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Journal of Medical Microbiology logoLink to Journal of Medical Microbiology
. 2012 Apr;61(Pt 4):489–499. doi: 10.1099/jmm.0.039511-0

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

Ajay P Nayak 1,2,✉, Brett J Green 1, Sherri Friend 3, Donald H Beezhold 1
PMCID: PMC3348537  PMID: 22160315

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.

Introduction

Bacterial haemolysins have a functional role in microbial pathogenesis through lysis of host cell membranes (Bhakdi et al., 1996; Chu et al., 1991; Seeger et al., 1991). Fungal haemolysins have been studied for their pleiotropic functions as virulence factors (Kumagai et al., 1999; Maličev et al., 2007; Rebolj et al., 2007; Žužek et al., 2006), in lipoprotein binding (Kudo et al., 2001, 2002) and fungal development (Berne et al., 2002, 2007), and their utility as markers of cholesterol micro-domains on eukaryotic cell membranes (Chowdhury et al., 2008; Rebolj et al., 2006). To date, limited information is available as to the function of these proteins in fungal biology.

Although Aspergillus fumigatus is the leading cause of invasive aspergillosis in immunocompromised individuals, Aspergillus terreus, as well as Aspergillus flavus, Aspergillus niger and rarely Aspergillus nidulans, have also been identified as aetiological agents (Balajee, 2009a; Perfect et al., 2001; Segal et al., 1998). A. terreus is an opportunistic fungal pathogen that has been identified to cause infections including onychomycosis (Hilmioğlu-Polat et al., 2005), otitis (Tiwari et al., 1995), endophthalmitis (Garg et al., 2003), peritonitis (Verghese et al., 2008), suppurative abscess (Sukroongreung & Thakerngpol, 1985) and osteomyelitis (Natesan et al., 2007). Resistance of A. terreus to amphotericin B, thermotolerance and production of accessory conidia have been suggested to aid in the rapid dissemination of the organism during invasive infections (Blum et al., 2008; Pore & Larsh, 1967). Collectively, these factors are believed to contribute to the very high mortality rates among immunocompromised patient populations and highlight the need for species-specific diagnostic methodologies.

Fungal haemolysins have been characterized from a variety of species (Ebina et al., 1982; Van Emon et al., 2003; Vesper & Vesper, 2004; Wartenberg et al., 2011). Aspergillus haemolysin (Asp-haemolysin) has been detected in the tissues of mice in an invasive aspergillosis animal model (Ebina et al., 1982). Stachylysin, a haemolysin derived from Stachybotrys chartarum, was reported to be detectable in sera of exposed animals and patients (Ebina et al., 1982; Van Emon et al., 2003). In a previous study, we used molecular methods to identify a haemolysin, terrelysin, produced by the opportunistic fungal pathogen A. terreus (Nayak et al., 2011a). In this study, we describe the generation of terrelysin-specific monoclonal antibodies (mAbs), and characterize the expression of native terrelysin during fungal growth.

Methods

Production of mAbs against recombinant terrelysin (rTerrelysin).

Three 8–10-week-old BALB/c mice (Jackson Laboratory) were housed in the animal facility at the National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV, USA. The facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and animals were free of viral pathogens, parasites, mycoplasma and Helicobacter spp. Animals were housed together in HEPA-filtered ventilated polycarbonate cages with autoclaved hardwood Beta-chip bedding and cotton fibre nesting material. The animals were provided with Teklad 7913 rodent chow (Harlan Laboratories) and autoclaved tap water ad libitum. All animal procedures and immunizations were reviewed and approved by the NIOSH Animal Care and Use Committee (ACUC).

Prior to immunization, blood samples were collected from the tail vein and the serum was stored at −20 °C. Each animal was immunized three times, every other week, with 25 µg purified rTerrelysin (Nayak et al., 2011a) emulsified 50 % (v/v) in TiterMax adjuvant. The mice were monitored for adverse health effects post-immunization. Between immunizations, serum was collected from the tail vein to screen for specific IgG antibody responses.

Following the development of sufficient IgG titres, the animals were euthanized by CO2 asphyxiation. The spleen was aseptically removed from each mouse and single-cell suspensions of the splenocytes were produced. Fusion of splenocytes with SP2/0-Ag 14 myeloma cells (ATCC CRL-1581) was performed by the method of Köhler & Milstein (1975). Hybridomas were selected by growing cells in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 1 mM sodium pyruvate, 100 U penicillin ml−1, 100 mg streptomycin ml−1, 0.292 mg l-glutamine ml−1, 100 mM sodium hypoxanthine, 16 mM thymidine, 10 % fetal calf serum (FCS) (HyClone) and 100 U IL-6 ml−1 (Boerhinger Mannheim). DMEM was also supplemented with azaserine for selective propagation of hybridomas. After 10–14 days of growth, medium from individual wells with hybridoma cell growth was replenished with fresh DMEM. The supernatants from individual colonies were tested in a screening ELISA to detect antibodies of varying IgG isotypes that reacted with rTerrelysin.

Supernatants of individual clones were tested twice to confirm reactivity of antibodies. Each positive well was then cloned twice by limiting dilution analysis and single positive clones were screened and selected for production of mAbs. Positive clones were frozen in 10 % DMSO, stored at −80 °C for 2 weeks and transferred to liquid nitrogen for long-term storage.

Purification, isotyping and quantification of rTerrelysin mAbs.

All rTerrelysin mAbs were concentrated and partially purified by ammonium sulfate precipitation (Andrew & Titus, 1997). Briefly, mAb supernatant was collected from individual hybridomas and centrifuged at 20 000 g for 30 min at 4 °C. The supernatant was collected and saturated ammonium sulfate was slowly added to the supernatant to 45 % saturation followed by incubation overnight at 4 °C. The tubes were centrifuged for 45 min at 20 000 g at 4 °C and the precipitate was collected and resuspended in PBS, pH 7.4. Concentration of purified antibodies and their isotype were determined using methods previously described (Nayak et al., 2011b).

Fungal cultures and extracts.

For time-point assays, fungal cultures were grown by inoculating 50 ml minimal medium (containing glucose, nitric salts and trace elements) (Hill & Kafer, 2001; Nayak et al., 2011b), with 2.5×107 viable A. terreus conidia. Viability was determined using the LIVE/DEAD BacLight Viability kit (Molecular Probes) (Chen & Séguin-Swartz, 2002). Following inoculation, flasks were incubated at either room temperature or 37 °C depending on the specific experimental design. A. terreus cultures were grown for up to 12 days, with an individual flask representing a 24 h time point. A. terreus cultures and mycelial pellets were collected in 50 ml polypropylene tubes and centrifuged at 4100 g for 10 min. The culture supernatant (CSN) and mycelial pellets were collected and stored at −80 °C; the lyophilized CSN residue was resuspended in PBS, and mycelial pellets were processed using a mortar and pestle in PBS containing Complete Mini Protease Inhibitor Cocktail (Roche Diagnostics). Mycelial slurry was then collected into 15 ml polypropylene tubes and incubated at 4 °C overnight on a shaker to facilitate the release of intracellular proteins into the lysis solution. The next day, mycelial extracts (ME) were centrifuged at 4100 g for 10 min, and the supernatant was collected and stored at −20 °C until analysis.

For cross-reactivity studies, ME were prepared from 29 fungal species, including 12 Aspergillus species, using the same method (Table 1). Fungi were grown until mycelial pellets had formed (3–4 days). Protein concentrations of CSN and ME were estimated using a NanoDrop ND-1000 spectrophotometer as previously described (Nayak et al., 2011b).

Table 1. Cross-reactivity profiles of terrelysin mAbs analysed by Western blotting.

A., Aspergillus; Ac., Acremonium; Al., Alternaria; Ch., Chaetomium; Cl., Cladosporium; Ex., Exserohilum; Fu., Fusarium; Ne., Nesosartorya; Pa., Paecilomyces; Pe., Penicillium; Sc., Scopulariopsis; Tr., Trichoderma. +, Positive reactivity.

Fungus 7D8 13G10 15B5 3B7 10G4 15C5 10G7
A. terreus FGSC 1156 + + + + + + +
A. terreus ATCC 1012 + + + + + + +
A. terreus NIOSH 17-30-31 − + + + + + +
A. terreus NIOSH 35-08-05 + + + + + + +
A. terreus NIOSH 35-08-06 − + + + + + +
A. candidus NIOSH 17-28-24 − − − − − − −
A. chevalieri NRRL 78 − − − − − − −
A. clavatus NIOSH 6-22-78 − − − − − − −
A. flavus NIOSH 15-41-07 − − − − − − −
A. fumigatus FGSC A1100 − − − − − − −
A. nidulans NIOSH 15-22-08 − − − − − + −
A. niger FGSC A1143 − − − − − − −
A. parasiticus ATCC 26690 − − − − − + −
A. penicilloides ATCC 16910 − − − − − − −
A. repens NRRL 13 − − − − − − −
A. ustus NRRL 275 − − − − − − −
Ac. strictum ATCC 46646 − − − − − − −
Al. alternata ATCC 11612 − − − − − + −
Ch. globosum NRRL 1870 − − − − − − −
Cl. cladosporioides NIOSH 17-28-17 − − − + − − −
Cl. sphaerospermum ATCC 11288 − − − − − − −
Ex. rostratum ATCC 26856 − − − − − + +
Fu. moniliforme NIOSH 32-40-16 − − − − − − −
Fu. oxysporum NIOSH 32-40-14 − − − − − − −
Ne. fischeri NIOSH 29-53-20 − − − − − − −
Pa. variotii ATCC 66705 − − − − − + +
Pe. chrysogenum NRRL 1951 − − − − − − −
Pe. expansum NRRL 973 − − − − − − −
Pe. melinii NIOSH 32-46-01 − − − − − − −
Pe. purpurogenum NRRL 1062 − − − − − − −
Sc. brumptii ATCC 16278 − − − − − − −
Tr. harzianum NIOSH 29-32-13 − − − − − + +
Tr. viride ATCC 16640 − − − − − − −
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ELISAs

Screening mAbs.

Hybridomas producing anti-terrelysin mAbs were identified by indirect ELISA. In brief, 96-well Immuno MaxiSorp microplates (Nunc) were coated overnight with rTerrelysin (1 µg ml−1) in 0.05 M carbonate coating buffer pH 9.6, and blocked with PBS containing 0.5 % Tween 20 and 5 % nonfat dry milk (PBSTM) for 1 h. CSN from each hybridoma was incubated in duplicate wells for 1 h at 37 °C, washed with PBS containing 0.5 % Tween 20 (PBST), and detected using alkaline-phosphatase-conjugated goat-anti-mouse IgG antibody (H+L) (Promega) diluted 1 : 5000 in PBSTM for 1 h at 37 °C. The wells were then washed in PBST and developed for 30 min using 4-nitrophenyl phosphate substrate (Sigma). Reactivity was determined by measuring A405. To compare the reactivity of identified mAbs, each mAb was purified, and used at a concentration of 1 µg ml−1 in assays with various dilutions of rTerrelysin bound to the assay plate. The plates were washed three times between individual steps of incubation with PBST.

Inhibition ELISA for analysis of native terrelysin.

An inhibition ELISA was performed over a period of 2 days. On day 1, the ‘assay plate’ was coated with 0.1 µg per well of rTerrelysin and a second ‘inhibition plate’ was blocked overnight with PBSTM to prevent protein binding. On day 2, both plates were washed with PBST and the ‘assay plate’ was blocked with PBSTM at room temperature for 2 h. In the inhibition plate, serial dilutions of ME and CSN samples (protein concentration range 5–0.078 mg ml−1) and serially diluted rTerrelysin standard (protein concentration range 78 µg ml−1–1 mg ml−1) were incubated with 40 ng anti-terrelysin mAb 10G4 ml−1. Negative control wells were incubated with PBS and positive control wells were incubated with mAb 10G4 alone. The inhibition plate was incubated on a shaker at room temperature for 1 h at 200 r.p.m. and for 30 min without shaking. Next, the assay plates were washed and incubated with 100 µl of the mixture from the inhibition plate at 37 °C for 1 h. Plates were washed and incubated with alkaline-phosphatase-conjugated goat anti-mouse IgG antibody (H+L) diluted 1 : 5000 in PBSTM for 1 h at 37 °C. Plates were washed and developed using 4-nitrophenyl phosphate substrate. Reactivity was measured as A405 and terrelysin concentrations were determined for each sample by comparison with the standard curve using regression analysis.

Western blot analysis of rTerrelysin and fungal ME.

Western blot analysis was performed for screening reactivity of individual mAbs to rTerrelysin and native terrelysin (A. terreus CSN and ME), and for cross-reactivity analysis. rTerrelysin (500 ng ml−1) and ME (2.5 mg ml−1) collected from 4 day cultures of A. terreus were individually separated using SDS-PAGE on 12 % polyacrylamide gels. For cross-reactivity testing, ME (2.5 mg ml−1) from 29 fungal species were separated on 12 % polyacrylamide gels. Proteins were transferred overnight to nitrocellulose membranes (0.22 µm, Bio-Rad) and the membranes were blocked using Tris-buffered saline (TBS) containing 0.1 % Tween 20 (TBST) and 3 % BSA (blocking buffer). Membranes were washed with TBST and transferred to a Bio-Rad Multi Screen apparatus. Individual lanes were incubated with 1 µg ml−1 of mAb diluted in blocking buffer and incubated on a rocker for 1 h. Membranes were washed three times with TBST and incubated for 1 h on a rocker with alkaline-phosphatase-conjugated goat anti-mouse IgG antibody (H+L) diluted 1 : 5000 in blocking buffer. Membranes were then washed with TBST and developed for 15–20 min using 1-Step NBT/BCIP (Promega) substrate solution. The reaction was stopped by washing the membranes with distilled water.

Epitope mapping.

Epitope mapping was performed using synthetic peptides synthesized by Sigma Genosys (JPT Peptide Technologies). For peptide scans, 68 peptides spanning the entire rTerrelysin sequence (including the Strep-tag II sequence) were synthesized as linear decapeptides overlapping by two amino acids. Peptides were covalently bound to a Whatman 50 cellulose support (PepSpots membrane) at the C-terminus, and the N-terminus was acetylated for higher stability. The membranes were processed for epitope mapping according to the manufacturer’s instructions.

In brief, the PepSpots membrane was rinsed in methanol for 5 min, washed three times with TBS for 10 min, then blocked overnight at 4 °C on a shaker with TBS containing 3 % BSA. The membrane was incubated with 5 µg ml−1 of rTerrelysin mAbs for 3 h at room temperature on a shaker. mAb 9B4, an IgG1 isotype which recognizes a Stachybotrys chartarum conidial surface protein, served as a negative control (Schmechel et al., 2006). The membrane was washed in TBST three times for 5 min each and then incubated with goat anti-mouse IgG horseradish-peroxidase-conjugated antibody (Promega) diluted 1 : 50 000 in blocking buffer for 1 h at room temperature on a shaker. The membrane was washed thoroughly in TBST three times for 5 min each and developed with ECL Western blotting substrate (Promega) according to the manufacturer’s instructions. After a brief incubation, excess substrate was discarded and the membrane was exposed to CL-XPosure clear blue X-Ray film (Thermo Scientific) and developed using an SRX-101A tabletop processor (Konica Minolta). For regeneration, the PepSpots membrane was washed twice with water for 10 min each and then incubated with regeneration buffer I (62.5 mM Tris containing 2 % SDS, pH 6.7; 100 mM 2-mercaptoethanol) at 50 °C using four 30 min incubations. The membranes were washed three times for 20 min with PBS (10×conc.), three times for 5 min with TBST, and three times with TBS for 10 min at room temperature. The membrane was analysed to ensure efficient removal of bound primary and secondary antibody prior to analysis of new mAbs.

Microscopic examination of A. terreus morphology.

In order to correlate the expression of terrelysin with growth changes in A. terreus, we cultivated the fungus in 24-well plates containing 2 ml minimal medium. Wells were inoculated with 1×106 viable A. terreus FGSC 1156 conidia and incubated on a shaker at either room temperature or 37 °C. Growth was monitored at various time points using an Olympus IX70 microscope; images were captured using a QImaging Retiga 2000R Fast camera and processed using the SimplePCI6 software (Hamamatsu Corp.).

Confocal scanning laser microscopy localization of native A. terreus terrelysin.

Immunolocalization of terrelysin was studied using a slight modification of previously described methods (Osmani et al., 1988; Xiang et al., 1995). Briefly, A. terreus FGSC 1156 cultures were grown on alcohol-sterilized coverslips in 6-well tissue culture plates that contained minimal medium. Cultures were incubated at 37 °C under static conditions for 24 h. Coverslips were fixed with 8 % formalin in buffered saline that contained 50 mM PIPES (pH 6.7), 25 mM EGTA, 1 % DMSO and 5 mM MgSO4 for 1 h at room temperature and the coverslips were then rinsed with MTSB (50 mM PIPES pH 6.7, 5 mM EGTA and 5 mM MgSO4). Cell wall digestion was carried out for 1 h at room temperature with an enzyme solution containing 2.5 % Driselase (Sigma), 1 % lysozyme from chicken egg white (Sigma) and 2 mM EGTA. Cells were rinsed with water and then treated with 0.1 % Triton X-100 in TBS pH 7.4 for 10 min. Cells were then rinsed in MTSB and TBS once each. Cells were then blocked with 3 % BSA in TBS (TBSB) overnight at 4 °C with gentle shaking. Next, the coverslips were incubated with mAb 15B5 (3 µg ml−1) in TBSB for 3 h with gentle shaking. mAb 9B4 (against a conidial surface protein of S. chartarum) served as a negative control and mAb 13E11, which reacts with an A. terreus exoantigen, leucine aminopeptidase, served as a positive control (Nayak et al., 2011b). Cells were washed thoroughly in TBST and stained with AlexaFluor 594-conjugated goat anti-mouse IgG antibody (Molecular Probes) diluted 1 : 50 in TBSB for 1 h at room temperature. Cells were washed in TBST and coverslips were placed on clean slides with ProLong Antifade Reagent with DAPI (Molecular Probes). Cells were observed with a Zeiss LSM-510 Meta Confocal Microscope System (Axioplan 2 Stand) and the images were acquired with Zeiss software version 3.2. All settings on the confocal laser microscope remained constant throughout the analysis.

Results

Isotyping and sensitivity screening of rTerrelysin mAbs

Thirty-two hybridomas recognized rTerrelysin during the initial screening. Sixteen clones survived multiple cloning steps and were further analysed in screening, cross-reactivity, and immunolocalization experiments. Nine clones (2G4, 3B2, 6D2, 7D8, 13G10, 15B5, 16C7, 9F4, 19E3) produced IgG1 isotype mAbs, four (3B7, 10G4, 15C5, 15E4) produced IgG2a isotype mAbs, and three (6E4, 10G7, 2D3) produced IgG2b isotype mAbs. These mAbs exhibited variable reactivity to immobilized rTerrelysin in ELISA (Fig. 1). mAbs 2G4, 9F4, 19E3, 6E4 and 15E4 showed the least reactivity (A405 ≤0.25 value) to rTerrelysin (data not shown). mAbs 3B2, 6D2, 7D8 and 16C7 showed moderate reactivity, while mAbs 13G10, 15B5, 3B7, 10G4, 15C5, 10G7 and 2D3 showed highest reactivity to rTerrelysin.

Fig. 1.

Fig. 1.

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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).

Western blot screening of mAbs to rTerrelysin and native terrelysin

Western blot analysis revealed reactivity to denatured rTerrelysin (16.5 kDa) for 11 of the 16 mAbs (Fig. 2a). The higher molecular mass bands observed in the lanes with purified rTerrelysin are likely to be aggregates formed during the storage of the protein at −20 °C. For native terrelysin, four of the 11 mAbs (3B2, 6D2, 16C7 and 2D3) did not show any reactivity with A. terreus ME (Fig. 2b). Seven mAbs (7D8, 13G10, 15B5, 3B7, 10G4, 15C5, and 10G7) reacted to a putative native terrelysin in ME and were chosen for further study.

Fig. 2.

Fig. 2.

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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.

Cross-reactivity and strain variations

Five strains of A. terreus, including the pathogenic strain FGSC 1156 and the commonly used environmental strain ATCC 1012, were analysed with each mAb by Western blotting. All mAbs, except 7D8, exhibited reactivity to a protein of the same molecular mass in ME derived from all strains (Table 1). Interestingly, mAb 7D8 reacted to ME from three strains, but two strains (NIOSH 17-30-31 and NIOSH 35-08-06) did not react.

ME from 11 other Aspergillus species, including the pathogens A. fumigatus, A. flavus, A. nidulans and A. niger, were also tested. mAb 15C5 showed maximum cross-reactivity among the mAbs tested, exhibiting cross-reactivity to ME from six different fungi, including A. nidulans and A. parasiticus. mAb 10G7 did not cross-react with any tested Aspergillus species, but it did react to ME from Exserohilum rostratum, Paecilomyces variotii and Trichoderma harzianum. mAb 3B7 cross-reacted to ME from Cladosporium cladosporioides. No cross-reactivity was observed with any mAbs against ME from four Penicillium species. Four mAbs (7D8, 13G10, 15B5 and 10G4) did not cross-react with any of the tested fungal species (Table 1).

Epitopes for rTerrelysin mAbs

Owing to their high specificity determined in cross-reactivity studies, three mAbs (15B5, 13G10 and 10G4) were tested to determine their epitopes using overlapping decapeptides spanning the entire sequence of rTerrelysin. For mAb 15B5, two spots were recognized, at positions 20 and 21 (Fig. 3). The sequences of spots 20 and 21 correspond to SFLYEGQFHS and LYEGQFHSPE, respectively. These data suggest that the epitope recognized by mAb 15B5 is LYEGQFHS.

Fig. 3.

Fig. 3.

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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.

After ensuring complete removal of bound mAb 15B5 and regeneration of the PepSpots membrane, the membrane was scanned using mAb 13G10. Four consecutive spots (52–55) reacted with mAb 13G10 and the resultant epitope was PSNEFE (Fig. 3). Subsequently, mAb 10G4 was found to recognize the same epitope as mAb 13G10 based on SPOTscan data (Fig. 3). SPOTscan of the membrane using control mAb 9B4 and secondary antibodies did not result in reactivity to any spots (data not shown).

Time-point kinetics of terrelysin expression

The expression of native terrelysin was examined using an inhibition ELISA to quantify the amount of terrelysin in CSN and ME at 24 h intervals (Fig. 4a). In ME, the highest relative concentrations of terrelysin were observed during the first few days of growth, ranging between 10 and 12 µg per mg total fungal protein. A 10-fold reduction was observed by day 4–5 (1 µg per mg total protein) and after day 6 very little terrelysin was quantified in ME. Lower levels of terrelysin were detected in the CSN, with a maximum at day 6 and then declining to nearly baseline by day 12.

Fig. 4.

Fig. 4.

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(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.

The morphological features that were observed at the same time points are depicted in Fig. 4(b). The early time points, when expression of terrelysin was the highest, involved the initial stages of conidial germination and hyphal extension (12–24 h). Hyphal aggregation was observed up to 96–120 h, when quantities of terrelysin were observed to decline. Beyond this time period, the morphological changes could not be differentiated. Collectively, these results suggest that terrelysin expression occurs during initial stages of fungal growth (days 0–4). When hyphal aggregation was complete, a concurrent reduction in the production of terrelysin was observed. Terrelysin was detected in the CSN at each measured time point, with peak concentrations observed on day 6.

As A. terreus is thermotolerant, this experiment was repeated at 37 °C to further compare morphological stages with terrelysin production. Again, cultures were grown for 12 days and ME and CSN were collected at 24 h intervals. Overal, the growth of A. terreus was accelerated and the morphological stages of conidial germination and hyphal extension occurred earlier compared to room temperature treatments (Fig. 5b). Terrelysin expression was highest on day 1 (12 µg per mg total protein) and had decreased threefold by day 2 (4 µg per mg total protein) (Fig. 5a). The concentration of terrelysin was further reduced eightfold by day 3 (0.5 µg per mg total protein). In CSN, a comparatively higher proportion of terrelysin was detected during initial growth at 37 °C compared to room temperature; however, there was a decline in the concentration of terrelysin in the culture supernatant at 37 °C.

Fig. 5.

Fig. 5.

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(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.

In summary, we observed that terrelysin expression is concurrent with conidial germination, hyphal extension and aggregation. Terrelysin concentration declined on formation of the mycelial pellets. Lower levels of terrelysin were detected in the CSN than in ME.

Immunolocalization of terrelysin

The cellular localization of terrelysin was examined by immunohistochemical staining methods using the terrelysin-specific mAb 15B5. Terrelysin was widely distributed within the hyphae (Fig. 6), with staining often of greater intensity near the hyphal tips. This pattern was distinctly different from that seen with the positive control, mAb 13E11, which stained a leucine aminopeptidase (a secreted protease) within the extracellular matrix of the hyphae (Nayak et al., 2011b). No staining was observed in the extracellular matrix with mAb 15B5.

Fig. 6.

Fig. 6.

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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.

Discussion

A. terreus is an emerging opportunistic pathogen reported to be an aetiological agent of fatal disseminated infections in immunocompromised populations (Baddley et al., 2003; Hachem et al., 2004; Iwen et al., 1998). Due to the ability of this filamentous fungus to grow at internal body temperatures and produce accessory conidia, A. terreus is capable of initiating an infection and disseminating within the body. Resistance to antibiotic treatment such as amphotericin B allows for a longer infection time until effective treatment. A combination of these factors facilitates rapid dissemination of the fungus and can result in mortality. To date, very little is known about the overall pathogenesis and involvement of specific virulence factors of A. terreus during infection.

In A. fumigatus infections, Asp-haemolysin was reported in tissues of experimental animals in an infection model (Ebina et al., 1982), and a role for haemolysins as potential virulence factors has been suggested (Berne et al., 2009; Ebina et al., 1982; Kumagai et al., 1999; Žužek et al., 2006).

Previously, the gene for terrelysin was cloned and used to produce a recombinant protein (Nayak et al., 2011a). In this study, rTerrelysin was used as the antigen for the development of terrelysin-specific mAbs, which were then utilized to characterize terrelysin expression during A. terreus growth. Four of the 11 mAbs generated to rTerrelysin (3B2, 6D2, 16C7 and 2D3) did not react to native terrelysin in A. terreus ME. Two possible reasons for this are (a) the mAbs recognized the N-terminal tag associated with rTerrelysin or (b) their epitopes may be modified by post-translational processes under natural conditions, as there is a putative N-glycosylation (N-X-S/T) site in the terrelysin sequence. One antibody (mAb 2D3) reacted with other purified recombinant proteins (rEnolase from Chaetomium globosum and rHev b 5, an allergen from the rubber tree Hevea brasiliensis) expressed in the same pASK-IBA6 vector (data not shown). It was concluded that mAb 2D3 recognizes an epitope in the N-terminal tag expressed by this vector. Epitope mapping showed that mAb 2D3 recognizes the Strep-tag II sequence WSHPQFEK (data not shown). The other three mAbs that did not react with native terrelysin did not show reactivity to other recombinant proteins; it is possible that their epitopes are conformational or blocked by post-translational modifications to the native protein.

In order to be useful as a diagnostic tool for the serological detection of terrelysin, it is essential that the mAb be species-specific. We tested five A. terreus strains, including the strain used for genomic sequencing (NIH 2624/FGSC A1156). All mAbs tested detected terrelysin expressed in this strain. Interestingly, mAb 7D8 showed reactivity to only three of the five strains of A. terreus tested; while low affinity might explain this inconsistent detection, genetic diversity within the epitope for this mAb is also a possibility. Although additional strains from different clinical and environmental isolates were not tested, the data suggest consistent expression of terrelysin across strains. Recently, a new species, Aspergillus alabamensis, which is phenotypically similar to but genetically different from A. terreus, was isolated from immunocompetent patient populations (Balajee et al., 2009b). It will be important to test the reactivity of these mAbs to A. alabamensis as well as other Aspergillus species within Section Terrei such as A. carneus and A. niveus, which can also cause infections under rare circumstances (Crissy et al., 1995; Wadhwani & Srivastava, 1984).

The terrelysin mAbs did not cross-react to ME from other clinically relevant Aspergillus species, including A. fumigatus, A. flavus and A. niger. Weak reactivity was observed for mAb 15C5 with A. nidulans and A. parasiticus in Western blots. This reactivity was restricted to several high-molecular-mass proteins of ~50 and ~60 kDa and probably represents non-specific staining. Using cross-reactivity data, we identified three mAbs (13G10, 15B5 and 10G4) that did not cross-react with any of the tested species. These three highly specific mAbs are important candidates for immunodiagnostic detection of A. terreus in clinical samples. Based on epitope mapping studies, we identified two different epitopes recognized by these antibodies. mAb 15B5 (IgG1) recognized the epitope LYEGQFHS while both mAbs 13G10 (IgG1) and 10G4 (IgG2a) recognized the epitope PSNEFE. These epitope sequences were not identified in homologous haemolysins derived from other fungal species with known aegerolysin sequences (Nayak et al., 2011a).

Expression of haemolysin in relation to growth has been studied previously for Pleurotus ostreatus and Pseudomonas aeruginosa (Berne et al., 2002, 2007; Rao et al., 2008). For P. ostreatus, a basidiomycete, the greatest expression of ostreolysin was observed during the initial stages of fungal fruiting. For the bacterium Ps. aeruginosa, it was observed that expression of its haemolysin, PA0122, is highest in the stationary phase of growth (Rao et al., 2008). In contrast, limited information is available for the expression of aegerolysins in ascomycetes. Recent studies have reported high Asp-haemolysin transcript levels in developing hyphae of A. fumigatus (Gravelat et al., 2008). In the present study, the expression of terrelysin was associated with particular morphological stages of A. terreus growth. The presence of terrelysin was highest during initial growth stages encompassing conidial germination, hyphal extension and hyphal aggregation (‘exponential phase’). During the stationary growth phase (lack of increase in mycelial pellet size) detection of terrelysin was significantly reduced. This was observed at earlier time points when cultures were grown at 37 °C rather than at room temperature, and the presence of terrelysin consistently correlated with the morphological changes. These findings are consistent with studies by Rutqvist (1965), which showed that the haemolytic principle could be purified from A. fumigatus mycelia only during a limited period of incubation at room temperature and that this period was earlier and shorter at 37 °C.

Using an inhibition ELISA, terrelysin was detected in CSN even though we did not identify a signal peptide on the terrelysin sequence using SignalP 3.0. This is in contrast to a report by Han et al. (2010), in which terrelysin was not identified in the secretome of A. terreus. Whether our detection of terrelysin in CSN is a result of active secretion, hyphal degradation, fragmentation or the involvement of unique secretory processes currently remains unknown and will be the focus of future research. Recently, Asp-haemolysin was identified as one of the most abundant proteins in the A. fumigatus secretome even though this protein does not possess a signal peptide (Wartenberg et al., 2011). Other studies have highlighted secretion of proteins with no predicted signal sequence and have proposed non-conventional secretory mechanisms (Medina et al., 2005; Nickel, 2010; Rubartelli & Sitia, 1997). At day 6 in room temperature cultures and at day 4 at 37 °C, small increases in levels of terrelysin were observed in CSN. Hyphal fragmentation was observed at these time points and this could, in part, explain the presence of terrelysin in CSN. Our immunolocalization studies demonstrate a uniform distribution of terrelysin within A. terreus hyphal cytoplasm, with greater reactivity localized at hyphal tips. The hyphal tips are a region of extensive metabolic activity and contain higher concentrations of proteins compared to regions adjacent to this zone of apical extension. Based on our morphological and immunohistochemical observations, it appears that terrelysin is expressed during early stages of growth, probably during the emergence of hyphae from conidia and active hyphal growth.

In summary, seven terrelysin mAbs were characterized and three were identified to be highly specific for A. terreus. These mAbs were used to show that terrelysin is expressed during conidial germination and early growth of A. terreus hyphae. Since terrelysin was detected in CSN, it is possible that it could be detected in the serum of infected patients; however, this aspect has not been confirmed and will be the focus of future research. Serological detection of terrelysin would make this protein useful as a biomarker; however, the early expression of terrelysin in the A. terreus growth cycle may limit its usefulness for detecting infection. The kinetics of terrelysin production in vivo is unknown and more studies are required.

Acknowledgements

The findings and the conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health. This work was supported in part by an InterAgency agreement with the National Institute of Environmental Health Sciences (Y1-ES0001-06).

Abbreviations:

CSN

culture supernatant

ME

mycelial extracts

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