Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

Abstract

BackgroundThe pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process.ResultsTo gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle.ConclusionsThe observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile environments.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

“…The alignment results were also edited using the Jalview 2.8 tool (49) for enhanced visual presentation. To find the metabolic enzymes responsible for the xylose and cellobiose degradation, we used the XYL1, XYL2, and XYL3 proteins of P. stipitis (50,51) as well as BGLs of A. niger (BGL1; Q30BH9) (52), A. fumigatus (BTGE; B0Y9Q9) (53), and Candida wickerhamii (BGLA; Q12602) (54) as the templates. We further applied the PSORTII tool (55) to predict the cellular localization of the BLASTPsearched putative BGL genes of Y. lipolytica.…”

Identification of Immunogenic Antigens from Aspergillus fumigatus by Direct Multiparameter Characterization of Specific Conventional and Regulatory CD4+ T Cells

“…Furthermore, T cell Ags in the metabolically active A. fumigatus morphotypes were largely overlapping, which is in line with recent results on the proteomic signature of A. fumigatus during early development. These studies showed that the majority of mycelial proteins are also present in all early, metabolically active morphotypes, and only their abundance varied (20,40,41).…”

A Review beyond the borders: Proteomics of microclonial black fungi and black yeasts

“…Tris or phosphate buffers at 10 -30 mM were used to keep the pH constant and/or to be compatible with DIGE-labeling [22]. Further, the addition of NaOH at 0.1 -1 M can enhance the protein solubility and destabilize the cell wall tremendously [23]. As a non-ionic detergent CHAPS is used at the concentrations of 1% -4%.…”