Immunosecretome analysis of Aspergillus fumigatus and generation of monoclonal antibodies

Abstract

The kingdom Fungi has approximately 100,000 species which have validly been described so far and out of these nearly 150 species have been recognized as primary pathogens of human and animals. Most of these fungi are termed as opportunistic pathogens except for Histoplasma, Coccidioides, Blastomyces, and Paracoccidioides which infect both immunocompromised as well as immunocompetent individuals. Among the opportunistic pathogens species of Aspergillus are found worldwide as a saprophyte and A. fumigatus is known to be the commonest species associated with human and animal mycoses. A. fumigatus has high sporulating capacity that results in the presence of high concentration of minute conidia in the air environment [1]. Aspergillus fumigatus is responsible for a range of diseases such as allergic diseases (causing asthma, allergic sinusitis, and alveolitis), invasive aspergillosis (IA), allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma caused due to inhalation of minute asexual spores present in the environment. In almost all the cases of aspergillosis immunocompromised condition of the host is mandatory which may be due to AIDS, prolonged use of chemotherapy in cancer patients or the use of immunosuppressant in the organ transplanted persons [2]. The cases of aspergillosis have increased in past three decades but the prognosis of such infections remains a challenge to clinicians pending prompt diagnosis and lack of broad spectrum antifungal agents. The diagnosis of such infections is largely dependent on the conventional methods that require isolation of the causal agent from the site of infected tissue/organ which is not always feasible. Further this requires longer time period to confirm the diagnosis. Most of the antifungal available in the market to combat such infections are fungistatic in nature and may result in the development of resistance in the causal organism. Therefore there is a renewed focus on the understanding of pathogenicity which is still in infant stage. For the early diagnosis people have used detection of antibodies in the secretions such as serum and urine but this method is not reliable because it is well known that antibodies once produced may remain elevated for longer periods even after the cure of the disease. This may result in incorrect/false diagnosis of the infection. Another option is the detection of antigens of the fungal pathogen that are released during aspergillosis which can be detected at an early stage with reliability. Platelia Aspergillus is a commercially available ELISA test to detect the released galactomannan by use of EB-A2 rat monoclonal antibody (BioRad, France). Platelia has been in use in Europe for more than 10 years and recently it was approved for use in the United States. But the release and kinetics of circulating galactomannan remains undefined and depends on growth phase, microenvironment, host immune status and pathology [3]. Other techniques such as detection of antibodies against Aspergillus spp., PCR technology for DNA and RNA analysis are also being used. Still from diagnostic point of view, improving the test accuracy remains a priority for patient care, therapeutic and diagnostic research [4]. PCR has the capability of rapid detection and molecular identification of opportunistic moulds besides Aspergillus at the genus level. The sensitivity of PCR is excellent, but its low level of specificity for invasive infections can be problematic, and falsepositive results are common [5]. A form of diagnosis based on clinical, laboratory, radiological, and immunological aspects should be sought, since the early recognition and treatment of ABPA can prevent the progression of the disease to pulmonary fibrosis [6]. During the course of infection the secreted proteins keep due importance when studying fungal pathogenicity since these molecules trigger the ongoing interaction with the host immune systems. The secretory proteins of A. fumigatus contain enzymes (such as proteases, peptidases and phospholipases), secreted toxins, adhesins and other molecules responsible to invade host immune systems and cause pathogenesis [7,8]. A. fumigatus secretes exoproteases and other cytoplasmic fungal products that are capable of compromising mucociliary clearance, breaching the airway epithelial barrier, and activating the innate immune system of the lung, including epithelial production of several cytokines [9]. The virulence caused by A. fumigatus may be augmented by its numerous secondary metabolites, including fumagillin, gliotoxin, helvolic acid etc [2]. Thus, the identification and study of secreted proteins may divulge unique infection mechanisms that could lead to new control measures for the aspergillosis. Further, the challenge remains in the field of treatment of disease associated with the Aspergillus spp. Many new antifungal agents with promising anti- Aspergillus activity have been introduced recently, but the mortality associated with invasive aspergillosis (IA) remains high. Currently there are four classes of antifungal agents with activity against Aspergillus: the polyenes, such as amphotericin B (AMB), and nystatin; the triazoles, itraconazole (ITC), voriconazole (VRC) and posaconazole (POS); the echinocandins such as caspofungin (CAS), micafungin (MICA) and anidulafungin; and the allylamines such as terbinafine (TRB) [10]. Most of the antifungals have side effects to the host. AMB the “gold” standard for antifungal therapy of invasive aspergillosis is associated with two major drawbacks, insolubility in water and severe side effects, particularly nephrotoxicty [2]. The introduction of lipid formulations of amphotericin B, as well as the triazoles, such as fluconazole and itraconazole has been found effective against fungal infections. Although these agents overcome the drawbacks associated with amphotericin B, they were limited by formulation, spectrum of activity, and the development of resistance. Despite considerable progress in the past decade, the morbidity and mortality of fungal infections are still high. Therefore there is a need for search and development of antifungal agents with new mechanisms of action that have a broad spectrum of activity (including resistant pathogens) and ease of administration. With publication of complete genome sequence of A. fumigatus [11] the study of pathobiology of this fungus becomes more feasible. The proteomics are the method of choice to get better understanding of the host-pathogen interactions. Immunoproteomics are the tool for the discovery of biomarkers for early diagnosis of aspergillosis [12]. Several proteomic studies on A. fumigatus have been carried out including optimization of sample preparation of 2-D gel for the fungi grown on different media [13], study of intracellular proteins [14], GPI- anchored proteins [15], conidial surface proteins [16], and functional proteomic study of β-glucosidase [17]. But the complete secretome analysis of A. fumigatus is not reported till date. Secretome-related studies are particularly relevant in understanding filamentous fungi because many fungi secrete a vast number of proteins to accommodate their saprotrophic lifestyle. The large numbers of extracellular hydrolytic enzymes are necessary to digest a plethora of potential substrates. Many of these proteins are of special interest in the study of host-pathogens interaction [18]. Secretome analysis of Aspergillus fumigatus and the immunogenic potential of the secretory proteins would be an asset for a long-term goal to understand its virulence factors, drug targets, and targets for immunodiagnosis of the diseases. Antibody generation is virtually universal during the interactions between the host and fungi. This occurs in response to normal exposure to environmental fungi during active infection caused by opportunistic and true pathogenic fungi. Antibody production influences immunity that can either be beneficial, neutral or detrimental to the host. The antibodies play direct role, such as neutralization of toxins and microorganisms, and indirect role with other components of the immune system, such as opsonization, complement activation, antibody-dependent cellular cytotoxicity, immunomodulation and the generation of protective memory antifungal immunity [19]. Hybridoma technology is being used to generate monoclonal antibodies against the antigenic proteins of A. fumigatus, which have the anti-Aspergillus activity in vitro and in murine model [20,21] or early diagnostic potential, indentifying the secreted galactomanan in the serum [22,23]. The objective of the present study was focused on the immunosecretome analysis of A. fumigatus and generation of monoclonal antibodies against the secretory proteins of A. fumigatus. In this piece of work, the secretory proteins were isolated from the culture filtrate and identified by 2D gel in combination with MALDI-TOF-TOF analysis. The immunosecretome analysis was carried out by western blotting of 2D gel transfer blot with pooled patient sera, immunized rabbit sera and immunized mouse sera followed by identification of the protein spots. The proteins identified through secretome analysis were characterized in silico for the identification of secretory signal by signalP analysis and for homology search with human proteins by NCBI BLAST search tool. The immunosecretome analysis is useful for identification of target molecules with early diagnostic potential as well as also useful in identification of virulence factors, required to understand the manifestations caused by aspergillosis. Hybridomas producing monoclonal antibody against secretory antigens of A. fumigatus were generated by hybridoma technology. The produced monoclonal antibodies were characterized for their diagnostic as well as for therapeutic potential.