Detection and quantification of proteome and phosphoproteome in wildtype and azole-resistant strains of A. fumigatus under voriconazole pressure
To investigate proteins and phosphorylation related to A. fumigatus azole resistance, we performed proteomic and phosphoproteomic analyses on the wildtype strain Af293 and azole-resistant strain Dt2441 (TR34/L98H genotype) cultured under voriconazole stress. Given that wildtype and drug-resistant A. fumigatus strains have varying tolerances to voriconazole, we cultured the two strains at their maximum voriconazole concentrations that allowed for biofilm formation, as described in a previous study26. The wildtype Af293 was cultivated in 0.0625 µg/ml voriconazole (Af293_VRC0.0625), while the azole-resistant Dt2441 was cultured at 1 µg/ml (Dt2441_VRC1). The analysis of the two strains(Dt2441_VRC1 vs. Af293_VRC0.0625) identified 3,628 unique proteins, among which 3,579 could be quantified. A comparison between the two strains revealed 294 differentially expressed proteins, with 148 upregulated and 146 downregulated (Table 1). The results obtained from IMAC-enriched samples for phosphorylated proteins showed a total of 8,972 phosphopeptides, with 5,714 quantifiable. As determined using Student's t-test, 425 phosphopeptides were differentially expressed between the two strains, with the majority (316 [74.4%]) exhibiting increased phosphorylation levels (Table 1). The analysis of phosphorylation sites indicated that 76.2% of residues were serine, 22.91% were threonine, and 0.68% were tyrosine (Fig. 1A). Furthermore, 74.37% of phosphoproteins had two or more phosphosites, with the protein A0A0J5PM23 containing as many as 95 phosphosites (Fig. 1B). Furthermore, phosphorylation motif analysis indicated that the most frequently occurring motifs of phosphorylation in the identified proteins were .R.R.._S_P....., ...R.._S_P....., and ......_S_PV..... In the upregulated phosphorylated peptides enrichments, the most frequently occurring motifs were ……_T_P….., .S…. _S_P….. and ……_S_P…..; whereas in the downregulated peptide enrichments, the most frequently occurring motifs were ……_S_P….., …R.. _S_......, and ……_S_..E… (Fig. 1C). A joint analysis of the proteome and phosphoproteome was conducted to eliminate the influence of protein changes on phosphorylation levels. Among the 938 correlated proteins, 133 proteins displayed differential expression only at the phosphorylation level, with 104 upregulated and 29 downregulated. Interestingly, two proteins were downregulated at the protein level but upregulated at the phosphoprotein level (Table 2).
In this study, 45 differentially expressed phosphoproteins, comprising 46 phosphopeptides, were chosen for targeted protein quantification through Parallel Reaction Monitoring (PRM). The results revealed that seven specific proteins, encompassing eight appropriate phosphopeptides (refer to Supplementary Table 1), were successfully validated using the PRM method.
Functional characterization of differentially expressed proteins and phosphoproteins
To characterize the functions and subcellular locations of the differentially expressed proteins (DEPs) in wildtype and azole-resistant strains, we conducted bioinformatic analyses of Gene Ontology (GO) and subcellular functional annotations. Among the differentially expressed proteins (DEPs), 111 were located in the cytoplasm, 105 were located in the nucleus, 60 were located in the mitochondria, and 42 were located in the plasma membrane (Fig. 2A). In contrast, more than half of the differentially expressed phosphoproteins were located in the nucleus, followed by cytoplasmic phosphoproteins (Fig. 2B). GO function annotation of the differentially expressed proteins revealed that the DEPs were primarily involved in cellular processes and metabolic processes for biological processes (BP), cell, organelle, and membrane for cellular components (CC), and catalytic activity and binding for molecular functions (MF) (Fig. 2C). The GO annotation for differentially expressed phosphoproteins yielded similar results (Fig. 2D).
We conducted KEGG enrichment analysis to identify pathways significantly represented in upregulated and downregulated proteins. Among the upregulated proteins, 'steroid biosynthesis' was most significantly enriched, involving three proteins: Y699_06061 (Cytochrome P450 sterol 14 alpha-demethylase), cyp51A, and AFUB_030680 (C-3 sterol dehydrogenase/C-4 decarboxylase). On the other hand, downregulated proteins showed the most significant enrichment in the 'FoxO signaling pathway,' which included two proteins: AFUB_007790 (Autophagy-related protein) and AFUB_046060 (catalase) (Fig. 2E). Interestingly, the KEGG enrichment analysis of differentially expressed phosphoproteins revealed a different trend. While 'steroid biosynthesis' remained significantly enriched in upregulated phosphopeptides, 'glucosinolate biosynthesis' emerged as the most enriched pathway in downregulated phosphopeptides (Fig. 2F).
In addition, we compared the proteome and phosphoproteome using GO enrichment and KEGG pathway enrichment. In contrast to the differentially expressed proteins, which were mainly associated with functions such as 'killing of cells of other organisms,' 'cytolysis' and 'cell killing,' and 'structural constituent of cell wall,' the differentially expressed phosphoproteins were primarily involved in 'signal transduction,' 'signaling,' and 'cell communication' (Fig. 2G). This finding was consistent with the KEGG enrichment comparison, which showed a significantly enriched pathway 'MAPK signaling pathway - yeast' for the phosphoproteome, while 'glycerolipid metabolism' was enriched for the proteome (Fig. 2H).
Regulation of proteins that were differentially expressed at the phosphorylation level in the comparison of wildtype and azole-resistant strains under voriconazole pressure.
Considering that phosphorylation serves as a reversible and dynamic post-translational modification that regulates protein function, we focused on proteins differentially expressed only at the phosphorylation level between Dt2441 (azole-resistant) and Af293 (wildtype) under voriconazole pressure. In Fig. 3A, a total of 37 proteins were differentially expressed exclusively at the protein level, 451 proteins were differentially expressed exclusively at the phosphoprotein level, and 63 proteins showed differential expression at both the protein and phosphoprotein levels.
Supplementary Table 2,3 presents the top 20 proteins with up-regulated phosphorylation levels and the top 15 proteins with down-regulated phosphorylation levels among the proteins whose expression levels remain unchanged. The results revealed that many of the proteins with significantly upregulated phosphorylation levels were ‘integral components of the membrane’, suggesting that the formation and stability of cell membranes might play a crucial role in A. fumigatus azole resistance. Conversely, among the proteins with significantly downregulated phosphorylation levels, several were related to drug transport, indicating that the drug resistance mechanism of TR34/L98H genotype A. fumigatus might not primarily rely on the work of drug efflux pumps.
GO enrichment analysis of these regulated phosphoproteins revealed their involvement in various processes, including positive regulation of immune system processes, signal transduction by protein phosphorylation, MAPK cascade, and protein modification by small protein conjugation or removal (Fig. 3B). On the other hand, KEGG annotation showed that the majority of proteins differentially expressed only at the phosphoprotein level were associated with pathways such as 'ribosome,' 'MAPK signaling pathway - yeast,' 'endocytosis,' 'oxidative phosphorylation,' and others (Fig. 3C).
MAPK pathway was found to be involved in azole resistance of A. fumigatus biofilms
Our primary interest lied in the phosphorylation regulation of proteins corresponding to the KEGG pathway 'MAPK signaling pathway - yeast' which might be involved in azole resistance of A. fumigatus biofilms. We identified 33 proteins with differential phosphorylation levels, among which 16 displayed decreased phosphorylation, 14 showed increased phosphorylation, and three exhibited both decreased and increased phosphorylation simultaneously. These proteins majorly belong to three functional classes including the conserved cell wall integrity (CWI), pheromone response/filamentous growth and high-osmolarity glycerol (HOG) (Table 3, Fig 4). After excluding the influence of protein expression changes, we found that three proteins (ROM1,2, Yck1,2, and PKC1) showed increased phosphorylation, while two proteins (Cdc24 and Wsc1,2,3) showed decreased phosphorylation. Specifically, Cdc24 contained two decreased phosphorylation sites at S705 and S750, and Wsc1,2,3 contained two decreased phosphorylation sites at S245 and S248.