Aspergillus fumigatus is known as the most common cause of IA in humans. The extracellular proteins of A. fumigatus, which functions in enabling the fungi to adhere to host tissues and take up nutrients from the hosts, play an important role in the interaction between fungi and hosts. The extracellular location of these proteins enables the proteins to interact easily with the host immune system. Accordingly, studies on the immunogenic nature of these extracellular proteins are of particular importance to understand the pathogenesis of A. fumigatus. The immunogenic proteins may represent candidate markers for the diagnosis of IA. In fact, preparations of A. fumigatus extracellular proteins have been used to detect antibodies in the sera of human patients or experimentally infected animals, and culture filtrates have also been used to raise polyclonal antibodies to detect A. fumigatus antigens in the sera or urine of patients or experimentally infected rats [26, 27]. Our group has previously observed that high titers of antibodies against extracellular proteins of A. fumigatus are often present in the sera of critically ill IA patients (unpublished data). However, knowledge of the extracellular proteins of A. fumigatus and the corresponding antibodies is limited. To investigate the immunodominant antigens, the extracellular proteins at different intervals were extracted from 4 media (Aspergillus minimal medium, YEPG, Czapek-Dox medium, and RPMI-1640), then probed with sera of IA patients. The results indicated that the protein yield reached a maximum at 14 days, and the YEPG culture supernatant contained the maximum number of proteins reacting with the sera in comparison to other media (unpublished data). Thus, the 14-day YEPG filtrate proteins were used in a subsequent study.
In the present study, the immunodominant proteins from the culture filtrate of A. fumigatus were detected using an immunoproteomic approach. The immunoreactive protein spots showing a significantly different pattern of recognition in sera from IA patients when compared with specimens from controls were characterized by MS. Of 17 identified proteins, 7 have been reported as antigens of Aspergillus and/or other fungi. For example, DppV, TR, FAD-dependant oxygenase, pectate lyase A, aspartyl aminopeptidase, and NAD-dependent malate dehydrogenase are already known as antigens or allergens of Aspergillus [28–31]. Fructose-bisphosphate aldolase was identified as an immunogen in patients with systemic candidiasis . Furthermore, diverse groups have reported that some metabolic enzymes interact specifically with human extracellar matrix proteins, such as fibronectin, laminin, and integrin-like vitronectin [33, 34], and are involved in adhesion and pathogenesis.
The immunodominant nature of these proteins is valuable for the diagnosis of invasive fungal infections. DppV, a member of the dipeptidyl-peptidase family in A. fumigatus, is identical to one of the principal antigens used in the diagnosis of IA. Moreover, DppV can generate protection responses, and improve the survival rate of Aspergillus-infected mice . DppV can also bind with collagen or other human proteins and degrade them, which can damage the host. Recombinant DppV has shown a great potential in the serodiagnosis of IA in immunocompromised and immunocompetent patients . NAD-dependent malate dehydrogenase, a key enzyme in glycometabolism that catalyze the reversible conversion between malate and oxaloacetate, was reported recently as an allergen of A. fumigatus and A. versicolor . Malate dehydrogenase was also shown to be a Paracoccidioides brasileinsis immunogenic protein  as well as a Candida albicans immunogen . Aspartyl aminopeptidase, an enzyme that specifically degrades only amino-terminal acidic amino acids from peptides, was recently reported as an antigen of A. fumigatus 
TR of A. fumigatus has been described as an extracellular antigenic protein by two recent studies [30, 31]. In one former study, the secreted fraction of two geographically different strains (190/96 and DAYA) of A. fumigatus were used to identify new immunogenic molecules reacting with pooled ABPA patient sera (IgG and IgE). TR was only detected on 2DE immunoblots of the secreted proteome of the DAYA strain probed with the IgE antibody fraction from pooled ABPA patients sera . This result suggested that TR might not be a good biomarker for ABPA. In another study, the immunosecretome of A. fumigatus was detected using pooled patient sera (total n = 22 patients [ABPA, n = 11; aspergilloma, n = 5; IA, n = 6]). The immunoreactive intensity of TR was lower than most other proteins . A possible explanation is that the anti-TR antibody titers were not high in pooled sera because most cases included in the study were not IA.
Although investigators in other laboratories recently noted the antigenic nature of TR [30, 31], no study has found shown diagnostic value for TR in non-neutropenic patients with IA. We showed that TR (spot no. 2A-2 M) had the strongest immunoreactivity with patient sera. TR, a component of the gliotoxin biosynthetic cluster, provides self protection to A. fumigatus against gliotoxin [37, 38]. This protein has been described as an extracellular protein of A. fumigatus by Singh and Kumar [30, 31]. However, Schrettl et al. showed that GliT is preferentially localized in the cytoplasm and nuclei by a GFP-GliT construct . To predict whether or not GliT is actively secreted into the culture supernatant, we used two bioinformatic tools (SignalP and WoLF PSORT) to analyze its localization. Our results support the findings of Singh and Kumar [30, 31]. Homology analysis indicated that TR had no match with human proteins, as Kumar et al.  reported. Muro et al.  also reported that the fungal TR has only 19% sequence similarity to human TR. Furthermore, sequence homology analysis showed that TR of A. fumigatus has low homology with most other Aspergillus species as well as most other fungi. Therefore, TR could be considered as a specific antigen of A. fumigatus and as a potential biomarker for the serological diagnosis of IA. In order to study its diagnostic potential, we cloned the TR gene and purified the recombinant protein. Immunoblots showed that recombinant protein could be recognized by the sera from all six IA patients. These results suggested that the TR of A. fumigatus could be developed as a biomarker for the diagnosis of IA, especially in critically ill patients.
One of the strengths of our study was that all patients included had histopathologic evidence and positive cultures. This enabled us to discriminate between invasive disease and colonization. However, we do realize that the study design has limitations. We did not further investigate the reactivity of individual patient serum with the extracellular fraction of A. fumigatus, thus we cannot provide data whether or not these proteins consistently react with individual IA patient serum. Moreover, the cases used in this study were limited in number, therefore the diagnostic value of the antigen identified should be validated in further prospective studies using large-scale serum specimens.