The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance.

The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.IMPORTANCEAspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkCA. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation.

In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion.

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of cell wall stress.

The protein kinase MpkA plays a prominent role in the cell wall integrity signaling (CWIS) pathway, acting as the terminal MAPK activating expression of genes which encode cell wall biosynthetic enzymes and other repair functions. Numerous studies focus on MpkA function during cell wall perturbation. Here, we focus on the role MpkA plays outside of cell wall stress, during steady state growth. In an effort to seek other, as yet unknown, connections to this pathway, an mpkA deletion mutant (ΔmpkA) was subjected to phosphoproteomic and transcriptomic analysis. When compared to the control (isogenic parent of ΔmpkA), there is strong evidence suggesting MpkA is involved with maintaining cell wall strength, branching regulation, and the iron starvation pathway, among others. Particle-size analysis during shake flask growth revealed ΔmpkA mycelia were about 4 times smaller than the control strain and more than 90 cell wall related genes show significantly altered expression levels. The deletion mutant had a significantly higher branching rate than the control and phosphoproteomic results show putative branching-regulation proteins, such as CotA, LagA, and Cdc24, have a significantly different level of phosphorylation. When grown in iron limited conditions, ΔmpkA had no difference in growth rate or production of siderophores, whereas the control strain showed decreased growth rate and increased siderophore production. Transcriptomic data revealed over 25 iron related genes with altered transcript levels. Results suggest MpkA is involved with regulation of broad cellular functions in the absence of stress.

Protein Engineering Strategies to Expand CRISPR-Cas9 Applications.

The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.

Insights regarding fungal phosphoproteomic analysis.

Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-ß-1,4-glucanase (GH12) from Aspergillus niveus.

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-ß-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan ß-1,3 or ß-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in ß-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.