Recombinant Candida glabrata Calpain-like protease palB/RIM13 (RIM13), partial

What is the functional role of palB/RIM13 protease in Candida glabrata signaling pathways?

PalB/RIM13 functions as a calpain-like cysteine protease that plays a critical role in pH-responsive signaling. It is a key component of the Pal signaling pathway where it mediates the proteolytic processing of the pH-responsive transcription factor PacC. This processing occurs in response to alkaline pH signals, enabling C. glabrata to adapt to different environmental pH conditions. The protease specifically cleaves within a highly conserved 24-residue "signaling protease box" that is critical for proper signal transduction .

Mutational analyses, particularly those affecting the predicted catalytic cysteine, strongly support the classification of PalB as a cysteine protease. These mutations provide valuable experimental tools for studying palB function in various genetic backgrounds .

How does the structure of palB/RIM13 relate to its proteolytic function?

While detailed structural information specific to C. glabrata palB/RIM13 is limited in the current literature, functional analyses through mutational studies have revealed critical insights:

Typical calpain-like proteases contain a catalytic domain with a catalytic triad, including the critical cysteine residue essential for proteolytic activity. Additional domains likely contribute to substrate recognition, particularly for specific targets like the PacC transcription factor within the highly conserved "signaling protease box" .

What experimental systems are optimal for recombinant expression of functional palB/RIM13?

Based on research protocols for similar fungal proteases, the following expression systems offer distinct advantages:

When expressing recombinant palB/RIM13, researchers should consider:

• Adding protease inhibitors during purification to prevent auto-proteolysis

• Including optimal cofactors (potentially calcium ions) for proper folding and activity

• Selecting appropriate affinity tags that don't interfere with the catalytic domain

How can palB/RIM13 activity be measured in laboratory settings?

Several methodological approaches can be employed to assess palB/RIM13 proteolytic activity:

• Analysis of PacC processing through Western blot detection of tagged PacC versions, as demonstrated in previous studies

• Fluorogenic substrate assays using peptides containing predicted cleavage sites

• FRET-based assays that detect changes in fluorescence upon substrate cleavage

• Mass spectrometry analysis of cleavage products to identify precise cleavage sites

When designing activity assays, researchers should consider the optimal pH conditions, as palB functions in response to alkaline pH signals .

What critical mutations affect palB/RIM13 function and their impact on signaling pathways?

Mutational analyses have identified numerous variants affecting palB function, with significant implications for pH signaling:

Mutations specifically affecting the catalytic cysteine region provide strong evidence for palB's function as a cysteine protease . Both truncating and missense mutations affecting the C-terminal region highlight this domain's critical role in protease function, potentially through substrate recognition or regulatory interactions .

Specific mutations (palB111, palB16, palB37, palB38, palB39, and palB70) allow growth on specific nitrogen sources like γ-aminobutyrate (GABA), providing valuable tools for functional studies . These mutations disrupt the normal proteolytic processing of PacC, preventing appropriate responses to alkaline pH signals.

How does palB/RIM13 contribute to C. glabrata's survival within host macrophages?

While direct evidence linking palB/RIM13 to macrophage survival is limited, pH signaling pathways are likely crucial for C. glabrata's adaptation during host infection. C. glabrata demonstrates remarkable ability to survive within macrophages through several mechanisms:

• Generation of strong stress responses against reactive oxygen species (ROS)

• Neutralization of the phagocytic environment

• Activation of multiple stress-response genes (Skn7p, Yap1p, Msn2p, and Msn4p) that encode detoxification proteins

These stress pathways are primarily regulated by stress-activated protein kinase Hog1, the Cap1 transcription factor, and DNA damage checkpoint kinase Rad53 . As a component of pH signaling pathways, palB/RIM13 may contribute to these adaptive responses, though specific interactions remain to be fully characterized.

Recent transcriptomic studies mapping C. glabrata responses during macrophage infection have identified dynamic chronological transcriptional changes, including the activation of specialized pathways at different infection timepoints . This suggests complex regulatory networks coordinate the fungal response to host environments.

What is the potential of palB/RIM13 as a target for novel antifungal therapeutics?

The increasing prevalence of drug-resistant C. glabrata infections highlights the urgent need for novel therapeutic targets. Several factors make palB/RIM13 a potentially valuable target:

• As a cysteine protease involved in essential signaling pathways , palB/RIM13 represents a druggable target class

• C. glabrata demonstrates increasing resistance to conventional antifungals including azoles, polyenes, and echinocandins

• Disruption of pH signaling could potentially attenuate C. glabrata's ability to adapt to varying host environments

Recent research has identified peptide derivatives with antifungal activity against Candida species. For example, a synthetic peptide derivative (Yhi1 2-13) demonstrated dose-dependent antifungal activity, blocking hyphal growth in C. albicans and causing crumpled growth in both C. albicans and C. glabrata . This highlights the potential for developing peptide-based inhibitors targeting specific fungal processes.

How can transcriptomic and proteomic approaches enhance understanding of palB/RIM13 function?

Advanced -omics technologies offer powerful approaches to investigate palB/RIM13 function within the broader cellular context:

• Genome-wide transcriptional profiling: RNA polymerase II (RNAPII) ChIP-seq comparing wild-type and palB/RIM13 mutant strains can identify genes regulated by the Pal signaling pathway under different pH conditions . This approach provides advantages over conventional mRNA measurements as it is less affected by transcript stability issues .

• Time-course analysis: Capturing dynamic transcriptional changes in response to pH shifts can reveal the temporal sequence of palB/RIM13-dependent signaling events. This is particularly valuable as C. glabrata mounts chronological transcriptional responses to environmental challenges .

• Proteomics for substrate identification: N-terminomics or degradomics approaches can identify specific protease substrates and cleavage sites, expanding our understanding beyond the known PacC substrate.

These methods can reveal not only direct targets of palB/RIM13 but also broader cellular processes affected by its activity, providing systems-level insights into its function in C. glabrata pathogenesis.

How does palB/RIM13 contribute to antifungal resistance mechanisms in C. glabrata?

C. glabrata exhibits various mechanisms of drug resistance that vary by geographical region . While direct evidence linking palB/RIM13 to antifungal resistance is limited, several potential connections exist:

• As a component of pH signaling pathways, palB/RIM13 may influence cell wall composition and membrane properties, which are important determinants of antifungal susceptibility

• C. glabrata resistance mechanisms include:

  • Overexpression of drug transporters

  • Gene mutations affecting thermotolerance

  • Increased adhesion factors leading to hypervirulence

  • Modifications in enzymes producing cell wall proteins that prevent drug activity

• The formation of biofilms, which palB/RIM13 may indirectly influence through signaling networks, contributes significantly to C. glabrata's evasion of host immune responses and antifungal resistance

Comparative studies examining palB/RIM13 expression and activity in drug-sensitive versus resistant C. glabrata isolates could provide valuable insights into its potential role in resistance mechanisms.

How can palB/RIM13 be utilized for developing improved diagnostic methods for C. glabrata infections?

Accurate diagnosis of C. glabrata infections remains challenging, especially in cases of invasive candidiasis without positive blood cultures . Given C. glabrata's inherent resistance to first-line antifungal drugs, rapid and precise identification is crucial for appropriate treatment .

PalB/RIM13-based diagnostic approaches could include:

• Development of PCR-based assays targeting the palB/RIM13 gene sequence for species-specific identification

• Creation of antibody-based detection methods (ELISA, lateral flow) for protein detection in clinical samples

• Activity-based probes that detect palB/RIM13 proteolytic signatures in patient specimens

Recent research has identified other C. glabrata-specific proteins, such as CgYHI1, that can serve as highly precise biomarkers for rapid diagnosis . Similar approaches could be applied to palB/RIM13, potentially as part of a multi-target diagnostic panel.

What interspecies interactions might involve palB/RIM13 during polymicrobial Candida infections?

Polymicrobial infections involving multiple Candida species present complex clinical challenges. Recent research has revealed interesting interspecies interactions:

• C. glabrata secretes a unique small protein (Yhi1) that induces hyphal growth in C. albicans, which is essential for host tissue invasion

• Synthetic peptide derivatives of this protein (Yhi1 2-13) demonstrated antifungal activity against both C. albicans and C. glabrata

While direct evidence linking palB/RIM13 to interspecies interactions is limited, its role in pH signaling could potentially influence how C. glabrata responds to other microbial species in shared host niches. Future research examining how palB/RIM13 activity affects the production or secretion of factors involved in interspecies communication would be valuable.

How does palB/RIM13 function integrate with other virulence mechanisms in C. glabrata?

C. glabrata possesses multiple virulence factors that enable host invasion and immune evasion:

• Production of extracellular enzymes:

  • Proteases

  • Phospholipases

  • Hemolysins

• Formation of biofilms that facilitate adherence and resistance

• Production of CgYapsins (encoded by CgYPS1-111 genes) that inhibit IL-1β production in macrophages, allowing fungal proliferation

• Strong antioxidant systems that neutralize reactive oxygen species (ROS) within phagocytes

As a component of signaling pathways, palB/RIM13 likely contributes to coordinating these virulence mechanisms in response to environmental cues. The identification of transcription factors like CgXbp1, which regulates both virulence-related genes and genes associated with drug resistance , suggests complex regulatory networks integrate multiple aspects of C. glabrata pathogenesis.

What are the key challenges in purifying enzymatically active recombinant palB/RIM13?

Researchers working with recombinant palB/RIM13 face several technical challenges:

• Maintaining proteolytic integrity: Preventing auto-proteolysis during expression and purification requires careful optimization of buffer conditions and potentially the use of protease inhibitors

• Ensuring proper folding: As a calpain-like protease, palB/RIM13 likely requires specific conditions for proper folding, potentially including cofactors like calcium ions

• Stabilizing the active conformation: The active conformation may be transient or dependent on specific regulatory interactions, making it challenging to capture in recombinant systems

• Developing appropriate activity assays: Identifying suitable substrates and establishing conditions that recapitulate the physiological activity of palB/RIM13 requires careful experimental design

When designing purification strategies, researchers should consider:

• Testing multiple expression systems to identify optimal conditions

• Including potential cofactors throughout purification

• Employing activity-based assays to monitor functional integrity during purification

How can CRISPR-Cas9 genome editing advance functional studies of palB/RIM13?

CRISPR-Cas9 technology offers powerful approaches for studying palB/RIM13 function:

• Precise genome editing: Creation of defined mutations in the catalytic domain or regulatory regions to systematically study structure-function relationships

• Domain swapping: Replacing specific domains with counterparts from related proteases to understand evolutionary conservation and specialization

• Conditional expression systems: Engineering strains with inducible palB/RIM13 expression to study temporal aspects of its function

• Fluorescent tagging: Creating fusion proteins with fluorescent tags to monitor subcellular localization and dynamics in real-time

• Systematic mutagenesis: Creating libraries of palB/RIM13 variants to identify critical residues beyond the catalytic site

When designing CRISPR-Cas9 experiments for C. glabrata, researchers should consider using appropriate selection markers and optimize transformation protocols for this specific Candida species.