Fungal burden, dimorphic transition and candidalysin: Role in Candida albicans-induced vaginal cell damage and mitochondrial activation in vitro.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.

An oxylipin signal confers protection against antifungal echinocandins in pathogenic aspergilli.

Aspergillus fumigatus is the leading causative agent of life-threatening invasive aspergillosis in immunocompromised individuals. One antifungal class used to treat Aspergillus infections is the fungistatic echinocandins, semisynthetic drugs derived from naturally occurring fungal lipopeptides. By inhibiting beta-1,3-glucan synthesis, echinocandins cause both fungistatic stunting of hyphal growth and repeated fungicidal lysis of apical tip compartments. Here, we uncover an endogenous mechanism of echinocandin tolerance in A. fumigatus whereby the inducible oxylipin signal 5,8-diHODE confers protection against tip lysis via the transcription factor ZfpA. Treatment of A. fumigatus with echinocandins induces 5,8-diHODE synthesis by the fungal oxygenase PpoA in a ZfpA dependent manner resulting in a positive feedback loop. This protective 5,8-diHODE/ZfpA signaling relay is conserved among diverse isolates of A. fumigatus and in two other Aspergillus pathogens. Our findings reveal an oxylipin-directed growth program-possibly arisen through natural encounters with native echinocandin producing fungi-that enables echinocandin tolerance in pathogenic aspergilli.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

The CHY-type zinc finger protein MoChy1 is involved in polarized growth, conidiation, autophagy and pathogenicity of Magnaporthe oryzae.

Polarized growth is critical for the development of filamentous phytopathogens, and the CHY-type zinc finger protein Chy1 regulates microtubule assembly to influence polarized growth and thereby affect plant infections. However, the biological role of a Chy1 homolog MoChy1 remains unknown in Magnaporthe oryzae. We found here that the MoChy1-GFP was distributed in the cytoplasm outside the vacuole in hyphae and localized mainly to the vacuole compartments as the appressorium matured. The Mochy1 mutants showed an extremely slow growth rate, curved and branched mycelium, reduced conidiation, and a smaller size in the appressorium. Meanwhile, the Mochy1 mutants showed increased sensitivity to benomyl, damaged microtubule cytoskeleton, and mislocalized polarisome protein MoSpa2 and chitin synthase MoChs6 in hyphae. Compared to Guy11, the Mochy1 mutants exhibited increased sensitivity to H2O2, impaired ability to eliminate host-derived ROS and reduced penetration into host plants, resulting in a strong reduction in pathogenicity of Mochy1 mutants. Furthermore, the Mochy1 mutants also exhibited defects in chitin distribution, osmotic stress tolerance, and septin ring organization during appressorium differentiation and fungal development. Nonselective autophagy was negatively regulated in Mochy1 mutants compared to Guy11. In summary, MoChy1 plays multiple roles in fungal polar growth and full virulence of M. oryzae.

The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched ß-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

The Rfg1 and Bcr1 transcription factors regulate acidic pH-induced filamentous growth in Candida albicans.

IMPORTANCE: Candida albicans is a human commensal and frequent pathogen that encounters a wide range of pH stresses. The ability of C. albicans to adapt to changes in extracellular pH is crucial for its success in colonization and pathogenesis. The Rim101 pH sensing pathway is well known to govern neutral-alkaline pH responses in this pathogen. Here, we report a novel Rfg1-Bcr1 regulatory pathway that governs acidic pH responses and regulates filamentous growth in C. albicans. In addition, the Rim101-Phr1 pathway, cAMP signaling pathway, transcription factors Efg1 and Flo8, and hyphal-specific G1 cyclin Hgc1 cooperate with this regulation. Our findings provide new insights into the regulatory mechanism of acidic pH response in C. albicans.

Hyphae-specific genes: Possible molecular targets for magnetic iron oxide nanoparticles alone and combined with visible light in Candida albicans.

Candida albicans readily develops resistance to fluconazole. Magnetic iron oxide nanoparticles (denoted as MION) and antimicrobial photodynamic therapy are attracting attention as therapeutic agents. This study aims to investigate the inhibitory efficacy of MION alone and combined with visible light against C. albicans and expression analysis of hyphal wall protein 1 (HWP1) and agglutinin-like sequence 1 (ALS1) genes in C. albicans. Antifungal susceptibility testing, photodynamic activity assay, reactive oxygen species (ROS) production assay and gene expression analysis were determined in C. albicans treated with MION alone and combined with visible light. MION at 1 × minimum inhibitory concentration (MIC) level (500 µg/mL) exhibited antifungal activity against C. albicans isolates. Further, 1 × MIC levels of MION alone and combined with visible light displayed remarkable fungicidal effects at 24 and 48 h after treatment. The MION combined with visible light caused the highest levels of ROS production by all C. albicans isolates. The relative RT-PCR data showed significant downregulation of HWP1 and ALS1 genes which are the key virulence genes in C. albicans. Differences in gene expression of  HWP1 and ALS1 were more significant in MION combined with visible light treatments than MION alone. Our study sheds a novel light on facile development of effective treatment of C. albicans especially fluconazole-resistant C. albicans infections. The hyphae-specific genes HWP1 and ALS1 could be probable molecular targets for MION alone and combined with visible light in C. albicans.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

Mechanistic model for nuclear migration in hyphae during mitosis.

Saccharomyces cerevisiae and Candida albicans, the two well-known human pathogens, can be found in all three morphologies, i.e., yeast, pseudohyphae, and true hyphae. The cylindrical daughter-bud (germ tube) grows very long for true hyphae, and the cell cycle is delayed compared to the other two morphologies. The place of the nuclear division is specific for true hyphae determined by the position of the septin ring. However, the septin ring can localize anywhere inside the germ tube, unlike the mother-bud junction in budding yeast. Since the nucleus often migrates a long path in the hyphae, the underlying mechanism must be robust for executing mitosis in a timely manner. We explore the mechanism of nuclear migration through hyphae in light of mechanical interactions between astral microtubules and the cell cortex. We report that proper migration through constricted hyphae requires a large dynein pull applied on the astral microtubules from the hyphal cortex. This is achieved when the microtubules frequently slide along the hyphal cortex so that a large population of dyneins actively participate, pulling on them. Simulation shows timely migration when the dyneins from the mother cortex do not participate in pulling on the microtubules. These findings are robust for long migration and positioning of the nucleus in the germ tube at the septin ring.

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

A C. albicans TRAPP Complex-Associated Gene Contributes to Cell Wall Integrity, Hyphal and Biofilm Formation, and Tissue Invasion.

While endocytic and secretory pathways are well-studied cellular processes in the model yeast Saccharomyces cerevisiae, they remain understudied in the opportunistic fungal pathogen Candida albicans. We previously found that null mutants of C. albicans homologs of the S. cerevisiae early endocytosis genes ENT2 and END3 not only exhibited delayed endocytosis but also had defects in cell wall integrity, filamentation, biofilm formation, extracellular protease activity, and tissue invasion in an in vitro model. In this study, we focused on a potential C. albicans homolog to S. cerevisiae TCA17, which was discovered in our whole-genome bioinformatics approach aimed at identifying genes involved in endocytosis. In S. cerevisiae, TCA17 encodes a transport protein particle (TRAPP) complex-associated protein. Using a reverse genetics approach with CRISPR-Cas9-mediated gene deletion, we analyzed the function of the TCA17 homolog in C. albicans. Although the C. albicans tca17Δ/Δ null mutant did not have defects in endocytosis, it displayed an enlarged cell and vacuole morphology, impaired filamentation, and reduced biofilm formation. Moreover, the mutant exhibited altered sensitivity to cell wall stressors and antifungal agents. When assayed using an in vitro keratinocyte infection model, virulence properties were also diminished. Our findings indicate that C. albicans TCA17 may be involved in secretion-related vesicle transport and plays a role in cell wall and vacuolar integrity, hyphal and biofilm formation, and virulence. IMPORTANCE The fungal pathogen Candida albicans causes serious opportunistic infections in immunocompromised patients and has become a major cause of hospital-acquired bloodstream infections, catheter-associated infections, and invasive disease. However, due to a limited understanding of Candida molecular pathogenesis, clinical approaches for the prevention, diagnosis, and treatment of invasive candidiasis need significant improvement. In this study, we focus on identifying and characterizing a gene potentially involved in the C. albicans secretory pathway, as intracellular transport is critical for C. albicans virulence. We specifically investigated the role of this gene in filamentation, biofilm formation, and tissue invasion. Ultimately, these findings advance our current understanding of C. albicans biology and may have implications for the diagnosis and treatment of candidiasis.

The posttranscriptional regulator CsrA affects multidrug resistance and biocontrol activity in Lysobacter enzymogenes.

AIMS: The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. METHODS AND RESULTS: In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. CONCLUSION: These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity.

Apoptotic Factors, CaNma111 and CaYbh3, Function in Candida albicans Filamentation by Regulating the Hyphal Suppressors, Nrg1 and Tup1.

The morphological switch from the yeast to hyphal form is a key virulence attribute of the opportunistic fungal pathogen, Candida albicans. Our recent report showed that deletion of the newly identified apoptotic factor, CaNma111 or CaYbh3, leads to hyperfilamentation and increased virulence in a mouse infection model. CaNma111 and CaYbh3 are homologs of the pro-apoptotic protease, HtrA2/Omi, and BH3-only protein, respectively. In this study, we examined the effects of CaNMA111 and CaYBH3 deletion mutations on the expression levels of the hypha-specific transcription factors, Cph1 (a hyphal activator), Nrg1 (a hyphal repressor), and Tup1 (a hyphal repressor). The protein levels of Nrg1 were decreased in Caybh3/Caybh3 cells while those of Tup1 were decreased in both Canma111/Canma111 and Caybh3/Caybh3 cells. These effects on Nrg1 and Tup1 proteins were retained during serum-induced filamentation and appear to explain the hyperfilamentation phenotypes of the CaNMA111 and CaYBH3 deletion mutants. Treatment with the apoptosis-inducing dose of farnesol decreased the Nrg1 protein levels in the wild-type strain and more evidently in Canma111/Canma111 and Caybh3/Caybh3 mutant strains. Together, our results suggest that CaNma111 and CaYbh3 are key regulators of Nrg1 and Tup1 protein levels in C. albicans.

Functional Dichotomy for a Hyphal Repressor in Candida albicans.

Nrg1 is a repressor of hypha formation and hypha-associated gene expression in the fungal pathogen Candida albicans. It has been well studied in the genetic background of the type strain SC5314. Here, we tested Nrg1 function in four other diverse clinical isolates through an analysis of nrg1Δ/Δ mutants, with SC5314 included as a control. In three strains, nrg1Δ/Δ mutants unexpectedly produced aberrant hyphae under inducing conditions, as assayed by microscopic observation and endothelial cell damage. The nrg1Δ/Δ mutant of strain P57055 had the most severe defect. We examined gene expression features under hypha-inducing conditions by RNA-sequencing (RNA-Seq) for the SC5314 and P57055 backgrounds. The SC5314 nrg1Δ/Δ mutant expressed six hypha-associated genes at reduced levels compared with wild-type SC5314. The P57055 nrg1Δ/Δ mutant expressed 17 hypha-associated genes at reduced levels compared with wild-type P57055, including IRF1, RAS2, and ECE1. These findings indicate that Nrg1 has a positive role in hypha-associated gene expression and that this role is magnified in strain P57055. Remarkably, the same hypha-associated genes affected by the nrg1Δ/Δ mutation in strain P57055 were also naturally expressed at lower levels in wild-type P57055 than those in wild-type SC5314. Our results suggest that strain P57055 is defective in a pathway that acts in parallel with Nrg1 to upregulate the expression of several hypha-associated genes. IMPORTANCE Hypha formation is a central virulence trait of the fungal pathogen Candida albicans. Control of hypha formation has been studied in detail in the type strain but not in other diverse C. albicans clinical isolates. Here, we show that the hyphal repressor Nrg1 has an unexpected positive role in hypha formation and hypha-associated gene expression, as revealed by the sensitized P57055 strain background. Our findings indicate that reliance on a single type strain limits understanding of gene function and illustrate that strain diversity is a valuable resource for C. albicans molecular genetic analysis.

Large-scale identification of genes involved in septal pore plugging in multicellular fungi.

Multicellular filamentous fungi have septal pores that allow cytoplasmic exchange, and thus connectivity, between neighboring cells in the filament. Hyphal wounding and other stress conditions induce septal pore closure to minimize cytoplasmic loss. However, the composition of the septal pore and the mechanisms underlying its function are not well understood. Here, we set out to identify new septal components by determining the subcellular localization of 776 uncharacterized proteins in a multicellular ascomycete, Aspergillus oryzae. The set of 776 uncharacterized proteins was selected on the basis that their genes were present in the genomes of multicellular, septal pore-bearing ascomycetes (three Aspergillus species, in subdivision Pezizomycotina) and absent/divergent in the genomes of septal pore-lacking ascomycetes (yeasts). Upon determining their subcellular localization, 62 proteins were found to localize to the septum or septal pore. Deletion of the encoding genes revealed that 23 proteins are involved in regulating septal pore plugging upon hyphal wounding. Thus, this study determines the subcellular localization of many uncharacterized proteins in A. oryzae and, in particular, identifies a set of proteins involved in septal pore function.