Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.

Introduction

Species of Aspergillus are found worldwide as a saprophyte and among these A. fumigatus is known to be the commonest associated with human mycoses. A. fumigatus has high sporulating capacity that results in the presence of high concentration of minute conidia in the air [1]. A. fumigatus is responsible for a range of diseases such as allergic diseases (asthma, allergic sinusitis, and alveolitis), invasive aspergillosis, 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 is mandatory which may be due to AIDS, prolonged use of chemotherapy in cancer patients or the use of immunosuppressant in the organ transplant cases [2]. A. fumigatus conidia inhaled by normal immunocompetent human are efficiently cleared by the innate immune response whereas in immunocompromised persons the conidia grow and release virulence factors. The factors developed in A. fumigatus for survival in the soil environment (natural habitat) become virulent when it accidentally infects humans and escapes from the innate immunity of hosts. There is a diversity of virulence factors in different forms of aspergillosis as they are under polygenic control, and depend on the stage specific protein biosynthesis [3].

The number of cases of aspergillosis has increased in past three decades but the prognosis of such infections remains a challenge to clinicians pending prompt diagnosis. 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 therefore the better and more reliable techniques for early diagnosis are needed. This demands the exhaustive knowledge of fungal antigens which may be detected in the body fluids of patients. Few antigens specific for the ABPA, aspergilloma, and invasive aspergillosis are known and being evaluated for the diagnosis [4]. 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 [3], [5]. 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 production of several cytokines [6]. 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.

With publication of whole genome sequence of A. fumigatus [7] the study of pathobiology of this fungus became more feasible. The proteomic approach for better understanding of the host–pathogen interactions is the method of choice and the immunoproteomics is the tool for the discovery of biomarkers for early diagnosis of aspergillosis [4]. Secretome analysis of A. fumigatus and the immunogenic potential of the secretory proteins might help in understanding its virulence factors, drug targets, and targets for immunodiagnosis of the diseases. The present study is an attempt to analyze the secretome of A. fumigatus by 2D gel in combination with MS/MS analysis on MALDI-TOF–TOF platform and immunosecretome by western blotting of 2D gel transfer blot with pooled patient sera, immunized rabbit sera and immunized mice sera that may be useful for identification of target molecules for early diagnosis as well as in identification of virulence factors required to understand the manifestations caused by aspergillosis.