Background: Aspergillus terreus is an emerging opportunistic fungal pathogen within the immunocompromised population. Currently there are no diagnostic markers available for specific identification of this organism. Fungal hemolysins have been identified as members of Aegerolysin family and are thought to have a putative role in pathogenesis. Detection of terrelysin in clinical samples from patients with invasive aspergillosis may serve as a biomarker of A. terreus infection. Methods: An Asp-hemolysin (Q00050) homologue was identified in the A. terreus genomic database (Q0CRX8) and the recombinant terrelysin (rTer) was produced in Escherichia coli using pASK-IBA6 vector with an N-terminal Strep-tag II and a Factor Xa cleavage site. The protein was expressed, purified on a Strep-Tactin sepharose column, and characterized using MALDI-TOF MS and MS/MS sequencing and Circular Dichroism (CD). Balb/c mice were immunized with rTer and anti-terrelysin monoclonal antibodies (mAbs) produced by fusion of splenic cells and Sp2/0-Ag14 plasmacytoma cell line. Initial characterization of mAbs was done using Western blot and inhibition ELISA analysis. Results: Purified rTer fusion protein was identified as a 16.4 kDa protein. cDNA and protein sequence analysis confirmed the terrelysin sequence and predicted an intron between nucleotides 87-119 of the database sequence (Q0CRX8). CD analysis revealed that rTer is predominantly a β -sheet protein. Conformational changes as a result of pH changes and thermal unfolding showed that the β -sheet conformation is preserved over a wide range of conditions. We generated 19 anti-terrelysin mAbs of isotypes IgG1 (12), IgG2a (4) and IgG2b (3). Western blot analysis showed that these antibodies recognize a 16 kDa protein in hyphal extracts of A. terreus. Using an inhibition ELISA, rTer could be detected when spiked into serum. Conclusions: We have successfully produced a recombinant terrelysin and developed mAbs that recognized the native molecule in A. terreus extracts. These reagents may be useful for the characterization of terrelysin as a potential biomarker of A. terreus infection.
Full conference title:
110th General Meeting American Society for Microbiology
- ASM 110th (2010)