Recombinant Schizosaccharomyces pombe Prohibitin-2 (phb2)

What is Prohibitin-2 (Phb2) in S. pombe and what are its fundamental characteristics?

Prohibitin-2 (Phb2) is a highly conserved mitochondrial protein in Schizosaccharomyces pombe that forms part of the prohibitin complex in the inner mitochondrial membrane. This protein is encoded by the phb2+ gene and is implicated in diverse cellular processes ranging from mitochondrial biogenesis to cell proliferation . Phb2 is characterized by its ability to form a high-molecular-weight complex with Phb1 in the inner mitochondrial membrane, where it plays crucial roles in maintaining mitochondrial function and integrity . The protein contains a transmembrane helix located at the N-terminus (predicted at amino acids 37-59 in the budding yeast homolog), which anchors it to the mitochondrial membrane with the majority of the protein facing the intermembrane space . This structural arrangement distinguishes it from Phb1, whose N-terminal helical region is shorter and may not constitute a true membrane-spanning domain, potentially explaining some of the functional differences between these two proteins .

How does S. pombe serve as a model organism for Phb2 research?

Schizosaccharomyces pombe, commonly known as fission yeast, serves as an excellent model organism for Phb2 research due to several advantageous characteristics. As a unicellular eukaryote, S. pombe has a fully sequenced genome (published in 2002), making genetic manipulations and analyses straightforward . Approximately 70% of S. pombe genes have orthologues in humans, including many involved in human diseases, making findings potentially translatable to human health applications . The well-characterized cell cycle and relatively simple genetic makeup of S. pombe facilitate clear interpretation of experimental results when studying complex cellular processes like those involving Phb2 .

For Phb2 research specifically, S. pombe offers several advantages: the protein is highly conserved between S. pombe and pathogenic fungi (including Cryptococcus, Aspergillus, and Candida species), allowing potential application of findings to clinically relevant pathogens . Additionally, the well-established genetic tools for S. pombe, including methods for gene overexpression and deletion, make it straightforward to manipulate Phb2 levels and study the resulting phenotypes . S. pombe has also become an important organism for studying cellular responses to DNA damage and DNA replication processes, which may intersect with Phb2 functions .

What is the relationship between Phb2 and antifungal drug resistance in basic terms?

In basic terms, both overexpression and deletion of the phb2+ gene in S. pombe lead to reduced susceptibility to multiple antifungal drugs, including clotrimazole, terbinafine, fluconazole, and amphotericin B . This counterintuitive finding—that both too much and too little Phb2 result in similar drug resistance phenotypes—suggests a complex relationship between Phb2 function and drug sensitivity mechanisms .

The current understanding is that any dysfunction of Phb2, whether through overexpression or deletion, disrupts normal mitochondrial function. This mitochondrial dysfunction leads to increased production of reactive oxygen species (ROS) and nitric oxide (NO) . These reactive molecules then activate the transcription factor Pap1, which is responsive to oxidative stress . The activation of Pap1 ultimately results in the upregulation of genes that confer resistance to antifungal drugs .

This mechanism has been experimentally supported by several key observations: (1) deletion of the pap1+ gene abolishes the drug resistance caused by Phb2 dysfunction; (2) both Phb2 overexpression and deletion significantly increase intracellular NO and ROS levels; and (3) Phb2 manipulation leads to increased mRNA expression of the pap1+ gene .

What is the detailed mechanism by which Phb2 dysfunction confers antifungal resistance?

The mechanism by which Phb2 dysfunction leads to antifungal drug resistance involves a complex cascade of cellular events centered around mitochondrial function, oxidative stress, and transcriptional regulation. When Phb2 is either overexpressed or deleted in S. pombe, the following sequence of events appears to occur:

First, Phb2 dysfunction causes significant mitochondrial damage. When Phb2 is overexpressed, mitochondria become fragmented and Phb2-RFP aggregates abnormally, consistent with previous reports showing that prohibitin depletion leads to fragmented and disorganized mitochondria . This structural perturbation of mitochondria represents the initial cellular insult.

Second, the compromised mitochondria produce elevated levels of both reactive oxygen species (ROS) and nitric oxide (NO). Studies using fluorescent probes such as DCFH-DA for ROS and DAF-FM DA for NO have demonstrated significant increases in both molecules in cells with either Phb2 overexpression or deletion . This is consistent with previous findings that prohibitin deficiency results in increased ROS production .

Third, the elevated ROS and NO levels activate the Pap1 transcription factor, which is crucial for the oxidative stress response in S. pombe. Pap1 accumulates in the nucleus in response to stress and activates numerous genes involved in stress tolerance . Both overexpression and deletion of the phb2+ gene significantly increase the mRNA expression of pap1+, indicating Pap1 activation .

Finally, activated Pap1 drives the expression of genes that confer resistance to multiple antifungal drugs. This is evidenced by the fact that deletion of the pap1+ gene abolishes the drug resistance phenotype caused by Phb2 dysfunction, and that direct overexpression of Pap1 exhibits drug resistance phenotypes similar to those seen with Phb2 dysfunction .

Why do both overexpression and deletion of Phb2 result in similar phenotypes?

The paradoxical finding that both overexpression and deletion of the phb2+ gene result in similar phenotypes of antifungal drug resistance represents a complex example of how protein homeostasis is critical for normal cellular function. This phenomenon can be explained by several potential mechanisms:

First, the Phb2 protein functions as part of a multiprotein complex with Phb1 in the inner mitochondrial membrane . The proper stoichiometry of this complex is likely crucial for its function. Both overexpression and deletion would disrupt this stoichiometry, albeit in different ways: deletion removes a necessary component, while overexpression may lead to an excess of unpaired Phb2 molecules that cannot form functional complexes and may instead form dysfunctional aggregates .

Second, Phb2 appears to have a distinct role from Phb1 in drug resistance pathways. While deletion of phb1+ resulted in drug resistance similar to that observed with phb2+ deletion, overexpression of phb1+ did not cause drug resistance, unlike phb2+ overexpression . This difference may be related to the structural distinctions between the two proteins: Phb2 has a well-defined transmembrane domain at its N-terminus that anchors it in the mitochondrial membrane, while the corresponding region in Phb1 is shorter and may not constitute a true membrane-spanning domain . These structural differences may explain why Phb2 has functions in drug resistance distinct from those of Phb1.

Third, experiments with truncated versions of Phb2 have shown that overexpression of just the N-terminal region (amino acids 1-393) is sufficient to cause drug resistance . This suggests that the N-terminal domain of Phb2, which includes the transmembrane helix, plays a critical role in the protein's function in drug resistance pathways.

In both cases—overexpression and deletion—the end result is mitochondrial dysfunction leading to increased ROS and NO production, which activates the Pap1 pathway and ultimately results in drug resistance .

How do the effects of Phb2 manipulation differ between S. pombe and S. cerevisiae?

The effects of Phb2 manipulation show interesting species-specific differences between the fission yeast S. pombe and the budding yeast Saccharomyces cerevisiae, despite the high conservation of the Phb2 protein. These differences provide valuable insights into the evolutionary divergence of cellular pathways and mechanisms of drug resistance across fungal species.

In S. pombe, deletion of the phb2+ gene results in resistance to multiple antifungal drugs, including clotrimazole, fluconazole, terbinafine, amphotericin B, and phenylglyoxal . Similarly, overexpression of phb2+ in S. pombe also confers resistance to these drugs .

These differences can be summarized in the following table:

The mechanisms underlying these species-specific differences remain unknown , but they likely reflect divergent evolutionary pathways in how these yeasts respond to environmental stresses and xenobiotics. The differences may be related to variations in mitochondrial function, ROS signaling pathways, or transcription factor networks between the two species. These discrepancies highlight the importance of considering species-specific effects when using yeast models to study drug resistance mechanisms and when attempting to translate findings across different fungal species.

How are genetic modifications of Phb2 performed in S. pombe?

Genetic modifications of Phb2 in S. pombe can be performed using several established techniques described in the literature. These methods allow researchers to create strains with various alterations to the phb2+ gene for functional studies.

For the isolation and identification of the phb2+ gene, researchers have used an S. pombe genomic library cloned into vectors such as pDB248 . Transformation of this library into wild-type cells (for example, strain HM123) followed by screening for phenotypes of interest, such as drug resistance, allows for the identification of genes involved in specific cellular processes . In the case of phb2+, transformants were replica-plated onto YPD plates containing clotrimazole or terbinafine to identify colonies showing resistance to these drugs .

For the creation of phb2+ deletion strains, the standard approach involves replacing the target gene with a selectable marker through homologous recombination. This typically requires constructing a deletion cassette containing the selectable marker (such as ura4+ or a drug resistance gene) flanked by sequences homologous to the regions upstream and downstream of the phb2+ gene. After transformation, cells in which successful homologous recombination has occurred can be selected using the appropriate medium or drug .

For overexpression studies, the phb2+ gene can be cloned into expression vectors with strong promoters suitable for S. pombe. The recovered plasmids can then be transformed into wild-type cells, and the phenotypes of the transformants can be analyzed to assess the effects of phb2+ overexpression .

To create strains expressing tagged versions of Phb2 for localization studies or protein interaction analyses, researchers can introduce sequences encoding tags such as GFP (green fluorescent protein) or RFP (red fluorescent protein) in frame with the phb2+ coding sequence. This can be done either by modifying the genomic copy of the gene or by expressing the tagged version from a plasmid .

What methods are used to assess drug resistance in Phb2-modified strains?

Researchers employ several methods to assess drug resistance in Phb2-modified S. pombe strains, with the streak assay and spot assay being the most commonly used approaches:

The streak assay is described as the simplest method for assessing strain growth. In this approach, each strain is streaked onto solid agar plates containing either YPD medium alone (as a control) or YPD supplemented with the drug of interest . The plates are then incubated at an appropriate temperature (typically 27°C for S. pombe) for several days (usually 4 days). After incubation, the number and size of the colonies that form are evaluated to assess drug resistance . Strains with increased resistance will form more and/or larger colonies on drug-containing media compared to sensitive strains.

The spot assay provides a more semi-quantitative approach for studying yeast cell growth. In this method, yeast cells are first grown to log phase in liquid medium and then resuspended in fresh medium to a standardized optical density (typically OD660 of 0.3, which corresponds to approximately 10^7 cells/ml) . A series of 10-fold serial dilutions of each culture is then prepared, and a small volume (5 μl) of each dilution is spotted onto solid agar plates containing either no drug or various concentrations of the drug being tested . The plates are incubated at the appropriate temperature (27°C for S. pombe, 30°C for S. cerevisiae) for several days, after which the number and size of the colonies at each spot are analyzed to assess drug resistance . This method allows for a more precise comparison of the relative drug sensitivities of different strains, as it can reveal differences in growth at various cell densities.

For more quantitative analyses, researchers may use liquid culture growth assays in which cells are grown in the presence of different drug concentrations, and growth is monitored by measuring optical density over time. This can allow for the determination of parameters such as minimum inhibitory concentration (MIC) or IC50 values (the drug concentration that inhibits growth by 50%).

How are NO and ROS levels measured in S. pombe with Phb2 dysfunction?

To measure nitric oxide (NO) and reactive oxygen species (ROS) levels in S. pombe strains with Phb2 dysfunction, researchers employ cell-permeant fluorescent probes that become activated in the presence of these reactive molecules.

For nitric oxide detection, the fluorescent NO probe DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) is used . This compound is cell-permeable and relatively non-fluorescent until it reacts with NO to form a fluorescent benzotriazole derivative. The experimental protocol likely involves incubating cells with the probe for a specified period, washing away excess probe, and then measuring fluorescence intensity using either fluorescence microscopy or a fluorometer. The intensity of the fluorescence signal correlates with the level of NO in the cells, allowing for quantitative comparisons between different strains or conditions.

For ROS detection, the fluorescent ROS probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) is employed . Similar to DAF-FM DA, DCFH-DA is cell-permeable and non-fluorescent until it is oxidized by ROS to form the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF). The protocol for ROS measurement likely follows a similar approach to NO measurement, with cells being incubated with the probe, washed, and then analyzed for fluorescence intensity.

Using these probes, researchers have demonstrated that both overexpression and deletion of the phb2+ gene significantly increase NO and ROS levels in S. pombe cells . This finding is crucial for understanding the mechanism by which Phb2 dysfunction leads to antifungal drug resistance, as the increased NO and ROS levels activate the stress-responsive transcription factor Pap1, which in turn upregulates genes involved in drug resistance .

It's worth noting that these fluorescent probes have some limitations, including potential artifacts from auto-oxidation and limited specificity for different ROS types. Therefore, complementary approaches such as gene expression analysis of oxidative stress response genes or direct biochemical assays for specific reactive species might also be employed for a more comprehensive assessment of cellular redox status.

What are the translational implications of S. pombe Phb2 research for pathogenic fungi?

The research on Phb2 in S. pombe has significant translational implications for understanding and potentially combating antifungal drug resistance in pathogenic fungi. These implications stem from several key observations and similarities between S. pombe and clinically relevant fungal pathogens.

First, the proteins involved in the Phb2-mediated drug resistance pathway—Phb1, Phb2, and Pap1—are highly conserved across diverse genera of human-pathogenic fungi, including Cryptococcus, Aspergillus, and Candida species . These fungi represent some of the most clinically significant fungal pathogens, causing a range of infections from superficial to life-threatening systemic mycoses. The high conservation of these proteins suggests that the mechanisms of Phb2-mediated drug resistance identified in S. pombe may operate similarly in these pathogenic fungi.

Second, the finding that Phb2 dysfunction confers resistance to multiple classes of antifungal drugs, including azoles (clotrimazole, fluconazole), allylamines (terbinafine), and polyenes (amphotericin B) , is particularly concerning from a clinical perspective. These drug classes represent the majority of currently available antifungal therapeutics, and multidrug resistance poses a significant challenge in treating fungal infections.

Third, the identification of the Pap1 pathway as a mediator of drug resistance provides a potential target for developing adjunctive therapies that could restore drug sensitivity . Compounds that inhibit Pap1 activation or its downstream effects might be used in combination with existing antifungal drugs to overcome resistance.

The authors of the study explicitly note that their findings "may pave the way for the development of novel therapeutic strategies to combat fungal diseases" . Potential strategies might include:

• Developing inhibitors of Phb2 function or expression to prevent its role in drug resistance.

• Targeting the Pap1 pathway to prevent activation of drug resistance mechanisms.

• Designing combination therapies that address both the primary antifungal target and the resistance mechanisms mediated by Phb2-Pap1.

• Using the knowledge of Phb2-mediated resistance to develop diagnostic tools that can predict drug resistance in clinical isolates.

What unresolved questions exist about the relationship between Phb2 and antifungal resistance?

Despite the significant advances in understanding the role of Phb2 in antifungal drug resistance, several important questions remain unresolved, presenting opportunities for future research in this field.

One major unresolved question is why the effects of Phb2 deletion differ so markedly between S. pombe and S. cerevisiae . While phb2 deletion in S. pombe confers resistance to most antifungal drugs tested, the same genetic modification in S. cerevisiae results in increased sensitivity to many of these drugs . Understanding the molecular basis for these species-specific differences could provide insights into the diverse mechanisms of drug resistance across fungal species and potentially identify species-specific vulnerabilities that could be exploited therapeutically.

Another important question concerns the precise molecular mechanism by which Phb2 dysfunction leads to increased NO and ROS production. While the study demonstrates that both overexpression and deletion of phb2+ result in elevated NO and ROS levels , the exact pathway leading from Phb2 dysfunction to increased production of these reactive molecules remains unclear. Elucidating this pathway could reveal additional targets for intervention to prevent drug resistance.

The relationship between Phb2 and Phb1 in mediating drug resistance also requires further investigation. The study found that while both phb1 and phb2 deletion resulted in drug resistance, only phb2 overexpression, not phb1 overexpression, caused resistance . The molecular basis for this functional distinction between these two components of the prohibitin complex is not fully understood.

Additionally, the specific genes regulated by Pap1 that directly contribute to drug resistance remain to be comprehensively identified. While the study establishes that Pap1 activation is necessary for Phb2-mediated drug resistance , the downstream targets of Pap1 that confer resistance to different classes of antifungal drugs have not been fully characterized.

Finally, the relevance of these findings to clinical antifungal resistance in pathogenic fungi requires validation. While the proteins involved are conserved across many fungal species , the extent to which this specific mechanism contributes to drug resistance in clinical isolates of pathogenic fungi has not yet been determined.

What innovative experimental approaches could advance Phb2 research?

Several innovative experimental approaches could significantly advance Phb2 research and address current knowledge gaps:

CRISPR-Cas9 Genome Editing: While traditional gene deletion and overexpression methods have been valuable, CRISPR-Cas9 technology could enable more precise modifications of the phb2+ gene. This approach would allow researchers to create specific point mutations or domain deletions to identify the critical regions of Phb2 involved in drug resistance. Additionally, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems could enable fine-tuned regulation of phb2+ expression, providing insights into how different expression levels affect drug sensitivity.

Proteomics and Interactomics: Advanced proteomic approaches could help identify the complete set of proteins that interact with Phb2 under different conditions. Techniques such as proximity-dependent biotin identification (BioID) or affinity purification coupled with mass spectrometry could reveal dynamic changes in the Phb2 interactome in response to drug exposure or oxidative stress. This would help elucidate the broader signaling networks involved in Phb2-mediated drug resistance.

Real-time Imaging of Mitochondrial Dynamics: Live-cell imaging techniques, including super-resolution microscopy, could provide detailed insights into how Phb2 dysfunction affects mitochondrial morphology, membrane potential, and dynamics in real-time. These approaches could help establish the temporal relationship between mitochondrial dysfunction, ROS/NO production, and Pap1 activation.

Single-cell Analysis: Single-cell RNA sequencing or single-cell proteomics could reveal cell-to-cell variability in the response to Phb2 manipulation. This would be particularly valuable for understanding heterogeneity in drug resistance within a population and could identify subpopulations with distinct resistance mechanisms.

Mathematical Modeling: Developing computational models of the Phb2-Pap1 pathway could help integrate the various experimental data and predict system behavior under different conditions. Such models could guide experimental design and help identify key control points in the pathway that might serve as potential therapeutic targets.

Comparative Genomics and Transcriptomics: Systematic comparison of gene expression patterns between S. pombe and S. cerevisiae in response to Phb2 manipulation could help explain the species-specific differences in drug sensitivity. This approach could also be extended to pathogenic fungi to assess the conservation of these pathways across species.

Small Molecule Screening: High-throughput screening of chemical libraries could identify compounds that specifically inhibit the Phb2-Pap1 pathway without affecting other cellular processes. Such compounds would not only serve as valuable research tools but might also represent starting points for the development of adjunctive therapies to combat antifungal resistance.