-1,2mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence. J. Biol. Chem. 280, 1051–1060 (2005). 124. Mora-Montes HM, Robledo-Ortiz CI,

González-Sánchez LC, López-Esparza A, López-Romero E, Flores-Carreón A: Purification and biochemical characterisation of endoplasmic reticulum a1,2-mannosidase from Sporothrix schenckii. Mem. Inst. Oswaldo Cruz 105, 79–85 (2010b).

113. Cambi A, Netea MG, Mora-Montes HM

101. Lima OC, Bouchara JP, Renier G et al.:

Immunofluorescence and flow cytometry analysis of fibronectin and laminin binding to Sporothrix schenckii yeast cells and conidia. Microb. Pathog. 37, 131–140 (2004).

Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages. Infect. Immun. 78, 1650–1658 (2010).

112. Aravalli RN, Hu S, Woods JP, Lokensgard

100. Lima OC, Figueiredo CC, Pereira BAS et al.:

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins. Braz. J. Med. Biol. Res. 32, 651–657 (1999).

118. McKenzie CG, Koser U, Lewis LE et al.:

111. Mora-Montes HM, Bates S, Netea MG et al.:

pathogenic fungi. Curr. Opin. Infect. Dis. 9, 141–145 (1996). Candida spp. and other microorganisms. Fungal Genet. Biol. 28, 135–145 (1999).

KB: Characterization of the PMT gene family in Cryptococcus neoformans. PLoS ONE 4, e6321 (2009).

biosynthesis in yeast. FASEB J. 7, 540–550 (1993).

97. Klotz SA, Smith RL: Gelatin fragments block

nn

Up-to-date review on the glycosylation pathways in C. albicans.

117. Willger SD, Ernst JF, Alspaugh JA, Lengeler

Describes the first correlation between expression of adhesins for fibronectin and virulence of S. schenckii.

109. Herscovics A, Orlean P: Glycoprotein

96. López-Ribot JL, Chaffin WL: Binding of

extracellular matrix component entactin to Candida albicans. Infect. Immun. 62,4564– 4571 (1994).

nn

108. Teixeira PA, de Castro RA, Nascimento RC,

95. Li F, Palecek SP: Distinct domains of the

Candida albicans adhesin Eap1p mediate cell-cell and cell-substrate interactions. Microbiology 154, 1193–1203 (2008).

Villagómez-Castro JC, Gow NAR, Flores-Carreón A, López-Romero E: Protein glycosylation in Candida. Future Microbiol. 4, 1167–1183 (2009).

107. Tronchin G, Esnault K, Renier G et al.:

94. Nobile CJ, Nett JE, Andes DR, Mitchell AP:

Function of Candida albicans adhesion Hwp1 in biofilm formation. Eukaryot. Cell 5, 1604–1610 (2006).

116. Mora-Montes HM, Ponce-Noyola P,

105. Walzer PD: Immunological features of

92. Taylor ML, Duarte Escalante E, Pérez A,

Comprehensive review on the Cryptococcus neoformans cell wall and capsule.

of

Aspergillus species to extracellular matrix protein: evidence for involvement of negatively charged carbohydrates on the conidial surface. Infect. Immun. 68, 3377–3384 (2000).

nn

ro

(2000). 91. WasyInka JA, Moore MM: Adhesion of

Excellent review on the Candida albicans innate immune recognition.

115. Doering TL: How sweet it is! Cell wall

biogenesis and polysaccharide capsule formation in Cryptococcus neoformans. Annu. Rev. Microbiol. 63, 223–247 (2009).


n

Describes the first biochemical characterization of an enzyme involved in the N-linked glycosylation pathway in S. schenckii.

125. O’Reilly MK, Zhang G, Imperiali B: In vitro

evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis. Biochemistry 45, 9593–9603 (2006). 126. Absmanner B, Schmeiser V, Kämpf M, Lehle

L: Biochemical characterization, membrane



synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases. Antonie Van Leeuwenhoek 88, 221–230 (2005). n

127. Herscovics A, Romero PA, Tremblay LO: The

specificity of the yeast and human class I ER a 1,2-mannosidases involved in ER quality control is not as strict previously reported. Glycobiology 12, 14G-15G (2002).

137. Goto M: Protein O-glycosylation in fungi:

nn

Comprehensive work on the O-linked glycan structures synthesized by fungi.

Curr. Opin. Microbiol. 2, 509–515 (1999).

139. Kan VL, Bennett JE: Efficacies of four

antifungal agents in experimental murine sporotrichosis. Antimicrob. Agents Chemother. 32, 1619–1623 (1988).

140. Silveira CP, Torres-Rodriguez JM, Alvarado-

Ramirez E et al.: MICS and minimun fungicidal concentrations of amphotericin B, itraconazole, posaconazole and terbinafine in Sporothrix schenckii. J. Med. Microbiol. 58, 1607–1610 (2009).

141. Milewski S, Mignini F, Micossi L, Borowski

mannosyltransferases in virulence and development. Cell. Mol. Life Sci. 65, 528–544 (2008).

A

132. Timpel C, Strahl-Bolsinger S, Ziegelbauer K,

Ernst JF: Multiple functions of Pmt1pmediated protein O-mannosylation in the fungal pathogen Candida albicans. J. Biol. Chem. 273, 20837–20846 (1998). 133. Prill SK, Klinkert B, Timpel C, Gale CA,

n

E: Antihistoplasmal in vitro and in vivo effect of Lys-Nva-FMDP. Med. Mycol. 36, 177–180 (1998).

142. Ram AF, Arentshorst M, Damveld RA,

vanKuyk PA, Klis FM, van der Hondel CA: The cell wall stress response in Aspergillus niger involves increased expression of the glutamine:fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall. Microbiology 150, 3315–3326 (2004).

Schroppel K, Ernst JF: PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol. Microbiol. 55, 546–560 (2005).

143. Naderer T, Wee E, McConville MJ: Role of

Comprehensive work on the C. albicans PMT family.

144. Buse MG: Hexosamines, insulin resistance,

134. Ruiz-Baca E, Villagómez-Castro JC,

Leal-Morales CA, Sabanero-López M, Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii:


hexosamine biosynthesis in Leishmania growth and virulence. Mol. Microbiol. 69, 858–869 (2008). and the complications of diabetes: current status. Am. J. Physiol. Endocrinol. Metab. 290, E1–E8 (2006). 145. Liu J, Richard BD, Chatham JC: Glutamine-

induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via www.futuremedicine.com

D-glucosamine auxotrophs of Saccharomyces cerevisiae: meiosis with defective ascospore wall formation. J. Bacteriol. 124, 1545–1557 (1975).

148. Milewski S: Glucosamine-6-phosphate

138. DiDomenico B: Novel antifungal drugs.

ut

131. Lengeler KB, Tielker D, Ernst JF: Protein-O-

147. Whelan WL, Ballou CE: Sporulation in

diverse structures and multiple functions. Biosci. Biotechnol. Biochem. 71, 1415–1427 (2007).

rP

Describes the generation in vivo of Man5-6GlcNAc2 by ER a1,2-mannosidase, supporting the hypothesis that these oligosaccharides break the reglycosylation and calnexin binding cycle during ER-associated degradation.

Summarizes our current understanding of the role of the HBP and O-GlcNAc on the regulation of cell function and survival and to present evidence that activation of these pathways represents a novel treatment strategy for severe injury and trauma. It is noteworthy that other metabolic-based treatments for severe injury such as glucose-insulin-potassium and glutamine also lead to increased HBP flux and O-GlcNAc levels.

of

Barreto-Bergter E: Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii. Glycobiology 15, 895–904 (2005).

ho

nn

nn

136. Pinto MR, Gorin PAJ, Wait R, Mulloy B,

130. Avezov E, Frenkel Z, Ehrlich M, Herscovics

A, Lederkremer GZ: Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ERassociated degradation. Mol. Biol. Cell 19, 216–225 (2008).

RB: Hexosamine biosynthesis and protein O-glycosylation: the first line of defense against stress, ischemia, and trauma. Shock 9, 431–40 (2008).

M, Bussey H: The Ktr1p, Ktr3p, and Kre2p/ Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J. Biol. Chem. 272, 15527–15531 (1997).

129. Mora-Montes HM, López-Romero E, Zinker

S, Ponce-Noyola P, Flores-Carreón A: Heterologous expression and biochemical characterization of an alpha1,2-mannosidase encoded by the Candida albicans MNS1 gene. Mem. Inst. Oswaldo Cruz 103, 724–30 (2008).

146. Chatham JC, Nöt LG, Fülöp N, Marchase

Describes the first biochemical characterization of enzymes involved in the O-linked glycosylation pathway in S. schenckii.

135. Lussier M, Sdicu AM, Bussereau F, Jacquet

128. Mora-Montes HM, López-Romero E, Zinker

S, Ponce-Noyola P, Flores-Carreón A: Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified a-mannosidase from Candida albicans. Glycobiology 14, 593–5988 (2004).

the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels. J. Mol. Cell Cardiol. 42, 177–185 (2007).

ro

association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an a1,2mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide. Biochem. J. 426, 205–217 (2010).

Review

nn

synthase- the multi-facets enzyme. Biochim. Biophys. Acta 1597: 173–192 (2002). Excellent review dealing with molecular and biochemical aspects of glucosamine6-P synthase.

149. Wojciechowski M, Milewski S, Mazerski J,

Borowski E: Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design. Acta Biochim. Pol. 52, 647–653 (2005). 150. Milewski S, Gabriel I, Olchowy J: Enzymes of

UDP-GlcNAc biosynthesis in yeast. Yeast 23, 1–14 (2006). 151. Floquet N, Moulleron S, Daher R, Maigret B,

Badet B, Badet-Denisot MA: Ammonia channeling in bacterial glucosamine-6phophate synthase (Glms): molecular dynamics simulations and kinetic studies of protein mutants. FEBS Lett. 581, 2981–2987 (2007). 152. Durand P, Golinelli-Pinpaneau B, Mouilleron

S, Badet B, Badet-Denisot MA: Highlights of glucosamine-6P synthase catalysis. Arch. Biochem. Biophys. 474, 302–317 (2008). 153. Richez C, Boetzel J, Floquet N et al.:

Expression and purification of active human internal His6-tagged l-glutamine:d-fructose6-P amidotransferase I. Protein Expr. Purif. 54, 45–53 (2007). 154. Sachadyn P, Jedrzejezak R, Milewski S, Kur J,

Borowski E: Purification to homogeneity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli. Protein Expr. Purif. 19, 343–349 (2000).

17

Review


155. Milewski S, Smith RJ, Brown AJP, Gooday

GW: Purification and characterization of glucosamine-6-P synthase from Saccharomyces cerevisiae. In: Advances in Chitin Science. Domard A, Jeuniaux J, Muzzarelli R, Roberts G (Eds). Jacques Andre Publishers, Lyon, France 96–101 (1996). 156. González-Ibarra J, Milewski S, Villagómez-

Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii. Arch. Biochem. Biophys. 272, 301–310 (1989). 161. Borgia PT: Roles of the arlA, tsE and bimG

genes of Aspergillus nidulans in chitin synthesis. J. Bacteriol. 174, 384–389 (1992).

Castro JC, Cano-Canchola C, López-Romero E: Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target. Med. Mycol. 48, 110–121 (2010).

162. Janiak MA, Hoffmann M, Milewska JM,

First report on the purification and partial characterization of native GlcN-6-P synthase from a truly dimorphic fungus.

163. Olchowy J, Gabriel I, Milewski S: Functional

158. Kornfeld R: Studies on l-glutamine

d-fructose-6-phosphate amidotransferase I. Feedback inhibition by uridine diphosphateN-acetylglucosamine. J. Biol. Chem. 242, 3135–3141 (1967). 159. Kikuchi H, Tsuiki S: Glucosaminephosphate

The hexosamine biosynthesis inhibitor azaserine prevents endothelial inflammation and dysfunction under hyperglycemic condition through antioxidant effects. Am. J. Physiol. Heart Circ. Physiol. 296, H815-H822 (2009). 168. Janiak MA, Milewski S: Mechanism of

antifungal action of kanosamine. Med. Mycol. 39, 401–408 (2001). 169. Zgódka D, Jedrzejczak R, Milewski S,

Boroski E: Amide and ester derivatves of N3-(4-methoxyfumaroyl)-(S)-2,3daminoprpanoic acid: the selective inhibitor of glucosaine-6-phosphate synthase. Bioorg. Med. Chem. 9, 931–938 (2001).

164. Kenig M and Abraham P: Antimicrobial

activities and antagonists of bacilysin and anticapsin. J. Gen. Microbiol. 94, 37–45 (1976).

170. Melcer A, Lacka I, Gabriel I, Wojciechowski

M, Liberek B, Wisniewski A, Milewski S: Rational design of N-alkyl derivatives of 2-amino-2-deoxy-D-glucitol-6P as antifungal agents. Bioorg. Med. Chem. Lett. 17, 6602–6606 (2007).

165. Milewski S, Chmara H, Borowski E:

Antibiotic tetaine a selective inhibitor of chitin and mannoprotein biosyntesis in Candida albicans. Arch. Microbiol. 145, 234–240 (1986).

166. Hull RL, Zraika S, Udayasankar J et al.:

Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and

Website 201. CABI Bioscience, CBS, 2009, Index

Fungorum www.indexfungorum.org

A

ut

ho

syntase of human liver. Biochim. Biophys. Acta 422, 241–246 (1976).

167. Rajapakse AG, Ming XF, Carvas JM, Yang Z:

of

Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase. J. Biol. Chem. 274, 4000–4008 (1999).

domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase. Biochem. J. 404, 121–130 (2007).

azaserine result in reduced islet amyloid formation in vitro. Am. J. Physiol. Cell Physiol. 293, 1586–1593 (2007).

ro

157. Milewski S, Kuszczak D, Jedrzejezak R:

Milewski S: Hydrophobic derivatives of 2-amino-2-deoxy-d-glucitol-6-phosphate: a new type of d-glucosamine-6-phosphate synthase inhibitors with antifungal action. Bioorg. Med. Chem. 11, 1653–1662 (2003).

rP

nn

160. Etchebehere LC, Costa-Maia MJ:

18

