1. Introduction
Superficial, non-life-threatening infections of the human skin, nails and mucosa are the most common fungal diseases in humans and affect around one quarter of the world population., Infectious diseases caused by fungi also contribute substantially to human morbidity and mortality. In particular, invasive fungal infections are associated with high mortality rates, which often exceed 50%. Altogether, 1.5 million people are estimated to be killed by invasive mycoses worldwide each year. More than 90% of these deaths are caused by fungi of the four genera:
Candida,
Aspergillus,
Cryptococcus, and
Pneumocystis [
1]. Immunocompromised patients are particularly vulnerable to these fungal killers, whereas invasive fungal infections are extremely rare in immunocompetent individuals [
2].
Candida species are a polyphyletic group, which is part of the commensal flora of the gastrointestinal tract in more than a half of the healthy population [
2]. Under certain conditions,
Candida species are capable of causing a range of infections from superficial to dangerous invasive infections, designated as invasive candidiasis. Systemic
Candida infections have a high clinical relevance: They account for more than 70% of all invasive fungal infections in immunocompromised and critically ill patients [
3] and cause 8% of all nosocomial blood stream infections in the United States [
4]. Worldwide,
Candida albicans remains the most frequently isolated agent of candidiasis, but non-
Candida albicans species have gained clinical importance [
5].
Candida albicans is undoubtedly the best studied pathogenic
Candida species and several virulence traits have been identified so far. Among them are their ability to grow in the yeast or hyphal form (dimorphism), the production of molecules, which mediate adhesion and invasion, the formation of biofilms, the secretion of hydrolases, and the acquisition of essential trace metals [
6].
In contrast to
Candida, filamentous fungi of the genus
Aspergillus are soil-borne fungi with a saprophytic life style [
7]. Their asexually produced spores are easily dispersed into the air and due to their small diameter they penetrate deep into the respiratory tract upon inhalation. Because of that, most invasive
Aspergillus infections disseminate from the lungs [
8]. Patients at risk for developing invasive aspergillosis include neutropenic and critically ill patients as well as patients on high-dose steroid therapy [
9].
A. fumigatus is the major cause of invasive aspergillosis in transplant patients (65%) followed by
A. flavus and
A. niger [
10]. Exposure to
Aspergillus conidia can also lead to chronic infections and allergic responses, which result in allergic bronchopulmonary aspergillosis (ABPA) and severe asthma [
9]. Due to its medical importance, the virulence traits of
A. fumigatus have been most intensively studied and are based on multiple factors. The acquisition of iron by siderophores and the defense against immune effector cells based on the pigment 1,8-dihydroxynaphtalene melanin are the most prominent examples [
11,
12].
The basidiomycetous yeast
Cryptococcus is more distantly related to the genera
Candida and
Aspergillus.
Cryptococcus infections occur by the inhalation of infectious cells and are considered a primary pulmonary illness. Nevertheless, disseminated infections often lead to inflammatory diseases of the central nervous system [
13]. Among the 37 recognized species of
Cryptococcus,
C. neoformans, and
C. gattii are the major pathogens to humans.
C. neoformans infections occur mostly in immunodeficient individuals, particularly in patients with AIDS.
C. gattii can also infect immunocompetent hosts and has traditionally been considered as “tropical” or “subtropical” fungus”. Despite that, endemic outbreaks were reported from Vancouver Island, Canada [
14]. The polysaccharide capsule is the major virulence factor of
Cryptococcus to evade host defenses [
15], but also the formation of melanin and urease activity function as virulence determinants [
16,
17].
Pneumonia caused by the opportunistic pathogenic fungus
Pneumocystis jirovecii is the most prevalent opportunistic infection in patients with AIDS. It causes little or no disease in healthy individuals. The fungus is most probably transmitted via aerosols from person-to-person and exists almost exclusively within the alveoli of the lung and does not invade the host cell. Since Pneumocystis species have not yet been isolated in pure culture, little is known about their biology and pathogenicity determinants [
18,
19].
In addition to the aforementioned fungi, several other species are able to cause severe diseases in humans. Their occurrence is either restricted to a specific region of the world or the frequency of infections is relatively rare.Nonetheless, mucormycosis has emerged as the third most common invasive infection after candidiasis and aspergillosis in patients with hematological malignancies and allogeneic stem cell transplantation. Mucormycosis is caused by filamentous fungi of the order Mucorales in the class Zygomycota. Medically most significant are species of the genera
Rhizopus,
Lichtheimia, and
Mucor [
20].
Another group of ascomycetes are termed the dimorphic fungal pathogens. They cause diseases in endemic regions of the world and include
Histoplasma capsulatum,
Blastomyces dermatidis,
Coccidioides immitis,
Paracoccidioides brasiliensis,
Sporothrix schenkii, and
Penicillium marneffei. A common feature of these species is that they grow as molds in soil at ambient temperature and convert to pathogenic yeasts after infectious spores are inhaled by humans [
21,
22].
The interplay between the fungal pathogen and the human host is only partially understood. It is evident that the human professional phagocyte population consisting of monocyte/macrophages, polymorphonuclear leukocytes (neutrophils/PMNs) and dendritic cells (DCs) plays a central role in the defense against fungi. Usually, macrophages are the first cells to encounter an invading fungus. They recognize fungal pathogens via pathogen-associated molecular patterns (PAMPs), phagocytose, and consecutively kill them intracellularly. In addition, they generate a proinflammatory response to activate further immune cells. Neutrophils are the most abundant phagocyte population, which is immediately recruited to the sites of infection. They have high phagocytic activity and are endowed with powerful oxidative and non-oxidative microbicidal components [
23,
24,
25]. Besides phagocytosis, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs). NETs are characterized by the release of extracellular DNA associated with histones and granular and cytoplasmic proteins, which exhibit antimicrobial activity [
26,
27,
28]. In contrast, dendritic cells (DCs) are important antigen-presenting cells that act as messengers between the innate and adaptive immune system. They have been shown to be important for the discrimination between different fungal morphotypes or growth stages [
29].
Little is known about the contribution of the adaptive immune system to confer resistance against fungal pathogens. It is generally accepted that the development of a specific T
h response contributes to the susceptibility to invasive mycoses. In contrast, there is a lack of clear evidence that antibodies confer protection against pathogenic fungi [
30].
In short, fungal infections are controlled primarily by the host innate immune system. Knowledge about the interplay between fungal pathogens and immune cells has increased recently due to the investigation of host-pathogen interaction transcriptomes [
31,
32,
33,
34,
35,
36,
37]. Transcriptomic profiles of the interaction of pathogenic fungi with epithelial or endothelial cells have also been examined [
38,
39,
40,
41,
42]. Due to technical challenges such as sample quantity, complexity, and heterogeneity, proteomic studies on this topic are still rare. Several proteomic data are available from
C. albicans, but two of the few examples from
A. fumigatus described the response of human bronchial epithelial cells and endothelial cells in response to this pathogenic mold [
43,
44]. Here, we review current efforts and strategies to investigate the proteomic changes during interaction of pathogenic fungi with immune effector cells in the human host. We also give a brief overview about the investigation of fungal-specific serum antibody signatures in patients with invasive mycoses.
2. Immunoproteomics
In clinical fungal infection studies, circulating serum antibodies are important molecular markers as they reflect a molecular imprint of antigens of infectious agents. In addition, antigens specific for certain fungal pathogens are promising candidates for diagnostic biomarkers and vaccination strategies.
The first proteomics study on immunoreactive protein antigens of a pathogen interacting with the host humoral immune response was reported for
Borrelia burgdorferi by Jungblut in 1999 [
45]. Later, in 2001, the term “immunoproteomics” arose to define studies on large sets of proteins involved in the humoral immune response [
46]. Over the years, the technical advances in the field of proteomics have markedly facilitated the detection of pathogen-specific antigens.
2.1. Gel-Based Immunoproteomics
The combinatorial approach of 2D-GE followed by immunoblotting is highly effective to isolate and identify antigenic proteins (immunoproteome). This approach has been defined as serological proteome analysis (SERPA) [
47]. The principle works as follows: Immunoreactive proteins are two-dimensionally separated, transferred onto a membrane, and probed with patient serum, which presumably contains certain pathogen-related antibodies. Although antigens are denatured by 2D-GE and only linear epitopes can be detected, post-translational protein modifications that could be part of epitopes and affect antigen-antibody recognition are still retained during the denaturation step.
In the last decade, several serological proteome analyses with focus on antigens of human pathogenic fungi have been conducted. Pitarch and co-workers decoded the serological responses of the host to the cell wall proteome as well as the intracellular proteome of
C. albicans to identify novel diagnostic, prognostic, and therapeutic candidate markers for systemic candidiasis [
48,
49,
50]. Although
C. albicans is a commensal in the human gut provoking a basic and persistent anti-Candida antibody level in the host, the authors found that a pattern of 22 IgG serum antibodies (mainly against glycolytic enzymes and heat shock proteins) can differentiate invasive candidiasis (IC) from non-IC patients by using unsupervised clustering analyses. The authors highlighted that the serum IgG antibody signature directed against heat shock protein 90 (Hsp90) and enolase 1 (Eno1) of
C. albicans can be applied for IC diagnosis in non-neutropenic patients. Later, the same group combined fingerprints of IgG antibodies to two distinct protein species of Eno1 and Pfk1 (phosphoglycerate kinase) to discriminate candidemia from non-infected patients [
51]. Similar studies have also been carried out for
Aspergillus fumigatus. Due to the allergy invoking capacity of
A. fumigatus, many studies focused on screening for immunoreactive anti-
Aspergillus IgE antibodies. Glaser
et al. [
52] detected specific IgE antibodies against the phialide cell wall protein PhiA in the sera of 94% of all investigated ABPA patients. This protein was identified as a major allergen and may be regarded as a potential tool for specific diagnosis of allergic sensitization against
A. fumigatus. The serological response to
A. fumigatus protein antigens in patients with invasive aspergillosis has been investigated as well [
12]. Even antibodies specific to an enzyme involved in the biosynthesis of the mycotoxin gliotoxin were proposed as a potential biomarker for the diagnosis of IA in non-neutropenic patients [
53].
Overall, the SERPA approach has a high resolution in protein separation and certain post-translational modifications of antigens remain retained, which can be visualized by suitable gel staining methods. However, this workflow is very time-consuming and requires great skill of the operator to ensure reproducibility. Moreover, only the most abundant and soluble proteins can be sufficiently resolved on the immunoblot and multiplexing [e.g., as applied for the difference gel electrophoresis technique (DIGE)] of different conditions, genotypes, and culturing time points is excluded for sera screening.
2.2. Gel-Free Immunoproteomics
The protein array is another high-throughput technology, which is applied for immunoproteomic studies. Complex protein samples from cells or tissues can be fractionated by multiple LC steps based on protein pI or hydrophobicity. A variety of technologies are available to spot protein sub-fractions onto the planar surfaces in ordered arrays [
54,
55,
56]. By applying patient antibodies to the protein arrays, protein antigen fractions are detected with the help of secondary labeled antibodies., After localization of interesting antigens on the array, the reactive antigen from the selected protein fraction has to be isolated and identified by further fractionation, immunoprecipitation, and MS detection. Instead of using protein sub-fractions, expressed recombinant proteins or peptides can also be used in this approach to produce protein arrays or multiplex bead arrays. Mochon
et al. [
57] reported on a
C. albicans protein microarray used for comparison of serological profiling of
C. albicans in different stages of candidemia. The authors selected a set of cell surface proteins according to the
Candida Genome Database (CGD,
http://www.candidagenome.org/) and expressed interesting candidates in
E. coli. Despite the fact that the immunocompetent host exists in a permanent host-pathogen interplay with the commensal
C. albicans, a set of 13 cell surface antigens mainly involved in either oxidative stress or drug resistance were identified [
57]. These candidates were specific for acute candidemia. Due to the cell free nature of
in vitro translated peptides, potential epitopes could get lost due to protein misfolding or a lack of post-translational modifications (e.g., glycosylations), which may affect the conformational structure of the native protein and its binding affinity.
Immunocapture MS is referred to as inverse immunoproteomics, since patient antibodies are firstly immobilized on the protein array to investigate antigen profiles. This approach is highly efficient allowing the simultaneous processing of large numbers of patient samples and an easy handling of native antigens in solution. Furthermore, low molecular weight (LMW) antigens (<20 kD) are more sensitively detected by this approach [
58].
Altogether, the SERPA approach and protein microarrays bring both advantages and disadvantages. SERPA requires less prior knowledge and is the ideal choice for the identification of potentially interesting fungal protein antigens. Protein/peptide microarrays, on the other hand, are more suitable for high-throughput screenings of serum samples and the generation of quantitative data. The combination of both methods has the highest potential for the diagnosis and immunotherapy of invasive fungal infections. In addition, LC-MS/MS-based approaches allow the identification of peptides presented on major histocompatibility complexes (MHCs) on the cell surface of immune cells.