Recombinant Candida glabrata Probable serine/threonine-protein kinase KKQ8 (KKQ8), partial

What is the role of serine/threonine protein kinases in Candida glabrata?

Serine/threonine protein kinases in C. glabrata play crucial roles in regulating multiple cellular processes including cell wall integrity, stress responses, and pathogenicity. These kinases function by phosphorylating serine or threonine residues on target proteins, thereby modulating their activity. In C. glabrata, these enzymes are involved in signaling cascades that respond to environmental stresses such as hypertonic conditions and cell wall disruption. For example, the Ste20 kinase in C. glabrata has been shown to be essential for maintaining a fully functional hypertonic stress response and intact cell wall integrity pathway . These kinases also participate in virulence mechanisms, as demonstrated by the attenuated virulence of ste20 mutants compared to reconstituted STE20 cells in infection models .

How do serine/threonine kinases in C. glabrata compare to those in related species?

Serine/threonine kinases in C. glabrata share significant homology with those in related yeast species but often display functional adaptations specific to C. glabrata's pathogenic lifestyle. For instance, C. glabrata Ste20 shares 53% identity and 58% predicted amino acid similarity with Saccharomyces cerevisiae Ste20 . Despite this homology, functional differences exist: while the C. glabrata Ste20 can complement both nitrogen starvation-induced filamentation and mating defects in S. cerevisiae ste20 mutants, C. glabrata Ste20 itself is not required for nitrogen starvation-induced filamentation in C. glabrata . This suggests evolutionary adaptation of these kinases to specific roles in C. glabrata's physiology and pathogenicity.

What structural features characterize serine/threonine kinases in C. glabrata?

Serine/threonine kinases in C. glabrata typically contain several conserved domains that define their function. For example, C. glabrata Ste20 contains a highly conserved p21-activated serine-threonine protein kinase domain, a binding site for G-protein beta subunits, and a regulatory Rho-binding domain that enables interaction with Cdc42 and/or Rho-like small GTPases . These structural elements are essential for the kinase's ability to participate in signaling cascades and interact with other cellular components. The conservation of these domains across fungal species highlights their fundamental importance in cellular signaling networks.

What genetic engineering techniques are most effective for studying serine/threonine kinases in C. glabrata?

The CRISPR-Cas9 system has emerged as a robust tool for generating loss-of-function mutants in C. glabrata, enabling detailed functional studies of serine/threonine kinases. A comprehensive approach includes: (i) using a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) employing an online program to select efficient guide RNAs for the target gene, and (iii) identifying mutant strains using the Surveyor technique and sequencing . This method allows for precise genetic manipulation to create knockout strains, enabling researchers to study the function of specific kinases through phenotypic analysis. Alternative approaches include traditional homologous recombination-based gene deletion strategies, which have been successfully used to generate ste20 null and disrupted strains for functional studies .

How can researchers effectively track the functional impact of serine/threonine kinase activity in C. glabrata?

Researchers can track kinase functionality using a combination of molecular and cellular approaches. One effective method involves fluorescent tracking of yeast division using carboxyfluorescein succinimidyl ester (CFSE) labeling combined with confocal microscopy and flow cytometry . This technique allows visualization of yeast cell division patterns and can reveal how kinase activity affects cellular processes. For instance, studies with Syk inhibitors have demonstrated that early Syk activity following macrophage phagocytosis is required for intracellular C. glabrata control . Additionally, researchers can employ phospho-specific antibodies to track kinase-mediated phosphorylation events, perform in vitro kinase assays to measure enzymatic activity, and utilize transcriptomic or proteomic approaches to identify downstream targets and pathways affected by kinase activity.

What are the optimal expression systems for producing recombinant C. glabrata serine/threonine kinases?

While the search results don't directly address expression systems for C. glabrata kinases, researchers typically employ heterologous expression systems such as E. coli, S. cerevisiae, or insect cells for recombinant protein production. For C. glabrata kinases, S. cerevisiae often serves as an optimal expression host due to its close phylogenetic relationship, similar codon usage, and capacity for post-translational modifications. When expressing these proteins, researchers should consider including affinity tags (His, GST, or FLAG) for purification, optimizing codon usage for the expression host, and ensuring proper folding through co-expression with chaperones if necessary. The choice of expression system should be guided by the specific research objectives, such as structural studies, enzyme activity assays, or interaction analyses.

How do serine/threonine kinases contribute to drug resistance mechanisms in C. glabrata?

Serine/threonine kinases play significant roles in drug resistance pathways in C. glabrata, particularly through the cell wall integrity (CWI) pathway. This pathway involves several kinases that respond to cell wall stress, including those induced by antifungal drugs. For instance, targeting the CWI pathway through deletion of PKC1 or activated MAP kinases (e.g., CgSLT2) induces echinocandin hypersensitivity, while increased activation of the pathway through over-expression of CgSLT2 reduces susceptibility to echinocandins . These kinases also mediate resistance by triggering compensatory mechanisms, such as increased production of other cell wall components like chitin, potentially compensating for the loss of beta-1,3-glucans targeted by echinocandins . Understanding these kinase-mediated pathways is crucial for developing strategies to overcome antifungal resistance.

What is known about the evolutionary conservation of KKQ8-like kinases across Candida species?

The evolutionary conservation of kinases across Candida species reflects their fundamental importance in cellular functions. While the search results don't specifically address KKQ8 conservation, they do indicate that C. glabrata kinases like Ste20 share significant homology with counterparts in related species such as S. cerevisiae . Genomic analyses of C. glabrata have revealed high levels of genetic heterogeneity in the population, with diverse strains, clades, and sequence types identified both inter- and intra-nationally . This genetic diversity likely extends to kinases, with potential implications for functional adaptation. The presence of well-conserved signaling pathways across fungal species suggests that while the specific sequence and regulation of kinases may vary, their core functions in cellular signaling are maintained throughout evolution.

How do serine/threonine kinases in C. glabrata influence host-pathogen interactions?

Serine/threonine kinases significantly impact host-pathogen interactions by regulating virulence factors and stress responses during infection. Research has shown that Syk, a host spleen tyrosine kinase, is required at an early stage for macrophage control of C. glabrata . From the pathogen perspective, C. glabrata kinases regulate processes critical for survival within the host environment. The ste20 mutants, while still capable of causing disease, demonstrate mild attenuation in virulence compared to reconstituted STE20 cells . This suggests that Ste20 and potentially other serine/threonine kinases contribute to C. glabrata's pathogenic potential. Additionally, the microevolution of C. glabrata within patients affects genes involved in drug resistance, including those regulated by kinase pathways, which may impact treatment outcomes .

How does the genetic diversity of C. glabrata populations impact kinase functionality?

The genetic diversity within C. glabrata populations can significantly influence kinase functionality through sequence variations that affect enzyme activity, substrate specificity, or regulation. Genome analysis of clinical isolates has revealed high genetic diversity, with at least 19 separate sequence types recovered from globally diverse locations and evidence of ancestral recombination . This diversity extends to genes under positive selection, particularly those encoding epithelial adhesins that facilitate fungal adhesion to human epithelial cells . Microevolution within patients further contributes to genetic diversity, with an enrichment for nonsynonymous and frameshift indels in cell surface proteins . Such genetic variations could affect kinase-dependent signaling pathways that regulate adhesion, stress responses, and drug resistance, potentially leading to differential virulence or treatment outcomes among C. glabrata strains.

What are the implications of mitochondrial genome diversity for kinase-dependent signaling in C. glabrata?

The C. glabrata mitochondrial genome shows remarkable diversity, with reduced conserved sequence and conserved protein-encoding genes in nonreference ST15 isolates . This mitochondrial diversity could impact kinase-dependent signaling in several ways. Mitochondria are central to cellular energy production and stress responses, processes that are often regulated by kinase signaling pathways. Variations in mitochondrial genes could affect ATP production, reactive oxygen species generation, and cellular stress responses, all of which may influence the activity and regulation of kinase-dependent pathways. Additionally, some kinases localize to mitochondria or regulate mitochondrial proteins, suggesting direct interactions between kinase signaling networks and mitochondrial function. The interplay between mitochondrial diversity and kinase signaling represents an important area for future research in understanding C. glabrata pathogenicity and drug resistance.

How might kinase inhibitors be developed as potential therapeutic targets against C. glabrata infections?

Developing kinase inhibitors as therapeutic agents against C. glabrata infections requires a multi-faceted approach focusing on selectivity and efficacy. Researchers would need to:

• Identify essential kinases unique to C. glabrata or with significant structural differences from human homologs

• Determine the three-dimensional structure of target kinases through X-ray crystallography or cryo-EM

• Perform virtual screening and structure-based drug design to identify potential inhibitor scaffolds

• Synthesize and test candidate compounds in vitro for inhibitory activity

• Evaluate efficacy in cellular and animal models of infection

The cell wall integrity pathway kinases, which have been implicated in echinocandin resistance, represent promising targets . A combination approach targeting both the fungal kinase and the antifungal resistance mechanism might prove most effective. For example, inhibitors of CgSLT2 could potentially sensitize resistant C. glabrata strains to existing echinocandin drugs, providing a synergistic therapeutic strategy .

What is the optimal workflow for expressing and purifying recombinant C. glabrata kinases for biochemical studies?

The optimal workflow for expressing and purifying recombinant C. glabrata kinases involves several key steps:

This workflow can be adapted based on specific research needs, such as structural studies requiring higher purity or functional assays requiring preserved enzymatic activity.

How should researchers design experiments to study the substrate specificity of C. glabrata serine/threonine kinases?

Researchers investigating substrate specificity of C. glabrata serine/threonine kinases should employ a multi-faceted experimental approach:

• In silico prediction: Utilize phosphorylation site prediction algorithms trained on fungal kinase datasets to identify potential substrates.

• Peptide array analysis: Screen synthetic peptide libraries representing potential phosphorylation motifs to define consensus sequence preferences.

• Kinase assays with candidate substrates:

  • Express recombinant proteins representing predicted substrates

  • Perform in vitro kinase assays using purified kinase, ATP, and potential substrates

  • Detect phosphorylation via 32P-ATP incorporation or phospho-specific antibodies

• Phosphoproteomic analysis:

  • Compare phosphoproteomes of wild-type and kinase-deficient strains

  • Identify differentially phosphorylated proteins as potential in vivo substrates

  • Validate using targeted approaches such as site-directed mutagenesis

• Genetic interaction studies:

  • Perform synthetic genetic array analysis to identify genes with functional relationships

  • Create double mutants to identify epistatic relationships indicative of pathway connections

This comprehensive approach enables identification of both direct substrates and broader signaling networks influenced by the kinase of interest.

What controls and validation steps are essential when studying the function of KKQ8 in C. glabrata drug resistance?

When studying KKQ8's potential role in drug resistance, researchers must implement rigorous controls and validation steps:

• Essential controls:

  • Positive control: Known kinase mutant with established drug resistance phenotype (e.g., CgSLT2 overexpression strain )

  • Negative control: Wild-type parental strain

  • Complementation control: KKQ8 deletion strain with reintroduced functional KKQ8

  • Kinase-dead control: KKQ8 mutant with catalytically inactive protein

• Validation approaches:

  • Multiple independent mutant clones to rule out off-target effects

  • Complementary approaches (CRISPR-Cas9 and traditional gene deletion)

  • Quantitative phenotype assessment using standardized drug susceptibility testing (CLSI or EUCAST methods)

  • Time-kill assays to distinguish between fungistatic and fungicidal effects

• Mechanistic validation:

  • Transcriptomic analysis to identify altered gene expression

  • Cell wall composition analysis (β-glucan, chitin content)

  • Stress response pathway activation assessment

  • Epistasis analysis with genes in known resistance pathways

• In vivo validation:

  • Testing in multiple infection models (e.g., Drosophila melanogaster and murine models)

  • Comparing virulence with drug treatment efficacy

These rigorous controls and validation steps ensure that any observed drug resistance phenotypes are specifically attributable to KKQ8 function rather than experimental artifacts or secondary mutations.

How do microevolutionary changes in kinase genes contribute to C. glabrata adaptation during infection?

Microevolutionary changes in kinase genes represent important mechanisms for C. glabrata adaptation during infection. Analysis of recurrent cases of candidiasis has revealed in-patient microevolution, with an enrichment for nonsynonymous and frameshift indels in cell surface proteins and genes involved in drug resistance . These genetic alterations can affect kinase functionality, substrate specificity, or expression levels, potentially impacting signaling pathways critical for pathogenicity and drug resistance. For example, mutations in the echinocandin target FKS1/2 coincide with changes in drug susceptibility . Given the central role of kinases in stress responses and cell wall integrity, microevolutionary changes in these genes likely contribute to C. glabrata's ability to adapt to host environments and antifungal treatments. Understanding these adaptive processes requires longitudinal studies of clinical isolates combined with functional characterization of observed genetic variations.

What role might serine/threonine kinases play in C. glabrata's stealth infection strategy?

Serine/threonine kinases likely play crucial roles in C. glabrata's distinctive "stealth" infection strategy. Unlike C. albicans, C. glabrata typically grows in yeast form and has evolved an infection strategy based on stealth and evasion without causing severe damage in murine models . This approach requires sophisticated regulation of virulence factors and stress responses, processes often controlled by kinase signaling networks. The pathogenicity of C. glabrata correlates with the number of epithelial adhesins (EPA) encoded in its genome, which facilitate adherence and colonization of human epithelial cells . Kinases likely regulate the expression and function of these adhesins in response to environmental cues. Additionally, the ability of C. glabrata to survive within macrophages depends on kinase-mediated stress responses, as demonstrated by studies showing that Syk kinase is required at an early stage for macrophage control of C. glabrata . Understanding the specific kinases involved in these processes could reveal key mechanisms underlying C. glabrata's successful colonization and persistence strategies.

How can systems biology approaches enhance our understanding of kinase networks in C. glabrata?

Systems biology approaches offer powerful tools for comprehensively mapping and understanding kinase networks in C. glabrata:

• Multi-omics integration: Combining transcriptomics, proteomics, phosphoproteomics, and metabolomics data can reveal the broader impact of kinase activity across cellular systems. This approach can identify both direct kinase substrates and downstream effects on cellular physiology.

• Network modeling: Constructing kinase signaling networks through computational approaches can predict pathway interactions and identify key regulatory nodes. These models can be validated and refined through targeted experimental interventions.

• Comparative genomics: Analyzing kinase conservation and divergence across Candida species and isolates can reveal evolutionary adaptations in signaling networks. The high genetic diversity observed in C. glabrata populations provides valuable material for such analyses.

• Temporal dynamics: Studying kinase activity across different infection stages can reveal how signaling networks adapt to changing host environments. Time-course experiments, particularly with fluorescent tracking methods , can capture these dynamic responses.

• Host-pathogen interaction modeling: Integrating fungal and host cell data can illuminate how kinase networks respond to and manipulate host defense mechanisms, particularly relevant for understanding C. glabrata's interactions with macrophages .

These systems-level approaches can transform our understanding from isolated kinase functions to comprehensive signaling networks that drive C. glabrata's pathogenicity, stress responses, and drug resistance mechanisms.