Recombinant Candida glabrata Pentafunctional AROM polypeptide (ARO1), partial

What is the Pentafunctional AROM polypeptide (ARO1) in Candida glabrata?

The ARO1 gene in Candida glabrata encodes an essential pentafunctional enzyme that catalyzes consecutive steps in the shikimate pathway. This multi-enzyme is crucial for the biosynthesis of chorismate, which serves as a precursor to folate and aromatic amino acids . The full protein consists of five distinct enzymatic domains that work in concert to convert precursors to essential metabolites. ARO1 is classified as a Drug:H+ antiporter (DHA) family protein and has been identified as essential for C. glabrata viability, making it a potential target for antifungal development .

What specific enzymatic activities are contained within the ARO1 pentafunctional enzyme?

The ARO1 pentafunctional enzyme catalyzes five consecutive reactions in the shikimate pathway, through distinct domains with the following activities:

• 3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase

• 3-Dehydroquinate (DHQ) synthase

• 3-Dehydroquinate dehydratase (DHQase)

• Shikimate dehydrogenase

• Shikimate kinase

Interestingly, molecular analysis has revealed that in some Candida species, not all five domains are functional. For example, in C. albicans, the DHQase domain contains catalytic site substitutions that render it inactive, with this function instead being performed by a separate enzyme called Dqd1 .

How does ARO1 contribute to Candida glabrata survival and virulence?

ARO1 plays a critical role in C. glabrata virulence through several mechanisms:

• Essential metabolic function: As a key enzyme in the shikimate pathway, ARO1 is required for the biosynthesis of aromatic amino acids and folate, making it essential for cell viability .

• Cell wall integrity: Studies have shown that disruption of the shikimate pathway can lead to changes in cell wall integrity and biofilm formation, which are important for pathogenesis .

• Host interaction: Research with infection models such as Galleria mellonella has demonstrated that ARO1 is necessary for full virulence, as mutants show attenuated virulence in these models .

The essential nature of ARO1 has been confirmed through genetic studies, including transposon mapping in haploid isolates of related Candida species .

What methods are most effective for heterologous expression of recombinant C. glabrata ARO1 protein?

For heterologous expression of recombinant C. glabrata ARO1, several approaches have proven successful:

E. coli Expression System:

• A fusion PCR strategy can be employed to amplify the ARO1 ORF and create a fusion with TrxA-6xHis-TCS (thioredoxin with histidine tag and thrombin cleavage site) .

• Cloning into pET vectors (such as pET32b+) with appropriate modifications to the thrombin cleavage site to reduce linker residues.

• Expression in BL21 (DE3) pLysS strain for better expression of the recombinant protein .

S. cerevisiae Expression System:

• ARO1 can be cloned into expression vectors like p426GPD under the control of a constitutive promoter .

• Transformation using the lithium acetate/PEG method as described by Gietz & Schiestl (2007) .

• Verification of transformants by PCR using specific primers targeting the integration junctions.

For protein purification, immobilized metal affinity chromatography (IMAC) utilizing the His-tag would be appropriate, followed by size exclusion chromatography to obtain the dimeric form of the protein.

How can researchers assess the enzymatic activities of individual domains in the ARO1 protein?

To assess the enzymatic activities of individual domains within the pentafunctional ARO1 protein:

What structural and functional differences exist in ARO1 between different Candida species?

ARO1 shows notable structural and functional differences between Candida species:

• Functional domain variations:

  • In C. albicans, the DHQase domain contains sequence substitutions in its catalytic site that render it inactive, while the other four domains remain functional .

  • This function is compensated by the type II DHQase Dqd1 enzyme, which showed robust activity against shikimate pathway intermediates in vitro .

• Sequence conservation:

  • Comparative genomic analyses show significant sequence variation between ARO1 homologs across fungal species.

  • For example, C. thermophilum AroM shows only 52% identity with C. albicans Aro1, indicating substantial evolutionary divergence despite conserved function .

• Structural organization:

  • Molecular imaging of C. albicans Aro1 revealed the architecture of all five enzymatic domains and their arrangement in the full-length protein .

  • ARO1 forms a flexible dimer allowing relative autonomy of enzymatic function for the individual domains .

• Evolutionary relationships:

  • Population genetics studies of C. glabrata isolates have revealed signatures of positive selection in several genes including those involved in epithelial adhesion, suggesting different selective pressures across lineages .

What role does ARO1 play in antifungal resistance mechanisms in C. glabrata?

ARO1's role in antifungal resistance in C. glabrata is multifaceted:

• Metabolic adaptation:

  • Transcriptomic analyses of C. glabrata biofilm cells have shown that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner, particularly affecting genes involved in ergosterol and ubiquinone biosynthesis .

  • As a metabolic enzyme involved in essential pathways, ARO1 likely contributes to these adaptive responses.

• Indirect effects on drug targets:

  • Studies have shown that microevolution within patients can affect genes involved in drug resistance, including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2 .

  • ARO1, as part of essential metabolic pathways, may indirectly influence these resistance mechanisms through metabolic regulation.

• Stress response integration:

  • Similar to other multidrug resistance transporters in C. glabrata (such as CgDtr1), ARO1 function may be integrated with stress response pathways that confer resistance to various stressors encountered during infection .

  • The shikimate pathway's products are essential for adaptation to oxidative and other stresses encountered in the host environment.

What experimental infection models are most suitable for studying ARO1 function in virulence?

Several infection models have proven valuable for studying ARO1 function in virulence:

Galleria mellonella (Greater Wax Moth) Model:

• This invertebrate model has been successfully used to study C. glabrata virulence factors .

• Advantages include:

  • Strong similarity between the larval innate immune system and mammalian innate immunity

  • Presence of hemocytes that function similarly to mammalian macrophages

  • Ability to assess fungal proliferation within hemolymph and hemocytes

  • Quantifiable endpoints including larval survival and fungal burden

Drosophila melanogaster (Fruit Fly) Model:

• This model has been used to test the virulence of CRISPR-Cas9-generated loss-of-function mutants in C. glabrata .

• Mutants in virulence genes showed reduced pathogenicity in this model .

Cell Culture Models:

• Hemocyte or macrophage cell cultures can be used to study fungal proliferation and interactions with immune cells .

• Methods include:

  • Co-culture of C. glabrata cells with hemocytes/macrophages at controlled multiplicity of infection (MOI)

  • Assessment of viable intracellular yeast cells after defined time points

  • Analysis of gene expression changes during internalization

How can CRISPR-Cas9 genome editing be optimized for studying ARO1 function in C. glabrata?

Optimizing CRISPR-Cas9 genome editing for ARO1 studies in C. glabrata requires several strategic approaches:

• Development of a recombinant strain expressing CRISPR-Cas9:

  • Generate a strain constitutively expressing the CRISPR-Cas9 system under appropriate promoters .

  • Consider using either S. cerevisiae or C. glabrata promoters for expression of Cas9 .

• Efficient guide RNA selection:

  • Utilize online programs specific for C. glabrata genome to design guide RNAs with minimal off-target effects .

  • Target sequences unique to ARO1 that are not present elsewhere in the genome.

• Delivery methods:

  • For complete gene deletion: Design guide RNAs targeting the 5' and 3' ends of the ARO1 gene.

  • For domain-specific mutations: Design guide RNAs targeting specific domains to introduce point mutations that affect only one enzymatic activity.

• Repair template design:

  • For homology-directed repair (HDR): Include homology arms of at least 40-50 bp flanking the Cas9 cut site.

  • For non-homologous end joining (NHEJ): Rely on the inherent NHEJ repair pathway in C. glabrata to generate loss-of-function mutations .

• Mutant identification:

  • Use the Surveyor technique and sequencing to identify and confirm mutants .

  • For essential genes like ARO1, consider conditional approaches such as using tetracycline-regulatable promoters.

• Validation of mutants:

  • Functional complementation studies by reintroducing wild-type ARO1 or domain-specific variants .

  • In vivo testing using appropriate infection models to assess virulence phenotypes .

What bioinformatic approaches can identify potential inhibitors targeting C. glabrata ARO1?

To identify potential inhibitors targeting C. glabrata ARO1, researchers can employ several bioinformatic approaches:

• Structural analysis and molecular modeling:

  • Generate homology models of C. glabrata ARO1 based on the available molecular images of fungal Aro1 proteins .

  • Identify unique structural features, particularly in the catalytic sites of each domain.

  • Focus on domains that are essential and unique to fungi compared to human enzymes.

• Virtual screening and docking:

  • Use the structural models to perform virtual screening of compound libraries against the active sites of each enzymatic domain.

  • Prioritize compounds that show high binding affinity to fungal ARO1 but not to human homologs.

  • Consider the dimeric structure of ARO1 and potential allosteric binding sites .

• Exploitation of known domain differences:

  • Target the four functional domains of ARO1 that have been shown to be individually essential for growth of Candida species .

  • Consider the sequence substitutions identified in specific domains (e.g., the inactive DHQase domain in C. albicans) for selective targeting .

• Machine learning approaches:

  • Train models using known inhibitors of related enzymes from other organisms.

  • Use sequence and structural features specific to C. glabrata ARO1 to refine predictions.

  • Implement deep learning methods to identify novel chemical scaffolds with potential inhibitory activity.

• Evolutionary analysis:

  • Analyze signatures of positive selection identified in population genomics studies .

  • Identify conserved residues across fungal species that might be critical for function.

Table: Comparison of ARO1 Domains Across Fungal Species