Identification of an Exceptionally Long Intron in the HAC1 Gene of Candida parapsilosis.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is well conserved in eukaryotes from metazoa to yeast. The transcription factor HAC1 is a major regulator of the UPR in many eukaryotes. Deleting HAC1 in the yeast Candida parapsilosis rendered cells more sensitive to DTT, a known inducer of the UPR. The deletion strain was also sensitive to Congo red, calcofluor white, and the antifungal drug ketoconazole, indicating that HAC1 has a role in cell wall maintenance. Transcriptomic analysis revealed that treatment of the wild type with DTT resulted in the increased expression of 368 genes. Comparison with mutant cells treated with DTT reveals that expression of 137 of these genes requires HAC1 Enriched GO term analysis includes response to ER stress, cell wall biogenesis and glycosylation. Orthologs of many of these are associated with UPR in Saccharomyces cerevisiae and Candida albicans Unconventional splicing of an intron from HAC1 mRNA is required to produce a functional transcription factor. The spliced intron varies in length from 19 bases in C. albicans to 379 bases in Candida glabrata, but has not been previously identified in Candida parapsilosis and related species. We used RNA-seq data and in silico analysis to identify the HAC1 intron in 12 species in the CTG-Ser1 clade. We show that the intron has undergone major contractions and expansions in this clade, reaching up to 848 bases. Exposure to DTT induced splicing of the long intron in C. parapsilosisHAC1, inducing the UPR.IMPORTANCE The unfolded protein response (UPR) responds to the build-up of misfolded proteins in the endoplasmic reticulum. The UPR has wide-ranging functions from fungal pathogenesis to applications in biotechnology. The UPR is regulated through the splicing of an unconventional intron in the HAC1 gene. This intron has been described in many fungal species and is of variable length. Until now it was believed that some members of the CTG-Ser1 clade such as C. parapsilosis did not contain an intron in HAC1, suggesting that the UPR was regulated in a different manner. Here we demonstrate that HAC1 plays an important role in regulating the UPR in C. parapsilosis We also identified an unusually long intron (626 bp) in C. parapsilosisHAC1 Further analysis showed that HAC1 orthologs in several species in the CTG-Ser1 clade contain long introns.

TPP riboswitch-dependent regulation of an ancient thiamin transporter in Candida.

Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species. Using an RFP/riboswitch expression system, we show that the functional elements of the riboswitch are contained within the native intron of DUR31 from Candida parapsilosis, and that the riboswitch regulates splicing in a thiamin-dependent manner when RFP is constitutively expressed. The DUR31 gene has been lost from Saccharomyces, and may have been displaced by an alternative thiamin transporter. TPP riboswitches are also present in other putative transporters in yeasts and filamentous fungi. However, they are rare in thiamin biosynthesis genes THI4 and THI5 in the Saccharomycotina, and have been lost from all genes in the sequenced species in the family Saccharomycetaceae, including S. cerevisiae.

A Plastic Vegetative Growth Threshold Governs Reproductive Capacity in Aspergillus nidulans.

Ontogenetic phases separating growth from reproduction are a common feature of cellular life. Long recognized for flowering plants and animals, early literature suggests this life-history component may also be prevalent among multicellular fungi. We establish the basis of developmental competence-the capacity to respond to induction of asexual development-in the filamentous saprotroph Aspergillus nidulans, describing environmental influences, including genotype-by-environment interactions among precocious mutants, gene expression associated with wild type and precocious competence acquisition, and the genetics of competence timing. Environmental effects are consistent with a threshold driven by metabolic rate and organism density, with pH playing a particularly strong role in determining competence timing. Gene expression diverges significantly over the competence window, despite a lack of overt morphological change, with differentiation in key metabolic, signaling, and cell trafficking processes. We identify five genes for which mutant alleles advance competence timing, including the conserved GTPase RasB (AN5832) and ambient pH sensor PalH (AN6886). In all cases examined, inheritance of competence timing is complex and non-Mendelian, with F1 progeny showing highly variable transgressive timing and dominant parental effects with a weak contribution from progeny genotype. Competence provides a new model for nutrient-limited life-cycle phases, and their elaboration from unicellular origins. Further work is required to establish the hormonal and bioenergetic basis of the trait across fungi, and underlying mechanisms of variable inheritance.

Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans.

Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.

Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

The APSES transcription factor Efg1 is a global regulator that controls morphogenesis and biofilm formation in Candida parapsilosis.

Efg1 (a member of the APSES family) is an important regulator of hyphal growth and of the white-to-opaque transition in Candida albicans and very closely related species. We show that in Candida parapsilosis Efg1 is a major regulator of a different morphological switch at the colony level, from a concentric to smooth morphology. The rate of switching is at least 20-fold increased in an efg1 knockout relative to wild type. Efg1 deletion strains also have reduced biofilm formation, attenuated virulence in an insect model, and increased sensitivity to SDS and caspofungin. Biofilm reduction is more dramatic in in vitro than in in vivo models. An Efg1 paralogue (Efh1) is restricted to Candida species, and does not regulate concentric-smooth phenotype switching, biofilm formation or stress response. We used ChIP-seq to identify the Efg1 regulon. A total of 931 promoter regions bound by Efg1 are highly enriched for transcription factors and regulatory proteins. Efg1 also binds to its own promoter, and negatively regulates its expression. Efg1 targets are enriched in binding sites for 93 additional transcription factors, including Ndt80. Our analysis suggests that Efg1 has an ancient role as regulator of development in fungi, and is central to several regulatory networks.

Evolution of mating within the Candida parapsilosis species group.

Candida orthopsilosis and Candida metapsilosis are closely related to Candida parapsilosis, a major cause of infection in premature neonates. Mating has not been observed in these species. We show that ∼190 isolates of C. parapsilosis contain only an MTLa idiomorph at the mating-type-like locus. Here, we describe the isolation and characterization of the MTL loci from C. orthopsilosis and C. metapsilosis. Among 16 C. orthopsilosis isolates, 9 were homozygous for MTLa, 5 were homozygous for MTLα, and 2 were MTLa/α heterozygotes. The C. orthopsilosis isolates belonged to two divergent groups, as characterized by restriction patterns at MTL, which probably represent subspecies. We sequenced both idiomorphs from each group and showed that they are 95% identical and that the regulatory genes are intact. In contrast, 18 isolates of C. metapsilosis contain only MTLα idiomorphs. Our results suggest that the role of MTL in determining cell type is being eroded in the C. parapsilosis species complex. The population structure of C. orthopsilosis indicates that mating may occur. However, expression of genes in the mating signal transduction pathway does not respond to exposure to alpha factor. C. parapsilosis is also nonresponsive, even when the GTPase-activating protein gene SST2 is deleted. In addition, splicing of introns in MTLa1 and MTLa2 is defective in C. orthopsilosis. Mating is not detected. The alpha factor peptide, which is the same sequence in C. parapsilosis, C. orthopsilosis, and C. metapsilosis, can induce a mating response in Candida albicans. It is therefore likely either that mating of C. orthopsilosis takes place under certain unidentified conditions or that the mating pathway has been adapted for other functions, such as cross-species communication.

Mutation of tagO reveals an essential role for wall teichoic acids in Staphylococcus epidermidis biofilm development.

The icaADBC-encoded polysaccharide intercellular adhesin (PIA) and wall teichoic acids (WTA) are structural components of Staphylococcus epidermidis biofilms. Deletion of tagO, which encodes the first enzymic step in WTA biosynthesis, had pleiotropic effects, including enhanced intercellular aggregation and autolytic activity, and impaired biofilm production. The biofilm-negative phenotype of the tagO mutant, named TAGO1, was associated with increased cell surface hydrophobicity, lower rates of primary attachment to polystyrene, and reduced icaADBC operon and PIA expression. Mild acid stress induced by growth in BHI glucose media reduced rates of stationary phase autolysis and enhanced aggregation by TAGO1, leading to formation of a pellicle, which unlike a biofilm was only loosely attached to the polystyrene surface. TAGO1 pellicles were dispersed by proteinase K and DNase I but not sodium metaperiodate, implicating protein and extracellular DNA (eDNA) and not PIA in this phenotype. Substantially increased levels of eDNA were recovered from TAGO1 culture supernatants compared with the wild-type. These data indicate that WTA are essential for the primary attachment and accumulation phases of the S. epidermidis biofilm phenotype. Furthermore, in the absence of WTA, proteins and eDNA can promote cell aggregation and pellicle formation, which also appear to limit interactions with artificial surfaces.

Anti-biofilm activity of sub-inhibitory povidone-iodine concentrations against Staphylococcus epidermidis and Staphylococcus aureus.

Biomaterial-related infections continue to hamper the success of reconstructive and arthroplasty procedures in orthopaedic surgery. Staphylococci are the most common etiologic agents, with biofilm formation representing a major virulence factor. Biofilms increase bacterial resistance to antimicrobial agents and host immune responses. In staphylococci, production of polysaccharide intercellular adhesin (PIA) by the enzyme products of the icaADBC operon is the best understood mechanism of biofilm development, making the ica genes a potential target for biofilm inhibitors. In this study we report that the antibacterial agent povidone-iodine (PI) also has anti-biofilm activity against Staphylococcus epidermidis and Staphylococcus aureus at sub-inhibitory concentrations (p < 0.001). Inhibition of biofilm by PI correlated with decreased transcription of the icaADBC operon, which in turn correlated with activation of the icaR transcriptional repressor in Staphylococcus epidermidis. These data reveal an additional therapeutic benefit of PI and suggest that studies to evaluate suitability of PI as biomaterial coating agent to reduce device-related infections are merited.

A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP.

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.