Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

Abstract

Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and five strains of A. terreus, showed that three mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 °C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest that terrelysin may be a candidate biomarker for A. terreus infection.

Fig. 1.

Reactivity of mAbs to rTerrelysin. mAbs with moderate reactivity (A₄₀₅ ≤1.0 when assayed with 1 µg mAb ml⁻¹) are shown in (a); mAbs with high reactivity (A₄₀₅ ≥1.0 when assayed with 1 µg mAb ml⁻¹) are shown in (b).

Fig. 2.

Western blot analysis of (a) rTerrelysin and (b) A. terreus ME with terrelysin mAbs. Lanes: 1, molecular mass markers, 2, mAb 2G4; 3, mAb 3B2; 4 , mAb 6D2; 5, mAb 7D8; 6, mAb 13G10; 7, mAb 15B5; 8, mAb 16C7; 9, mAb 9F4; 10, mAb 19E3; 11, mAb 3B7; 12, mAb 10G4; 13, mAb 15C5; 14, mAb 15E4; 15, mAb 6E4; 16, mAb 10G7; 17, mAb 2D3. Positive reactivity is identified by presence of an immunoreactive band at ~17 kDa.

Fig. 3.

Epitope mapping of anti-rTerrelysin mAbs. SPOTs membrane scans for mAbs 15B5, 13G10 and 10G4. Each spot represents a decapeptide of rTerrelysin sequence. Decapeptides were sequential, with an overlap of two amino acids, and spanned the entire sequence of rTerrelysin, including the N-terminal purification Strep-tag II and the Factor Xa cleavage site.

Fig. 4.

(a) Kinetics of terrelysin expression at room temperature. ELISA inhibition analysis was performed using mAb 10G4. •, CSN; ○, ME. Data points represent samples collected on different days after inoculation and error bars represent sem calculated from three separate experiments. (b) Morphological changes and progression of A. terreus culture growth at room temperature. Black arrows indicate germinating conidia and hyphal extension in early cultures. Bars represent 10 µm for 0–24 h and 100 µm for 48–120 h.

Fig. 5.

(a) Kinetics of terrelysin expression at 37 °C. Details as for Fig. 4(a). (b) Morphological changes and progression of A. terreus culture growth at 37 °C. Black arrows indicate germinating conidia and hyphal extension in early cultures. Bars represent 10 µm for 12–24 h and 100 µm for 36–72 h.

Fig. 6.

Immunolocalization of terrelysin in A. terreus hyphae. Immunolocalization of the antigens was determined with AlexaFluor 594-labelled goat anti-mouse IgG secondary antibodies (red) and nuclear staining was identified via DAPI staining (blue). mAb 13E11, which reacts with A. terreus leucine aminopeptidase, served as a positive control and Stachybotrys-specific mAb 9B4 served as a negative control. Black arrows indicate staining by anti-terrelysin 15B5 mAb at hyphal tips. Bars represent 10 µm. DIC, differential interference contrast.