Recombinant Neosartorya fumigata Transcription elongation factor spt5 (spt5), partial

What is Neosartorya fumigata and how does it relate to Aspergillus fumigata?

Neosartorya fumigata is the teleomorphic (sexual) state of Aspergillus fumigatus, which belongs to the genus Aspergillus section Fumigati. This filamentous fungus is predominantly identified in laboratories by morphological features. It's important to note that accurate identification of Neosartorya species can be challenging based solely on phenotypic characteristics, as several members of the Aspergillus section Fumigati appear morphologically similar but are genetically distinct . This taxonomic relationship is significant for researchers working with recombinant proteins from this organism, as literature may reference either name depending on which form was studied. The accurate identification of these organisms may be clinically meaningful due to potential differences in antifungal susceptibilities and virulence factors .

What is transcription elongation factor Spt5 and its biological significance?

Spt5 is a transcription elongation factor that represents the only known RNA polymerase-associated factor conserved across all three domains of life (Bacteria, Archaea, and Eukarya) . This remarkable evolutionary conservation underscores its fundamental importance in transcription regulation. Functionally, Spt5 stimulates transcription elongation by enhancing the processivity of RNA polymerase. In eukaryotic systems, Spt5 forms a complex with Spt4 (known as Spt4/5 or DSIF in humans), which has both positive and negative effects on transcription elongation . The high degree of conservation suggests that Spt5 from Neosartorya fumigata likely plays a critical role in fungal transcription elongation similar to its homologs in other organisms.

What is the domain architecture of Spt5 protein?

Spt5 contains several conserved domains that are functionally significant. In eukaryotes like Neosartorya fumigata, Spt5 typically contains:

• The NusG N-terminal (NGN) domain - This domain functions as the primary effector domain that mediates interaction with RNA polymerase and is essential for elongation activity .

• Multiple Kyprides–Ouzounis–Woese (KOW) domains - Eukaryotic Spt5 variants include four to six copies of this domain .

• C-terminal hepta- and octa-peptide repeats (in eukaryotes) - These regions are subject to posttranslational phosphorylation that regulates Spt5 activity .

This domain organization is significant as the NGN domain has been identified as containing a hydrophobic pocket that serves as the binding site for RNA polymerase, specifically interacting with the RNAP clamp coiled-coil motif .

What expression systems are recommended for producing recombinant Neosartorya fumigata Spt5?

Based on similar recombinant protein production methods, E. coli expression systems are commonly employed for recombinant fungal proteins. For instance, recombinant Neosartorya fumigata Major allergen Asp f 2 is successfully expressed in E. coli with an N-terminal 6xHis-SUMO tag . For Spt5, a similar approach would be recommended:

• Use an E. coli expression system with a suitable vector containing an N-terminal affinity tag (such as 6xHis or 6xHis-SUMO) to facilitate purification.

• Express the protein in a soluble form in Tris-based buffer, potentially with glycerol for stability.

• Purify using affinity chromatography followed by size exclusion chromatography to achieve >90% purity as determined by SDS-PAGE .

For functional studies of the Spt4/5 complex, it may be necessary to co-express Spt4 and Spt5, as demonstrated in studies of the archaeal Spt4/5 complex .

What are effective purification strategies for recombinant Spt5?

A multi-step purification protocol is recommended for obtaining high-purity recombinant Spt5:

• Immobilized metal affinity chromatography (IMAC) using the His-tag

• Tag cleavage with a specific protease if the experimental design requires tag removal

• Size exclusion chromatography to remove aggregates and further increase purity

• Ion exchange chromatography as a polishing step if necessary

The final preparation should be assessed for purity using SDS-PAGE, with a target purity of greater than 90% . For storage, a Tris-based buffer with 50% glycerol has been effectively used for similar recombinant proteins from Neosartorya fumigata .

How can researchers investigate the structure-function relationship of Neosartorya fumigata Spt5?

To elucidate the structure-function relationship of Neosartorya fumigata Spt5, researchers can employ a multi-faceted approach:

• Domain deletion analysis: Generate constructs expressing truncated versions of Spt5 to identify the minimal functional domains. As demonstrated in studies with archaeal Spt5, the NGN domain is likely the primary effector domain mediating interaction with RNA polymerase and essential for elongation activity .

• Site-directed mutagenesis: Target specific residues in the hydrophobic pocket of the NGN domain that are predicted to interact with RNA polymerase based on homology with characterized Spt5 proteins .

• X-ray crystallography: Determine the three-dimensional structure of Neosartorya fumigata Spt5 alone or in complex with Spt4 and/or RNA polymerase. This approach has been successfully used with the archaeal Spt4/5 complex from Methanococcus jannaschii .

• In vitro transcription assays: Establish recombinant transcription systems to assess the effect of Spt5 on transcription elongation, similar to assays developed for archaeal RNA polymerase . These assays should utilize synthetic DNA-RNA elongation scaffolds and measure the formation of run-off transcripts in the presence and absence of Spt5.

What experimental systems can be used to study Spt5's effect on transcription elongation?

Researchers can establish in vitro transcription elongation assays using:

• Synthetic DNA-RNA elongation scaffolds: These consist of a template DNA strand, an RNA primer, and optionally a non-template DNA strand. As observed with archaeal systems, the addition of the non-template strand enhances processivity .

• Reconstituted transcription system: Using recombinant RNA polymerase from Neosartorya fumigata or a related fungal species, along with purified recombinant Spt5.

• Quantitative analysis: Measure the formation of run-off transcripts both in the absence and presence of Spt5. Based on studies with archaeal Spt4/5, one might expect stimulation of approximately 1.75-2.5 fold in transcript production .

A detailed protocol might include:

• Assembling elongation complexes with radiolabeled RNA primers

• Adding purified Spt5 at varying concentrations

• Initiating elongation by adding nucleotides

• Analyzing transcription products by denaturing gel electrophoresis

• Quantifying stimulation by comparing run-off transcript production with and without Spt5

How can researchers accurately differentiate between Neosartorya fumigata and related species?

Accurate identification of Neosartorya species is crucial as phenotypic identification of filamentous fungi may not reliably distinguish morphologically similar, but genetically distinct, members of the genus Aspergillus section Fumigati . Researchers should employ:

• Multilocus sequence typing: Sequence and analyze specific genetic markers including:

  • Internal transcribed spacer 1 and 2 regions of ribosomal DNA (rDNA)

  • β-tubulin gene

  • Rodlet A gene

• Mating studies: For teleomorphic identification, researchers can perform mating studies with reference strains to confirm identification. This is particularly important for distinguishing between closely related Neosartorya species that cannot be differentiated solely by morphological characteristics .

• Colony morphology analysis: Neosartorya species often present as whitish, fast-growing, slowly sporulating colonies that may only produce conidiophores with conidia after prolonged incubation on laboratory medium .

• Growth on differential media: Culturing isolates on multiple media types such as Potato Dextrose Agar (PDA), Czapek-Dox (CZD) supplemented with 20% dextrose, Malt Extract Agar (MEA), and Sabouraud Dextrose Agar (SDA) can help identify morphological characteristics specific to Neosartorya fumigata .

What are the implications of species misidentification in Neosartorya research?

Misidentification of Neosartorya species can have significant implications for research:

• Genetic variability: Different Neosartorya species can have distinct genetic profiles that affect protein structure and function, potentially leading to misleading experimental results if species are incorrectly identified .

• Antifungal susceptibility differences: Various Neosartorya species show different minimum inhibitory concentrations (MICs) to antifungal agents. For example, N. pseudofischeri isolates have demonstrated higher MICs to voriconazole compared to A. fumigatus Af293 . This variability could impact studies investigating drug resistance mechanisms or therapeutic targets.

• Pathogenicity factors: Different species may possess unique virulence factors or pathogenicity mechanisms. For instance, N. udagawae causes infections with distinct clinical manifestations compared to typical invasive aspergillosis caused by A. fumigatus .

• Research reproducibility: Using inconsistently identified isolates across different studies leads to poor reproducibility and conflicting results in the scientific literature.

How does Spt5 from Neosartorya fumigata compare to homologs in other organisms?

Spt5 is remarkable for being the only known RNA polymerase-associated transcription factor universally conserved across all three domains of life . A comparative analysis would likely reveal:

• Domain conservation: The NGN domain, which mediates interaction with RNA polymerase, is highly conserved in structure and function across bacteria (NusG), archaea (Spt5), and eukaryotes (Spt5) .

• Functional conservation: Across species, Spt5 stimulates transcription elongation by enhancing the processivity of RNA polymerase. In archaeal systems, Spt4/5 stimulates the formation of run-off transcripts by approximately 1.75-2.5 fold . Similar stimulation might be expected from Neosartorya fumigata Spt5.

• Structural variations: While eukaryotic Spt5 typically forms a complex with Spt4 and contains multiple KOW domains (four to six) and C-terminal repeat regions subject to phosphorylation, bacterial NusG is generally a single protein with fewer domains .

• Interaction interfaces: The hydrophobic pocket on the Spt5 NGN domain that serves as the binding site for RNA polymerase appears to be a conserved feature, as is the interaction with the RNAP clamp coiled-coil motif .

What insights from model organisms can be applied to Neosartorya fumigata Spt5 research?

Studies in model organisms provide valuable insights for Neosartorya fumigata Spt5 research:

• Archaeal Spt4/5 studies: Research on archaeal Spt4/5 complexes, particularly from Methanococcus jannaschii, offers a framework for understanding the core structure and function of Spt5. The archaeal studies demonstrate that the NGN domain is the primary effector domain mediating RNA polymerase interaction and elongation activity .

• Human DSIF studies: Research on human Spt4/5 (DSIF) shows both positive effects on processivity and involvement in promoter-proximal stalling of RNA polymerase II, suggesting complex regulatory roles that might also be present in N. fumigata Spt5 .

• Bacterial NusG research: Studies on bacterial NusG have demonstrated pleiotropic effects on transcription elongation and termination, providing a basis for understanding the fundamental roles of this protein family .

• Experimental approaches: Methods for in vitro transcription elongation assays using synthetic DNA-RNA elongation scaffolds have been established in archaeal systems and can be adapted for studying N. fumigata Spt5 .

What is the significance of studying Spt5 in the context of Neosartorya fumigata infections?

Studying Spt5 in Neosartorya fumigata has several important implications:

• Transcriptional regulation during infection: Understanding how Spt5 regulates gene expression in N. fumigata may reveal mechanisms controlling virulence factor production during host invasion.

• Antifungal resistance mechanisms: Transcriptional responses to antifungal drugs may involve Spt5-mediated regulation. Neosartorya species have demonstrated variable susceptibility to antifungal agents, with some showing relatively higher minimum inhibitory concentrations to various agents compared to typical A. fumigatus isolates .

• Species identification: Research on species-specific variations in Spt5 could potentially contribute to molecular methods for distinguishing between morphologically similar Neosartorya species, addressing the challenge that phenotypic identification may not reliably identify genetically distinct members of the Aspergillus section Fumigati .

• Potential therapeutic target: Given its essential role in transcription, Spt5 might represent a novel target for antifungal drug development, particularly if fungal-specific features can be identified.

How can Spt5 research contribute to understanding chronic fungal infections?

Research on Spt5 in Neosartorya fumigata may provide insights into chronic fungal infections:

• Persistent transcriptional programs: Spt5's role in regulating gene expression might influence adaptation to long-term host colonization. Infections due to some Neosartorya species like N. udagawae have been observed to be chronic, with a median duration of 35 weeks, compared to a median of 5.5 weeks for infections caused by A. fumigatus sensu stricto in patients with chronic granulomatous disease .

• Stress response regulation: Spt5's involvement in transcriptional responses to various stresses may explain how these fungi persist despite host immune responses and antifungal therapy.

• Biofilm formation: If Spt5 regulates genes involved in biofilm formation, this could explain the difficulty in eradicating chronic Neosartorya infections, as biofilms are known to enhance resistance to both immune clearance and antifungal agents.

• Host-pathogen interaction: Understanding how Spt5-regulated genes respond to the host environment may reveal mechanisms by which Neosartorya infections spread across anatomical planes in a contiguous manner and become refractory to standard therapy, as observed in clinical cases .

What are common challenges in expressing and purifying functional Spt5, and how can they be addressed?

Researchers may encounter several challenges when working with recombinant Neosartorya fumigata Spt5:

• Protein solubility: Spt5 may form inclusion bodies when expressed in E. coli. To address this:

  • Optimize expression temperature (typically lower temperatures of 16-18°C)

  • Use solubility-enhancing fusion tags like SUMO or MBP

  • Consider co-expression with Spt4, as the Spt4/5 complex may be more soluble than Spt5 alone

  • Use a Tris-based buffer with 50% glycerol for storage, similar to other recombinant proteins from Neosartorya fumigata

• Protein activity: Ensuring the recombinant protein retains its native activity:

  • Express the full-length protein (if known) or conduct domain analyses to determine the minimal functional unit

  • Consider that the NGN domain alone may be sufficient for some functional studies based on research with archaeal Spt5

  • Verify protein folding using circular dichroism or limited proteolysis

• Complex formation: If studying the Spt4/5 complex:

  • Either co-express both proteins or reconstitute the complex in vitro from separately purified components

  • Verify complex formation using size exclusion chromatography or native PAGE

What controls are essential for validating Spt5 activity in transcription elongation assays?

When establishing in vitro transcription elongation assays to study Spt5 function, several controls are essential:

• Negative controls:

  • Assays without Spt5 to establish baseline transcription elongation rates

  • Heat-inactivated Spt5 to confirm that the observed effects are due to the active protein

  • Mutant Spt5 with alterations in the hydrophobic pocket of the NGN domain that disrupts RNA polymerase binding

• Positive controls:

  • If available, a well-characterized transcription elongation factor with known stimulatory effects

  • Comparison of 10-subunit vs. 12-subunit RNA polymerase reactions, as the latter has been shown to have higher processivity in archaeal systems

• Experimental variations:

  • Tests with and without the non-template strand, as its presence enhances processivity in transcription systems

  • Concentration gradient of Spt5 to establish dose-dependent effects

  • Various DNA templates to ensure the effect is not template-specific

• Technical replicates:

  • At least three independent experiments to ensure reproducibility

  • Statistical analysis to quantify the significance of observed stimulation