Date of Graduation
School of Medicine
Microbiology, Immunology, and Cell Biology
Donald H. Beezhold.
Exposure to fungi can lead to diverse conditions from allergy to colonization to invasive and disseminated infection, depending on the status of the host immune system. Recent data suggests that fungal infections and especially Aspergillus species has been on the rise. Though mostly affecting immunocompromised individuals, more recent reports have identified infections in immunocompetent populations as well. Aspergillus terreus is of particular interest, since it causes rapid disseminating infections with high mortality rates, owing to its natural resistance to antifungal drugs and production of vegetative aleurioconidia during infection. These attributes coupled with poor current diagnostics and increasing costs involved with controlling the infection, have generated considerable interest in developing novel strategies for diagnosis of A. terreus infections. Fungal hemolysins have been proposed as biomarkers of exposure because of an association between exposures to the black mold Stachybotrys chartarum and an outbreak of idiopathic pulmonary hemorrhage. Using polyclonal antibodies, the hemolytic antigen was detected at concentrations of 371 ng/ml in patient sera. Our initial experiments were designed to develop monoclonal antibodies (mAbs) towards a stachylysin hemolytic preparation. IgM mAbs were developed with limited cross-reactivity within Stachybotrys species. ELISA and FHIA analysis showed that most stachylysin was present in hyphae. Using proteomic analysis, we could not confirm the identity of stachylysin because the genome of S. chartarum has not been sequenced. To better characterize hemolysins as biomarkers, the clinically relevant fungus (A. terreus) with a sequenced genome was chosen for analysis. Initial attempts at the characterization of a hemolytic fraction from A. terreus resulted in development of multiple A. terreus -specific IgG1 mAbs to proteolytic enzymes including, leucine aminopeptidase and dipeptidyl peptidase V. We were unable to identify the hemolysin of interest using this mAb approach. Alternatively, expression of recombinant terrelysin (rTerrelysin) using the pASK-IBA6 vector in Escherichia coli yielded a protein with secondary structure similar to other Aegerolysin family proteins. Highly specific mAbs (10G4, 15B5 and 13G10) to the recombinant protein consistently recognized native terrelysin in different strains of A. terreus. Expression kinetics suggested that terrelysin is produced in high concentrations immediately after germination but levels decrease with increasing fungal biomass, and not present at the later time points when the previous hemolytic preparations were made. These data suggest interpretation of previous studies on detection of 'hemolysins' in exposed individuals and experimental animals are likely an error. The mAbs developed in this study for terrelysin, leucine aminopeptidase, and dipeptidyl peptidase V have potential for development of rapid and specific diagnostic assays. Tools developed in this study could be used for analysis in various animal models to determine combinatorial assays using mAbs developed to different proteins of A. terreus.
Nayak, Ajay Padmaj, "Characterization of terrelysin, a potential biomarker for Aspergillus terreus" (2012). Graduate Theses, Dissertations, and Problem Reports. 3598.