Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection

Abstract

Aspergillus fumigatus is one of the most common airborne molds capable of causing mycoses and allergies in humans. During infection, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established for surface proteomics (surfomics). Biotinylation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptides is a particularly efficient method to identify the surface-exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 231 different biotinylated surface proteins (including several well-known proteins such as RodA, CcpA, and DppV; allergens; and heat shock proteins [HSPs]), as well as some previously undescribed surface proteins. The dynamic change of the surface proteome was illustrated by detection of a relatively high number of proteins exclusively at one developmental stage. Using immunofluorescence microscopy, we confirmed the surface localization of several HSPs of the HSP70 family, which may have moonlighting functions. Collectively, by comparing our data with data representative of previously published A. fumigatus surface proteomes, our study generated a comprehensive data set corresponding to the A. fumigatus surfome and uncovered the surface-exposed regions of many proteins on the surface of conidia or hyphae. These surface-exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, our data sets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. IMPORTANCE Aspergillus fumigatus is the most important airborne human-pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergy-inducing infections in individuals with atopic allergy. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of A. fumigatus infection remain major challenges. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between A. fumigatus and other organisms, sometimes even including the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.

CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus

“…Shm2 is a ubiquitous, 51.9 kDa protein found in all pro-and eukaryotes, and plays a central role in providing one-carbon units for biosynthetic processes (25). Shm2 is present on the surface of A. fumigatus germinating conidia and hyphae (26), was found to trigger IgG responses in sera of patients with invasive aspergillosis (9), and is a strong elicitor of memory T cell responses (9). FIGURE 2 | Cytokine profiles of A. fumigatus antigen-stimulated mDCs and moDCs.…”

Re-direction of phagosomes to the recycling expulsion pathway by a fungal pathogen

“…This way, we identified a candidate protein with an apparent molecular mass of 70 kDa after co-incubation of A549 cells with protein extract of germlings and a weaker signal for this protein with swollen conidia (Sc) (Figures 1C and 1D). In our previous proteome analysis of surface proteins of A. fumigatus during germination (Jia et al, 2020), six proteins with molecular masses between 65 and 75 kDa were detected in extracts of germinating conidia, including four 70 kDa heat-shock proteins.…”

“…(Afu2g09960/AFUB_025800), and TktA (Afu1g13500/AFUB_012990) were detected, but only HscA was predominantly identified in samples of germlings (Jia et al, 2020).…”

“…A. fumigatus conidia from WT and knockout strains were collected in water from AMM agar plates after 5 days of growth at 37°C, and were counted using a CASY ® Cell Counter, as previously described (Jia et al, 2020). For germination assays, 10 9 A. fumigatus resting conidia were incubated at 37°C in RPMI 1640 (GIBCO) to produce swollen conidia (4 h), germlings (8 h), and hyphae (14 h), as described previously (Jia et al, 2020). For conidia production assay, 10 5 conidia were spread on AMM agar plates.…”

Chaperone Networks in Fungal Pathogens of Humans

“…In the context of fungal pathogens, the Hsp70s have high levels of expression at elevated temperatures and also influence morphological transitions [ 41 , 42 , 43 ]. Hsp70s are found localized to the cell surfaces of C. albicans [ 44 ], C. neoformans [ 45 ], and A. fumigatus [ 46 , 47 ]. Accordingly, Hsp70s are immunogenic proteins in systemic infections caused by C. neoformans [ 48 , 49 ] or C. albicans [ 50 ].…”