Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis.
This manuscript is part of the series of works presented at the “V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi” (Oaxaca, Mexico, 2012).
Sporothrix schenckii es el agente etiológico de la esporotricosis, una micosis subcutánea endémica en América Latina. Las proteínas de la pared celular (PC), localizadas en la superficie celular, inducen respuestas de inmunidad celular y humoral, y son candidatas potenciales tanto para objetivos diagnósticos como para la generación de vacunas en la prevención de las infecciones fúngicas. En la presente revisión se destaca el uso potencial de las proteínas de la PC de S. schenckii como inductoras de una respuesta inmunitaria protectora y terapéutica frente a la esporotricosis. Muchos de los hongos patógenos presentan componentes de la pared celular que se han caracterizado como inductores de respuestas inmunológicas celulares y humorales protectoras frente al patógeno a partir del cual se obtienen. El aislamiento y caracterización de los componentes proteicos inmunodominantes de la pared celular de S. schenckii llegan a ser pertinentes para su uso como inductores del desarrollo de respuestas inmunitarias protectoras y terapéuticas frente a la esporotricosis.
Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012).
Sporothrix schenckii is a dimorphic fungus and the etiological agent of sporotrichosis.25 Natural infection and disease occur after the mycelial form of the fungus penetrates the host through skin abrasions produced by fungal-contaminated plants or animals (or, more rarely, after inhalation) and converts into the yeast morphotype. Acute or chronic cutaneous and lymphocutaneous lesions are the most common clinical manifestations of sporotrichosis.50 Frequently, the occurrence of systemic and disseminated cutaneous forms is associated with immunosuppression.39 Hence, the efficiency of the host immune responses, along with the virulence and pathogenicity of the strain, determines the extent of fungal invasion.26,45 Sporotrichosis has been considered as an occupational disease since it commonly affects gardeners, florists, horticulturists, etc. These individuals are exposed to plants and soil that constitute the ecological niche of the pathogen.25,36 Sporotrichosis has a cosmopolitan distribution and is especially endemic in Latin American areas with tropical and subtropical climates.25,38 However, S. schenckii is not the only etiologic agent of sporotrichosis. Other members of the Sporothrix species complex, such as S. schenckii sensu stricto, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, Sporothrix luriei, and Sporothrix albicans,25,27,28 may also be relevant in this context. However, until now, only S. schenckii sensu stricto, S. brasiliensis, and S. globosa have sparked medical interest. Furthermore, other three Sporothrix environmental species, namely, Sporothrix stylites, Sporothrix humicola, and Sporothrix lignivora, have recently been described.11
Adhesion of the fungus to host cells plays a central role in pathogenesis.20 Due to its location, composition, and immunogenicity, the CW is the major fungal structure involved in the interaction with the host, and its immunogenicity is mediated by a variety of molecules that includes glycoproteins, polysaccharides, lipids, and pigments.34 Interestingly, some of these CW components have also the potential to modify the course of the fungal disease in favor of the host.31 Various S. schenckii CW and secretory molecules are highly immunogenic and thus are potential targets for the development of vaccines and serological tests for the diagnosis and treatment of sporotrichosis.40–42,48
Cell wall immunogenicityThe CW of S. schenckii contains alkali-soluble and -insoluble glycans that are found in similar proportions in its two morphotype phases.25,35,41 One of these components is the peptide rhamnomannan, a polymer whose chains are constituted of α-1,6-linked mannosyl and α-d glucuronic acid units.18,49 This macromolecule has been isolated from the CW of yeast-like cells of S. schenckii and has a polysaccharide composition of d-mannose (50%), l-rhamnose (33%), and galactose (1%), as well as a peptide fraction of nearly 16%.24 The peptide-rhamnomannan can be separated into two fractions depending on its affinity to the lectin concanavalin A (Con-A). The fraction that binds Con-A is relevant to the diagnosis of sporotrichosis in humans, as the peptide is recognized by 100% of sera of patients suffering cutaneous sporotrichosis.33 In other clinical forms, the same fraction demonstrates 90% of sensitivity and 86% of global efficiency.1 These percentages may vary depending on the fractions extracted from different strains.2 The fraction that binds Con-A is also useful for the diagnosis of feline sporotrichosis, with high sensitivity and specificity.14 These results suggest that the identification of the immunoreactive components of the CW can guide the design of tools for a highly sensitive and specific diagnosis of this mycosis in its different clinical forms.
Two glycoproteins of 60 and 70kDa, named as Gp60 and Gp70, respectively, have been demonstrated to be the most immunogenic components of S. schenckii CW.41 Two-dimensional immunoblots have revealed that Gp60, which has an isoelectric point between 4.5 and 5.1, is present only in the yeast morphotype. More recently, it has been demonstrated that the three clinically relevant species (i.e., S. schenckii sensu stricto, S. brasiliensis, and S. globosa) of the Sporothrix complex secrete a 60kDa protein that is recognized only by sera of mice infected with the most virulent strains.13 In contrast, Gp70, which has an isoelectric point of 4.1 and contains approximately 5.7% of its molecular mass of N-linked glycans, is present in both fungal morphotypes. This glycoprotein is potentially involved in adhesion during host–pathogen interaction since incubation of yeast-like cells of S. schenckii with anti-Gp70 heteroclonal antibodies decreases the fungal ability to adhere to components of the dermal extracellular matrix from mouse tails.42 Studies using a soluble antigen mixture derived from S. schenckii and sera from patients with extracutaneous sporotrichosis have revealed that these patients present elevated titers of antibodies that recognize between 15 and 20 proteins with molecular weights ranging from 22 to 70kDa. However, recognition was reduced to 8–10 proteins when sera from patients with cutaneous sporotrichosis were used. Notably, the immunodominant antigens were the 40 and 70kDa proteins identified with all serum samples.43
The relevance of Gp60 and Gp70 expression in both morphotypes of S. schenckii in the context of natural and experimental infections has not yet investigated. Likewise, the relationship of these glycoproteins to the host immune response and the evolution of sporotrichosis are unknown. A better understanding of these aspects would facilitate the design of more effective immunoprotective strategies as Gp60 and Gp70 are the most immunogenic glycoproteins in S. schenckii and because their presence in the CW is related to dimorphic transition, which is a relevant attribute of fungal pathogenicity.
Immunity against cell wall componentsThe pathogenicity and virulence of microorganisms trigger a variety of innate and adaptive immune responses in the host that, under optimal conditions, delay the spread of the invading microorganism and may lead to its eradication. In fungi, CW components have already been proven to induce protective responses in several mycoses such as blastomycosis, histoplasmosis, paracoccidioidomycosis, coccidioidomycosis, and candidiasis. In all cases, cellular and humoral responses are important to achieve a protective state.34 To date, there are few studies addressing similar strategies for protection against sporotrichosis.
Cellular immunityThe cellular immune response, or Th1 response, is characterized by an increase in the levels of cytokines such as IFN-γ, which activates effector cells of the immune system, and IL-12, which induces increased production of IFN-γ and activates CD8+ lymphocytes. The relevance of this response against S. schenckii has been demonstrated by the higher susceptibility to this fungal infection of athymic (nu/nu) versus normal mice.44 Furthermore, when athymic mice are reconstituted with thymocytes, the animals acquire higher resistance compared to control groups.12 By contrast, mice injected with a peptide fraction of S. schenckii display a delayed hypersensitivity response, although spleen lymphocytes from mice systemically infected with this fungus are not able to proliferate in vitro when challenged with the same antigen.5 Further evidence supporting the relevance of the cellular immune response against this fungus is the protection achieved after reconstitution with lymph node cells from pre-immunized mice to BALB/c mice infected subcutaneously, which display a lower fungal burden compared to control group mice. Moreover, the protective or anti-fungal effect is abrogated when, prior to the passive transfer, primed cells are incubated with monoclonal antibodies specific for CD4 lymphocytes and macrophages. Incubation of lymph node cells with S. schenckii stimulates the production of IFN-γ, which is a potent inducer of fungicidal activity in macrophages.46 These studies highlight the protective importance of cellular immunity against fungal infection, and other studies using more specific cell wall antigens also confirm this view. Thus, the mannose protein MP65 from the CW of Candida albicans has been demonstrated to exert a potent lymphoproliferative effect,17 and the 70kDa heat-shock protein, which is also present on the surface of this organism, is a potent inducer of cellular immunity via the stimulation of IFN-γ production.3
In the same line, the 120kDa protein from the CW of Blastomyces dermatitidis induces strong cellular and humoral responses in humans and dogs with a blastomycosis infection.21 Similarly, HIS-62 and HIS-80, two CW and membrane proteins from Histoplasma capsulatum, are the main inducers of cellular immune response in patients with histoplasmosis.16 Also, a 60kDa heat shock protein isolated from Paracocciodioides brasiliensis induces protection against experimental lung infections caused by this fungus. This protective response is most likely mediated by the production of IFN-γ and IL-12.7
Humoral immunityTh2 response is characterized by the presence of IL-4, which favors and maintains the development of subpopulations of Th2 lymphocytes, and IL-10, which inhibits the synthesis of IL-12. Additionally, antibody production is another distinctive feature of the Th2 response. Although humoral immune response to S. schenckii is poorly studied, it has been identified by ELISA and western blot analyses in patients with sporotrichosis.43 Interestingly, mice with experimental sporotrichosis display Th1 response during the entire infective period and develop a Th2 response only during the most advanced phases.26 In this sense, sera from BALB/c mice infected with S. schenckii exhibit a Th2 response that is represented by IgG1 and IgG3 isotypes specific for a soluble 70kDa protein.29 Recently, similar results have been obtained using other virulent strains of the Sporothrix complex to infect BALB/c mice which produce antibodies specific for a 60kDa secretory protein. This finding suggests that this protein might be involved in fungal virulence13 and is probably related to the Gp60 protein from the CW, as previously described by Ruiz-Baca et al.41 Infections with other pathogenic fungi have also demonstrated that the host can manifest a significant humoral response. CW proteins from C. albicans in a 20–260kDa range, the 120kDa glycoprotein from Coccidioides immitis, and the WI-1 protein from B. dermatitidis are all efficient inducers of humoral response.6,22,34
Suitability of cell wall components as immunoprotective agentsIt is well established that subcutaneous inoculation of S. schenckii yeast-like cells into BALB/c mice prior to a subsequent lethal intravenous infection with this same fungus, results in a protective response against infection and a concomitantly lower fungal burden. This protection may be induced in athymic mice once they are implanted with lymph node cells from immunized mice.46
In contrast with other dimorphic fungi such as H. capsulatum, P. brasiliensis, Coccidioides spp., B. dermatitidis, or C. albicans, there is little information regarding specific CW molecules from S. schenckii with the ability to modify the course of the infection. Therefore, the immunogenic Gp70 and Gp60 proteins of this pathogen are attractive research targets. Gp70 has adhesive properties and hence it could be defined as virulence attribute. This characteristic is suggested by the fact that pre-incubation of S. schenckii cells with anti-Gp70 antibodies decreases fungal adhesion to the extracellular matrix of the mouse tail dermis. It has been suggested that this protein may be a potential target for treating this infection.42 The injection of monoclonal antibodies directed against a 70kDa protein of S. schenckii induces a passive immune state that is effective in BALB/c mice subsequently infected with yeast-like cells by intraperitoneal via.30 The relevance of humoral immunity against S. schenckii infection and protection of the host is demonstrated by the fact that similar results can be obtained using monoclonal antibodies specific for this fungus. However, the implicated mechanisms with the use of monoclonal antibodies during mycotic infections have not been elucidated. It is possible that these antibodies function as opsonins and facilitate phagocytosis of the fungus,10 as it has been suggested in BALB/c mice after the use of monoclonal antibodies specific to a 70kDa (Gp70) cellular surface glycoprotein of P. brasiliensis, which alter the pulmonary granuloma development. The Gp60 glycoprotein from the CW of S. schenckii is one of the immunodominant proteins and is a possible fungal virulence factor. Consequently, Gp60 is considered an ideal candidate to be used as an inducer of potent cellular and humoral responses that may result in immune protection.13,41 This assumption is based on the immune protection obtained after immunization with the 60kDa heat-shock proteins (Hsp60) isolated from the CW of H. capsulatum or P. brasiliensis, during experimental pulmonary histoplasmosis15 and experimental paracoccidioidomycosis in BALB/c mice,7 which showed increased survival times and decreased fungal burdens. Active immunization with a mixture of Hsp60 and an immunodominant H. capsulatum antigen increases the survival rates mediated by CD4+ lymphocytes, IFN-γ, IL-12, and IL-10, during the induction phase of vaccination.9 Furthermore, passive immunization with monoclonal antibodies directed against Hsp60 from H. capsulatum decreases the organ fungal burden, modify the course of experimental histoplasmosis, and increases the survival rates of C57BL/6 mice.19 The immunoprotective antifungal effect of Th1 response, with elevated IFN-γ and IL-2 levels, has also been observed after a lethal infection with C. immitis of mice immunized with a 63kDa urease, the homologous of the H. capsulatum 60kDa heat-shock protein, or DNA encoding the C. immitis urease.23
A 43kDa (Gp43) glycoprotein from the CW of P. brasiliensis is the main antigen used in a variety of diagnostic serological tests.8 Monoclonal antibodies directed against this glycoprotein reduce the fungal burden in the lung and spleen of mice infected with this fungus.4 The use of a DNA vaccine encoding an epitope of Gp43 (known as P10), before and during the infection with P. brasiliensis in mice, decreases the fungal load in the lung, spleen, and liver, and increases IFN-γ production.37 Likewise, the use of antibodies directed against H2B from H. capsulatum, a cell surface histone-like protein, decreases the inflammation and fungal load in organs from C57BL/6 mice with experimental histoplasmosis, which results in higher survival rates, as compared with the control group.32 These protective effects might be associated with increased production of IFN-γ. Comparable results have been obtained using a fraction of the CW proteins (TX114-DF) from Coccidioides posadasii in C57BL/6 mice. This protein fraction contains a 43kDa immunodominant antigen that induces T cell proliferation and the secretion of high levels of IFN-γ in in vitro assays, indicating a strong Th1 response.47
Certain fungal antigens, such as the 120kDa adhesin from B. dermatitidis (known as WI 1), elicit immunoprotective Th1 and Th2 responses. This protein increases the survival rates of infected C57BL/6 mice. Noteworthy, mice produce elevated titers of a mixture of antibodies such as IgG2a and IgG3, which are associated with cellular immunity, but also IgG1 and IgG2b, which are more closely related to humoral immune response.51 During C. albicans infections, the use of specific antibodies against β-1,2-mannotriose, a CW glycan, conjugated with peptide fractions of the fructose bisphosphate aldolase, methylhydrotriglutamate-monocysteinemethyltransferase, and phosphoglycerate kinase, induces a protective response in infected C57BL/6 and BALB/c mice, measured by the increase of its survival rates.52
Conflict of interestsThe authors declare that they have no conflict of interests.
This paper constitutes a partial fulfillment of the requirements of the Graduate Program in Biomedical Sciences of the UJED. CAAF thanks the scholarship No. 201509 granted by the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico.
