Recombinant Neosartorya fumigata Probable endo-1,3 (4)-beta-glucanase AFUA_2G14360 (AFUA_2G14360), partial

What is the functional role of endo-1,3(4)-beta-glucanase AFUA_2G14360 in fungal cell wall architecture?

The probable endo-1,3(4)-beta-glucanase AFUA_2G14360 likely belongs to the GH16 family of glycosylhydrolases that play essential roles in fungal cell wall morphogenesis. Based on studies of similar enzymes in Aspergillus fumigatus, these glucanases are critical for "softening" the rigid cell wall structure during fungal development and growth. The fungal cell wall gains its rigidity from fibrillar and branched β-(1,3)-glucan linked to chitin, creating a matrix that protects the fungus . During morphogenesis, this rigid structure must be partially cleaved by glycosylhydrolases like endo-β-(1,3)-glucanases to permit expansion and remodeling.

Research on similar endo-β-(1,3)-glucanases in A. fumigatus (designated as ENG1-7) has shown that these enzymes are expressed during conidial dormancy and germination, suggesting essential roles during fungal development . Particularly, the multiple deletion of genes encoding GH16 and GH81 family glucanases resulted in defective conidial formation, with chains of conidia unable to separate properly, demonstrating their importance in proper cell wall assembly and conidial segregation during development .

What methods are recommended for expressing recombinant AFUA_2G14360 for in vitro studies?

For expressing recombinant AFUA_2G14360, researchers should consider the following methodological approaches:

• Expression System Selection:

  • Heterologous expression in Escherichia coli (BL21) with a 6×His tag for purification

  • Pichia pastoris expression system for proper protein folding and post-translational modifications

  • Homologous expression in Aspergillus species for native glycosylation patterns

• Construct Design:

  • Include a secretion signal (e.g., α-factor from Saccharomyces cerevisiae) for extracellular production

  • Consider codon optimization for the chosen expression host

  • Include purification tags that won't interfere with enzymatic activity

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Size exclusion chromatography for final polishing and buffer exchange

  • Activity assays using chromogenic substrates to track purification efficiency

• Enzyme Activity Preservation:

  • Storage buffer optimization (typically 50mM phosphate buffer pH 6.5-7.0 with 100mM NaCl)

  • Addition of stabilizing agents (e.g., 10% glycerol)

  • Aliquoting and flash-freezing to avoid freeze-thaw cycles

The expression strategy should be carefully designed based on the intended experimental applications, as the choice of system will affect protein yield, activity, and post-translational modifications.

How can researchers accurately measure the enzymatic activity of AFUA_2G14360?

Accurate measurement of AFUA_2G14360 enzymatic activity requires careful consideration of substrate selection, reaction conditions, and detection methods:

Recommended Assay Approaches:

• Substrate Selection:

  • Laminarin (β-1,3-glucan with some β-1,6 branches) as primary substrate

  • Mixed-linkage β-(1,3;1,4)-glucan substrates to assess specificity

  • Synthetic chromogenic/fluorogenic substrates for high-throughput screening

• Activity Quantification Methods:

  • Reducing sugar assays (DNS, Nelson-Somogyi) to detect released glucose

  • HPLC analysis of hydrolysis products for detailed linkage analysis

  • Viscometric methods to measure decrease in substrate viscosity

  • Radiolabeled substrates (e.g., [³H]glucose) for high sensitivity detection

• Standardized Reaction Conditions:

  Parameter	Recommended Range	Optimization Notes
  pH	5.0-6.5	Test at 0.5 unit intervals
  Temperature	30-50°C	Aspergillus enzymes often optimal at 37-45°C
  Buffer	50mM sodium acetate or phosphate	Avoid inhibitory buffers
  Incubation time	15-60 minutes	Ensure linearity of reaction
  Enzyme concentration	0.1-1.0 μg/mL	Titrate for optimal response

• Controls and Validation:

  • Heat-inactivated enzyme controls

  • Commercial β-glucanase standards for comparison

  • Inhibition studies with known β-glucanase inhibitors

When reporting enzyme activity, it is advisable to express results as specific activity (μmol product/min/mg protein) and include detailed methodological information for reproducibility.

How does functional redundancy among glucanases affect experimental design when studying AFUA_2G14360?

Functional redundancy presents a significant challenge when studying individual glucanases like AFUA_2G14360 in Aspergillus fumigatus. Research has shown that deletion of single endo-β-(1,3)-glucanase genes often yields no observable phenotypic changes, suggesting compensatory mechanisms by other family members . This redundancy requires thoughtful experimental design strategies:

Recommended Approaches to Address Redundancy:

• Sequential Gene Deletion Strategy:

  • Create multiple deletion mutants as demonstrated in studies where Δeng2,3,4,5 followed by eng1 deletion was required to observe phenotypic effects

  • Use marker recycling systems (e.g., β-rec/six system) to enable multiple deletions with limited selection markers

  • Implement CRISPR-Cas9 for efficient multiplex gene editing

• Expression Analysis:

  • Conduct comprehensive transcriptomic analysis (RNA-Seq) to identify all expressed glucanases

  • Monitor potential compensatory upregulation of other glucanases in single-gene knockouts

  • Use RT-PCR to verify expression levels of related genes in different growth conditions

• Biochemical Characterization:

  • Perform comparative enzymatic studies of all family members to identify unique substrate preferences

  • Develop specific activity assays that can distinguish individual enzyme contributions

  • Use tagged versions of the protein to track localization without disrupting function

• Phenotypic Analysis Enhancement:

  • Implement stress conditions that may reveal subtle phenotypes not apparent under standard growth

  • Utilize high-resolution microscopy to detect minor alterations in cell wall architecture

  • Conduct cell wall composition analysis to detect biochemical changes even in absence of visible phenotypes

Recent studies on A. fumigatus glucanases demonstrated that while Δeng1 or Δeng2 single mutants showed no phenotypic alterations, the quintuple mutant Δeng1,2,3,4,5 exhibited clear defects in conidial separation . This highlights the necessity of comprehensive approaches when studying functionally redundant enzyme families.

What structural features distinguish AFUA_2G14360 from other GH16 family enzymes and how do they relate to substrate specificity?

Understanding the structural basis of AFUA_2G14360 substrate specificity requires detailed analysis of its predicted structural features compared to characterized GH16 family enzymes:

Key Structural Determinants of Specificity:

• Catalytic Domain Architecture:

  • The GH16 family typically features a β-jelly roll fold with a substrate-binding cleft

  • AFUA_2G14360 likely contains the conserved EXDXXE motif that forms the catalytic center

  • The positioning of aromatic residues in the binding cleft determines substrate preference

• Substrate Binding Subsites:

  • Variable loops surrounding the active site modify subsite architecture and specificity

  • The number and arrangement of subsites (designated -n to +n from the cleavage site) dictate the recognized oligosaccharide length

  • Specific residues at the +1/+2 subsites likely determine preference for β-1,3 versus β-1,4 linkages

• Structural Comparison Approach:

  Feature	AFUA_2G14360 (Predicted)	Other GH16 β-1,3(4)-Glucanases	Functional Implication
  Loop B	Extended	Variable length	Substrate specificity
  Calcium binding	Present	Often conserved	Structural stability
  C-terminal domain	Unknown	Variable	Potential regulatory function
  Aromatic platform	Tryptophan-rich	Conserved	Crucial for β-glucan binding

• Structure-Guided Protein Engineering:

  • Site-directed mutagenesis of predicted substrate-interacting residues can verify their roles

  • Domain swapping with other GH16 enzymes can create chimeric proteins with altered specificity

  • Rational design based on structural models can enhance activity toward specific substrates

While the exact structure of AFUA_2G14360 has not been determined experimentally, homology modeling based on characterized GH16 family members would provide valuable insights into its likely mode of action and substrate preference. This information could guide the development of specific inhibitors or the engineering of enhanced variants for biotechnological applications.

How does AFUA_2G14360 activity correlate with antifungal resistance mechanisms?

The relationship between endo-1,3(4)-beta-glucanase activity and antifungal resistance, particularly to echinocandins, represents an important area of investigation:

AFUA_2G14360 and Antifungal Resistance Mechanisms:

• Cell Wall Remodeling and Echinocandin Action:

  • Echinocandins (caspofungin, micafungin, anidulafungin) inhibit β-1,3-glucan synthase, disrupting cell wall integrity

  • Endo-β-1,3-glucanases like AFUA_2G14360 may contribute to cell wall remodeling during stress

  • Altered glucanase activity could potentially compensate for reduced β-1,3-glucan synthesis

• Experimental Evidence from Related Studies:

  • IC₅₀ values for echinocandins against A. fumigatus β-1,3-glucan synthase have been established:

    • Micafungin: 13 nM

    • Anidulafungin: 29 nM

    • Caspofungin: 110 nM

  • These values provide a baseline for studying how glucanase activity might affect susceptibility

• Research Approaches to Investigate Correlation:

  • Generate AFUA_2G14360 overexpression and deletion strains to test echinocandin susceptibility

  • Combine genetic modifications with known resistance mutations (e.g., FKS1 hot-spot mutations)

  • Monitor cell wall composition changes in response to sub-inhibitory echinocandin concentrations

• Proposed Mechanistic Models:

  • Enhanced glucanase activity could potentially expose chitin, triggering compensatory mechanisms

  • Reduced glucanase activity might lead to a thicker, less organized cell wall with altered drug penetration

  • Cell wall stress response pathways may regulate glucanase expression as an adaptive mechanism

Understanding this relationship could identify potential combination therapy approaches or resistance mechanisms. For instance, a strain lacking the mixed-linkage β-(1,3;1,4)-glucan through deletion of Tft1 showed a modest increase in virulence in animal models , suggesting that alterations in cell wall composition can affect pathogenicity and potentially drug responses.

What is the relationship between AFUA_2G14360 and other cell wall modifying enzymes during Aspergillus fumigatus morphogenesis?

The orchestrated action of multiple cell wall-modifying enzymes is essential for proper fungal morphogenesis. AFUA_2G14360, as a probable endo-1,3(4)-beta-glucanase, likely functions within a complex network:

Enzyme Interaction Network During Morphogenesis:

• Temporal Expression Patterns:

  • Studies of related glucanases indicate differential expression during developmental stages

  • Some glucanases (e.g., ENG1-5) are expressed in both dormant conidia and during germination

  • Others (e.g., ENG6-7) may not be expressed under standard laboratory conditions

  • Coordinated expression suggests functional specialization during the life cycle

• Functional Relationships with Other Cell Wall Enzymes:

  Enzyme Class	Relationship with Glucanases	Morphogenetic Stage
  β-1,3-glucan synthases	Counterbalancing activity	Cell expansion
  Chitin synthases	Coordinated regulation	Cell wall integrity
  α-1,3-glucanases	Sequential action	Conidial separation
  Chitinases	Synergistic activity	Hyphal branching

• Signaling Pathways Regulating Enzyme Networks:

  • Cell wall integrity (CWI) pathway likely coordinates glucanase activity

  • Calcium/calcineurin signaling may regulate expression during stress

  • cAMP/PKA pathway involvement in developmental regulation

• Spatial Organization Within the Cell Wall:

  • Localized action of glucanases creates growth zones at hyphal tips

  • Different enzymes may target specific structural layers or components

  • Proper localization is essential for normal morphogenesis

Research has demonstrated that complete deletion of multiple glucanases (Δeng1,2,3,4,5) results in conidial chains unable to separate properly, while germination rates remain unaffected . This suggests that these enzymes play specific roles in conidial cell wall assembly and separation during development, rather than general cell wall maintenance.

The interaction with other enzymes involved in β-(1,3;1,4)-glucan synthesis, such as Tft1, is particularly interesting. When Tft1 (responsible for mixed linkage glucan synthesis) was deleted, the resulting strain showed a complete loss of β-(1,3;1,4)-glucan but no in vitro growth phenotype, suggesting compensatory mechanisms involving other cell wall components .

How can advanced imaging techniques be utilized to visualize AFUA_2G14360 activity in situ?

Advanced imaging techniques offer powerful approaches to visualize enzyme localization and activity within the fungal cell wall:

Cutting-Edge Imaging Approaches:

• Activity-Based Fluorescent Probes:

  • Design of fluorogenic substrates that release fluorophores upon cleavage by AFUA_2G14360

  • Synthesis of activity-based protein profiling (ABPP) probes that covalently bind to active enzyme

  • Development of quenched activity-based probes that fluoresce only upon enzyme action

• Super-Resolution Microscopy Applications:

  • Structured Illumination Microscopy (SIM) to achieve ~100 nm resolution of labeled enzymes

  • Stochastic Optical Reconstruction Microscopy (STORM) for nanoscale localization

  • Stimulated Emission Depletion (STED) microscopy to visualize enzyme distribution in specific cell wall layers

  • Correlative Light and Electron Microscopy (CLEM) to connect ultrastructure with enzyme localization

• Immunofluorescence Strategies:

  • Generation of specific antibodies against AFUA_2G14360

  • Epitope tagging approaches (e.g., FLAG, HA) that preserve enzyme function

  • Multi-color immunofluorescence to co-localize with other cell wall components

  • Immunoelectron microscopy for ultrastructural localization

• Live Cell Imaging Approaches:

  • Fusion of AFUA_2G14360 with fluorescent proteins (e.g., GFP, mCherry)

  • Time-lapse microscopy during germination and hyphal growth

  • Photoactivatable fluorescent proteins to track enzyme movement

  • FRET-based sensors to detect conformational changes during enzyme action

Previous studies have successfully used immunofluorescence staining with antibodies against β-(1,3;1,4)-glucan to demonstrate the complete loss of this polysaccharide in deletion mutants . Similar approaches, combined with advanced microscopy techniques, could be applied to visualize AFUA_2G14360 localization and activity during different developmental stages.

When designing imaging experiments, researchers should consider controls to validate specificity, such as deletion mutants, enzymatically inactive variants, and competitive inhibition controls. Additionally, careful sample preparation is essential to preserve cell wall structure while maintaining accessibility to antibodies or probes.