Identification of novel allergens of <i>Aspergillus fumigatus</i> using immunoproteomics approach

Gautam, Poonam; Sundaram, Curam Sreenivasacharlu; Madan, Taruna; Gade, W. N.; Shah, Ashok; Sirdeshmukh, Ravi; Sarma, P. Usha

doi:10.1111/j.1365-2222.2007.02765.x

Cited by 59 publications

(44 citation statements)

References 32 publications

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“…Recently, novel biomarkers that can be used as drug targets or markers of effective treatment have been identified by microarray genomic analysis or other methods of RNA analysis (10,37,40). Proteomic analysis relying on two-dimensional (2D) gel electrophoresis has been used to identify novel allergens, evaluate changes in levels of expressed protein in response to amphotericin B, as well as build a reference map of different compartments of the cell (3,15,16,22,23,26,43). This conventional approach provides a basis for proteomic analysis but it is limited by the number of protein spots that can be resolved as well as the ability to excise, identify, and quantify the proteins (4).…”

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confidence: 99%

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin

Cagas¹,

Jain²,

Li³

et al. 2011

Antimicrob Agents Chemother

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The proteomic response of Aspergillus fumigatus to caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 g/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy.

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confidence: 99%

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin

Cagas¹,

Jain²,

Li³

et al. 2011

Antimicrob Agents Chemother

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“…A mass tolerance of 50 ppm and a requirement of at least five peptides matched were also used for protein identification. Only those proteins that showed significant matches with A. fumigatus proteins were considered in the present study (17).…”

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confidence: 99%

“…Fixed and variable modifications were considered (Cys as an S-carboamidomethyl derivate and methionine as oxidized methionine), and one missed cleavage site was allowed. A Mascot score above 58 was taken as the cutoff, molecular mass was unrestricted, and sequence coverage was Ͼ25% (17). A mass tolerance of 50 ppm and a requirement of at least five peptides matched were also used for protein identification.…”

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confidence: 99%

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B

Gautam

Shankar

Madan

et al. 2008

Antimicrob Agents Chemother

105

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Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.Invasive aspergillosis, caused primarily by Aspergillus fumigatus, has emerged as the leading cause of mortality among immunocompromised patients with underlying hematological diseases or bone marrow transplantation (7, 38). Amphotericin B (AMB), a broad-spectrum fungicidal agent, has been widely used to treat patients with invasive aspergillosis. AMB is reported to be fungicidal (MFC/MIC Յ 4) against all A. fumigatus and Aspergillus flavus isolates but not on Aspergillus terreus isolates (25). Its therapeutic use is limited by its toxicity (nephrotoxicity, cytotoxicity, and hepatotoxicity, etc.) in the host (13) and development of resistance in fungal isolates (42). However, use of lipid formulations of AMB, administration by the inhalation route, and development of less toxic analogues have facilitated better therapeutic outcomes (35). In general, it is known that AMB intercalates with ergosterol of the fungal cell membrane and forms pores resulting in leakage of fungal cell components, which leads to death via osmotic collapse (15, 22).It also promotes oxidative damage to cell membranes through generation of reactive oxygen species (ROS) (43) and damage to DNA resulting in loss of cell viability, a characteristic of apoptosis (31).Earlier efforts reported genome-wide expression analysis to understand the mechanism of action and specific effects of AMB and other antifungal drugs on nonfilamentous fungal species, such as Saccharomyces cerevisiae and Candida albicans (1,24,46). However, in an earlier study of interaction of A. fumigatus with voriconazole (11), decreased mRNA expression of ergosterol biosynthesis genes was observed, which was in contrast with previous reports of S....

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“…In addition, there are some reports dealing with the role of a cell wall glycoprotein of 70 kDa in adhesion of S. schenckii to host tissues and extracellular matrix components as well as fungal pathogenesis (Nascimento et al 2008, Ruiz-Baca et al 2009, Teixeira et al 2009). 2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al 2002, Gautam et al 2007, Young et al 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii.…”

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confidence: 99%

“…2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al 2002, Gautam et al 2007, Young et al 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii.…”

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confidence: 99%

2D-immunoblotting analysis of Sporothrix schenckii cell wall

Ruiz-Baca

Mora-Montes

López-Romero

et al. 2011

Mem. Inst. Oswaldo Cruz

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We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting) with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70) was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells.In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.Key words: sporotrichosis -Sporothrix schenckii -cell wall -glycoproteins -2D-immunoblotting analysisThe dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America, whose prevalence has significantly increased during the last few years mainly in immunocompromised patients (Callens et al. 2006, Lopes-Bezerra et al. 2006). The S. schenckii cell wall is composed of alkali-soluble and insoluble glucans, which are found in both morphological phases of the fungus (Previato et al. 1979, Lopes-Bezerra et al. 2006. Despite significant progress in the knowledge of structural polysaccharides, little is known regarding the identity and characteristics of cell wall proteins. Thus far, a peptide-rhamnomannan and peptide-rhamnogalactan have been isolated and characterized from wall preparations of yeast-like cells (Lloyd & Bitoon 1971, Nakamura 1976, Lopes-Bezerra et al. 2006). In addition, there are some reports dealing with the role of a cell wall glycoprotein of 70 kDa in adhesion of S. schenckii to host tissues and extracellular matrix components as well as fungal pathogenesis (Nascimento et al. 2008, Ruiz-Baca et al. 2009, Teixeira et al. 2009).2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al. 2002, Gautam et al. 2007, Young et al. 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii. Antibodies raised against the whole fungal cell allowed the identification of morphology-specific cell wall glycoproteins as well as glycoproteins present in both fungal phases.Organism and culture conditions -S. schenckii ATCC 58251 was used in this study. For mycelia propagation, 2-l Erlenmeyer flasks containing 600 mL of YPG medium (0.3% yeast extract, 1% peptone and 2% glucose, pH 4.5) were inoculated with 1 x 10 6 conidia mL -1 and incubated for 24 h at 28ºC with shaking (120 rpm). To obtain the yeast-like form, the pH of the medium was adjusted to 7.2 and the flasks were shaken for six days at 37ºC. Hyphae were collected by filtration in a Büchner filter and yeast-like cells were harvested by centrifugation at...

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Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

Cited by 59 publications

References 32 publications

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B

2D-immunoblotting analysis of Sporothrix schenckii cell wall

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