scholarly journals The  Candida albicans  Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

2021 ◽  
Author(s):  
Jeffrey Hollomon ◽  
Zhongle Liu ◽  
Scott Rusin ◽  
Nicole P. Jenkins ◽  
Allia K. Smith ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Kinase Activity ◽  
Immune Cell ◽  
Hyphal Growth ◽  
Regulation Of Transcription ◽  
Multiple Phenotypes ◽  
Amino Acid Utilization ◽  
Functional Variant ◽  
Kinase Substrates

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In  Candida albicans , the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog  3-MB-PP1 . Consistent with  ssn3  null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential.  Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate  C. albicans  morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent activity, suggesting that Ssn3 kinase activity negatively regulates Flo8. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids.  Here, we show that  FLO8  and  STP2 , a transcription factor involved in amino acid utilization, are required for  ssn3 ?/? phenotype, but that loss of the Ssn3 phosphosites identified in Flo8 was not sufficient to phenocopy the  ssn3 ?/? mutant. These data highlight the spectrum of processes affected by the modulation of Ssn3 activity and underscore the importance of considering Ssn3 function in the control of transcription factor activities.

scholarly journals The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

PLoS Genetics ◽  
2022 ◽  
Vol 18 (1) ◽  
pp. e1009622
Author(s):  
Jeffrey M. Hollomon ◽  
Zhongle Liu ◽  
Scott F. Rusin ◽  
Nicole P. Jenkins ◽  
Allia K. Smith ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Kinase Activity ◽  
Immune Cell ◽  
Morphological Changes ◽  
Hyphal Growth ◽  
Regulation Of Transcription ◽  
Multiple Phenotypes ◽  
Amino Acid Utilization ◽  
Hypha Formation

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

scholarly journals Role of Transcription Factor CaNdt80p in Cell Separation, Hyphal Growth, and Virulence in Candida albicans

2010 ◽  
Vol 9 (4) ◽  
pp. 634-644 ◽  
Author(s):  
Adnane Sellam ◽  
Christopher Askew ◽  
Elias Epp ◽  
Faiza Tebbji ◽  
Alaka Mullick ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Cell Separation ◽  
Hyphal Growth ◽  
Functional Domain ◽  
Transcription Factor Family ◽  
Content Type ◽  
Genes Encoding

ABSTRACT The NDT80/PhoG transcription factor family includes ScNdt80p, a key modulator of the progression of meiotic division in Saccharomyces cerevisiae. In Candida albicans, a member of this family, CaNdt80p, modulates azole sensitivity by controlling the expression of ergosterol biosynthesis genes. We previously demonstrated that CaNdt80p promoter targets, in addition to ERG genes, were significantly enriched in genes related to hyphal growth. Here, we report that CaNdt80p is indeed required for hyphal growth in response to different filament-inducing cues and for the proper expression of genes characterizing the filamentous transcriptional program. These include noteworthy genes encoding cell wall components, such as HWP1, ECE1, RBT4, and ALS3. We also show that CaNdt80p is essential for the completion of cell separation through the direct transcriptional regulation of genes encoding the chitinase Cht3p and the cell wall glucosidase Sun41p. Consistent with their hyphal defect, ndt80 mutants are avirulent in a mouse model of systemic candidiasis. Interestingly, based on functional-domain organization, CaNdt80p seems to be a unique regulator characterizing fungi from the CTG clade within the subphylum Saccharomycotina. Therefore, this study revealed a new role of the novel member of the fungal NDT80 transcription factor family as a regulator of cell separation, hyphal growth, and virulence.

scholarly journals The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

Genetics ◽  
2021 ◽  
Author(s):  
Raha Parvizi Omran ◽  
Bernardo Ramírez-Zavala ◽  
Walters Aji Tebung ◽  
Shuangyan Yao ◽  
Jinrong Feng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Systemic Infection ◽  
Hyphal Growth ◽  
Growth Conditions ◽  
Hyphal Development ◽  
Zinc Cluster ◽  
Function Blocks ◽  
External Signals

Abstract Zinc cluster transcription factors are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster transcription factor regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under non-hyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing transcription factors Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

Generation and functional in vivo characterization of a lipid kinase defective phosphatidylinositol 3-kinase Vps34p of Candida albicans

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Juliane Günther ◽  
Monika Nguyen ◽  
Albert Härtl ◽  
Waldemar Künkel ◽  
Peter F. Zipfel ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Transport ◽  
Kinase Activity ◽  
Hyphal Growth ◽  
Lipid Binding ◽  
Homologous Region ◽  
Null Mutant ◽  
Lipid Kinase ◽  
In Vivo Characterization

The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans has lipid kinase and autophosphorylation activity and is involved in virulence and vesicular protein transport. In order to characterize the roles of lipid kinase activity, a chimeric Vps34 protein was created which lacks lipid kinase but retains autophosphorylation activity. To this end, six amino acids within the putative lipid-binding site of Vps34p were replaced by the homologous region of the PI 3-kinase-like C. albicans Tor protein. The resulting chimeric Vps34T protein was recombinantly expressed in Escherichia coli and shown to lack lipid kinase activity. The corresponding chimeric VPS34TOR gene was inserted into the genome of C. albicans, and this lipid-kinase-defective strain had a distinctive phenotype compared to those of the wild-type strain SC5314 and the vps34 null mutant. The lipid-kinase-defective strain was non-virulent, and showed altered hyphal growth, reduced adherence, as well as defective vacuole morphology and endosomal vesicle transport. These results demonstrate an important role for the lipid kinase activity of Vps34p in virulence and vesicular protein transport. On the other hand, the lipid-kinase-defective strain and the vps34 null mutant differ in their temperature- and osmotic-stress response. This indicates a possible role for activities different from the lipid kinase function of Vps34p.

scholarly journals Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Sophia Ruben ◽  
Enrico Garbe ◽  
Selene Mogavero ◽  
Daniela Albrecht-Eckardt ◽  
Daniela Hellwig ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Transcriptional Control ◽  
Cell Damage ◽  
Iron Acquisition ◽  
Hyphal Growth ◽  
Fungal Virulence ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Full Expression

ABSTRACT The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans. However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1. In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1. In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies. IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

scholarly journals The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

2020 ◽  
Author(s):  
Raha Parvizi Omran ◽  
Chris Law ◽  
Vanessa Dumeaux ◽  
Joachim Morschhäuser ◽  
Malcolm Whiteway
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Transcriptional Profiling ◽  
Hyphal Growth ◽  
Growth Conditions ◽  
Hyphal Development ◽  
Zinc Cluster ◽  
Genes Encoding

AbstractZinc cluster transcription factors are essential fungal specific regulators of gene expression. In the dimorphic pathogen Candida albicans, they control processes ranging from metabolism and stress adaptation to mating, virulence, and antifungal resistance. Here, we have identified the gene CaORF19.1604 as encoding a zinc cluster transcription factor that acts as a regulator of filament development. Hyperactivation of CaORF19.1604, which we have named RHA1 for Regulator of Hyphal Activity, leads to a wrinkled colony morphology under non-hyphal growth conditions, to pseudohyphal growth and filament formation, to invasiveness and enhanced biofilm formation.  Cells with activated Rha1 are sensitive to cell wall modifying agents such as Congo red and the echinocandin drug caspofungin but show normal sensitivity to fluconazole. RNA-sequencing-based transcriptional profiling of the activated Rha1 strain reveals the up-regulation of genes for core filamentation and cell-wall-adhesion-related proteins such as Als1, Als3, Ece1, and Hwp1. Upregulation is also seen for the genes for the hyphal-inducing transcription factors Brg1 and Ume6 and genes encoding several enzymes involved in arginine metabolism, while downregulation is seen for the hyphal repressor Nrg1. The deletion of BRG1 blocks the filamentation caused by activated Rha1, while null mutants of UME6 result in a partial block. Deletion of RHA1 can partially reduce healthy hyphal development triggered by environmental conditions such as Spider medium or serum at 37°C.In contrast to the limited effect of either single mutant, the double rha1 ume6 deletion strain is totally defective in both serum and Spider medium stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization in even brg1 ume6 double mutants. Our results thus suggest that in response to external signals, Rha1 functions to facilitate the switch from an Nrg1 controlled yeast state to a Brg1/Ume6 regulated hyphal state.Author SummaryCandida albicans is the predominant human fungal pathogen, generating a mortality rate of 40% in systemically infected patients. The ability of Candida albicans to change its morphology is a determinant of its tissue penetration and invasion in response to variant host-related stimuli. The regulatory mechanism for filamentation includes a complex network of transcription factors that play roles in regulating hyphae associated genes. We identify here a new regulator of filamentation from the zinc cluster transcription factor family. We present evidence suggesting that this transcription factor assists the Nrg1/Brg1 switch regulating hyphal development.

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