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Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

Rodrigues AM, Fernandes GF, Araujo LM, Della Terra PP, dos Santos PO, Pereira SA, Schubach TM, Burger E, Lopes-Bezerra LM, de Camargo ZP - PLoS Negl Trop Dis (2015)

Bottom Line: Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans.Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii.We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.

Methodology: We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20).

Principal findings: Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis.

Conclusions: A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

No MeSH data available.


Related in: MedlinePlus

Identification of IgG-reactive proteins from the S. brasiliensis proteome via 2D immunoblotting.(A) Proteins from isolate CBS 132990 were fractionated using 13 cm pH 4–7 strips (left to right) in the first dimension and 10% SDS-PAGE in the second dimension. (B) Immunoproteomics of S. brasiliensis during feline sporotrichosis. Serum-derived antibodies (pooled sera; n = 10) in naturally infected cats mainly recognized all six isoforms of 3-carboxymuconate cyclase (gp60) in the S. brasiliensis proteome (a1-a6; GenBank: KP233225). Immunoblotting is as described in Methods.
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pntd.0004016.g005: Identification of IgG-reactive proteins from the S. brasiliensis proteome via 2D immunoblotting.(A) Proteins from isolate CBS 132990 were fractionated using 13 cm pH 4–7 strips (left to right) in the first dimension and 10% SDS-PAGE in the second dimension. (B) Immunoproteomics of S. brasiliensis during feline sporotrichosis. Serum-derived antibodies (pooled sera; n = 10) in naturally infected cats mainly recognized all six isoforms of 3-carboxymuconate cyclase (gp60) in the S. brasiliensis proteome (a1-a6; GenBank: KP233225). Immunoblotting is as described in Methods.

Mentions: We previously reported that the 60-kDa and 70-kDa proteins in S. brasiliensis and S. schenckii, respectively, are related to virulence profiles and are the main antigenic molecules during murine [3] and human [45] sporotrichosis. We also determined that this protein undergoes post-translational modification and is present as isoforms and glycoforms in the S. brasiliensis and S. schenckii proteomes [45]. We therefore investigated whether antibodies present in cat sera recognize all six isoforms in the S. brasiliensis proteome, as previously shown with human antibodies [45]. S. brasiliensis proteins were therefore resolved via 2D electrophoresis and immunoblotted with pooled sera from cats with sporotrichosis (n = 10) and optimal antibody titers according to ELISA. Serum-derived antibodies in naturally infected cats mainly recognized all six isoforms of gp60 (Fig 5). The present results confirm that S. brasiliensis 3-carboxymuconate cyclase is a highly polymorphic protein [45] with sizes of 55–62 kDa and with isoelectric points of 4.45–4.80. 2D immunoblotting revealed less diversity and more weakly reacting spots than 1D immunoblotting, perhaps due to protein loss during sample preparation for 2D electrophoresis (compared to the crude extracts used in 1D immunoblotting and ELISA) and serum dilution during pooling.


Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

Rodrigues AM, Fernandes GF, Araujo LM, Della Terra PP, dos Santos PO, Pereira SA, Schubach TM, Burger E, Lopes-Bezerra LM, de Camargo ZP - PLoS Negl Trop Dis (2015)

Identification of IgG-reactive proteins from the S. brasiliensis proteome via 2D immunoblotting.(A) Proteins from isolate CBS 132990 were fractionated using 13 cm pH 4–7 strips (left to right) in the first dimension and 10% SDS-PAGE in the second dimension. (B) Immunoproteomics of S. brasiliensis during feline sporotrichosis. Serum-derived antibodies (pooled sera; n = 10) in naturally infected cats mainly recognized all six isoforms of 3-carboxymuconate cyclase (gp60) in the S. brasiliensis proteome (a1-a6; GenBank: KP233225). Immunoblotting is as described in Methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549111&req=5

pntd.0004016.g005: Identification of IgG-reactive proteins from the S. brasiliensis proteome via 2D immunoblotting.(A) Proteins from isolate CBS 132990 were fractionated using 13 cm pH 4–7 strips (left to right) in the first dimension and 10% SDS-PAGE in the second dimension. (B) Immunoproteomics of S. brasiliensis during feline sporotrichosis. Serum-derived antibodies (pooled sera; n = 10) in naturally infected cats mainly recognized all six isoforms of 3-carboxymuconate cyclase (gp60) in the S. brasiliensis proteome (a1-a6; GenBank: KP233225). Immunoblotting is as described in Methods.
Mentions: We previously reported that the 60-kDa and 70-kDa proteins in S. brasiliensis and S. schenckii, respectively, are related to virulence profiles and are the main antigenic molecules during murine [3] and human [45] sporotrichosis. We also determined that this protein undergoes post-translational modification and is present as isoforms and glycoforms in the S. brasiliensis and S. schenckii proteomes [45]. We therefore investigated whether antibodies present in cat sera recognize all six isoforms in the S. brasiliensis proteome, as previously shown with human antibodies [45]. S. brasiliensis proteins were therefore resolved via 2D electrophoresis and immunoblotted with pooled sera from cats with sporotrichosis (n = 10) and optimal antibody titers according to ELISA. Serum-derived antibodies in naturally infected cats mainly recognized all six isoforms of gp60 (Fig 5). The present results confirm that S. brasiliensis 3-carboxymuconate cyclase is a highly polymorphic protein [45] with sizes of 55–62 kDa and with isoelectric points of 4.45–4.80. 2D immunoblotting revealed less diversity and more weakly reacting spots than 1D immunoblotting, perhaps due to protein loss during sample preparation for 2D electrophoresis (compared to the crude extracts used in 1D immunoblotting and ELISA) and serum dilution during pooling.

Bottom Line: Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans.Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii.We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.

Methodology: We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20).

Principal findings: Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis.

Conclusions: A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

No MeSH data available.


Related in: MedlinePlus