Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by Candida species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in Candida albicans and Candida glabrata that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.

Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata . We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.

A new toolkit for gene tagging in Candida albicans containing recyclable markers.

Gene manipulation and epitope tagging are essential tools for understanding the molecular function of specific genes. The opportunistic human pathogen Candida albicans is a diploid fungus that utilizes a non-canonical genetic code. Since selection markers available in this organism are scarce, several tools based on recyclable markers have been developed for gene disruption, such as the Clox system. This system relies on the Cre recombinase, which recycles selection markers flanked by loxP sites with high efficiency, facilitating single marker or multi-marker recycling. However, PCR-based modules for epitope tagging, such the pFA-modules, mainly use limited non-recyclable auxotrophic markers. To solve this problem, we have used a Gibson assembly strategy to construct a set of new plasmids where the auxotrophic markers of the pFA vectors were swapped with five recyclable marker modules of the Clox system, enhancing the versatility of the pFA plasmids. This new toolkit, named pFA-Clox, is composed of 36 new vectors for gene disruption and epitope tagging (GFP, 3xGFP, mCherry, 3xHA, 5xmyc and TAP). These plasmids contain the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting functional analysis of laboratory strains as well as clinical isolates of C. albicans. In summary, we have adapted the Clox system to the pFA-backbone vectors. Thus, the set of primers used for the amplification of previously published pFA modules can also be utilized in this new pFA-Clox system. Therefore, this new toolkit harbors the advantages of both systems, allowing accelerated gene modification strategies that could reduce time and costs in strain construction for C. albicans.

The anillin-related Int1 protein and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans.

Variation in cell ploidy is a common feature of Candida albicans clinical isolates that are resistant to the antifungal drug fluconazole. Here, we report that the anillin-related protein Int1 interacts with septins for coupling cytokinesis with nuclear segregation. Loss of Int1 results in a rapid disassembly of duplicated septin rings from the bud neck at the onset of actomyosin ring contraction. Strikingly, this has no major impact on cytokinesis and septum formation. However, Int1 genetically interacts with the Sep7 septin, maintaining the diffusion barrier at the bud neck and guarantying a faithful nuclear segregation. Indeed, int1ΔΔ sep7ΔΔ mutant cells, in contrast to int1ΔΔ cdc10ΔΔ, undergo a premature activation of mitotic exit prior to the alignment of the mitotic spindle with the division axis, producing large multinucleated cells. Some of these multinucleated cells arise from trimeras similar to those observed upon fluconazole exposure. Finally, the defects in nuclear segregation could be in part due to the inability to maintain the Lte1 mitotic exit activator at the cortex of the daughter cell. These results suggest that Int1 and Sep7 play a role in maintaining genome stability by acting as a diffusion barrier for Lte1.

A single nucleotide polymorphism uncovers a novel function for the transcription factor Ace2 during Candida albicans hyphal development.

Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages.

Eng2 is a component of a dynamic protein complex required for endocytic uptake in fission yeast.

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.

Characterization of glycoside hydrolase family 5 proteins in Schizosaccharomyces pombe.

In yeast, enzymes with ß-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-ß(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1(+) (locus SPBC1105.05), exg2(+) (SPAC12B10.11), and exg3(+) (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against ß(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1(+) showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-ß(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.