scholarly journals The Ndr/LATS Kinase Cbk1 Regulates a Specific Subset of Ace2 Functions and Suppresses the Hypha-to-Yeast Transition in Candida albicans

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Rohan S. Wakade ◽  
Laura C. Ristow ◽  
Mark A. Stamnes ◽  
Anuj Kumar ◽  
Damian J. Krysan
Keyword(s):  
Candida Albicans ◽  
Regulatory Network ◽  
Transcriptional Analysis ◽  
Large Set ◽  
Phase Growth ◽  
Content Type ◽  
Hyphal Morphogenesis ◽  
Specific Subset ◽  
Wide Range ◽  
Yeast Phase

ABSTRACT The regulation of Ace2 and morphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans. The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast-phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hypha transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hypha-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hypha-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans. IMPORTANCE The regulation of Ace2 and morphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis: suppression of the hypha-to-yeast transition. Very few regulators of this transition have been described, and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.

scholarly journals The Ndr/LATS kinase Cbk1 regulates a specific subset of Ace2 functions and suppresses the hyphae-to-yeast transition in Candida albicans

2020 ◽  
Author(s):  
Rohan S. Wakade ◽  
Laura C. Ristow ◽  
Mark A. Stamnes ◽  
Anuj Kumar ◽  
Damian J. Krysan
Keyword(s):  
Candida Albicans ◽  
Regulatory Network ◽  
Transcriptional Analysis ◽  
Minor Role ◽  
Large Set ◽  
Phase Growth ◽  
Hyphal Morphogenesis ◽  
Specific Subset ◽  
Wide Range ◽  
Yeast Phase

AbstractThe Regulation of Ace2 and Morphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans. The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hyphae transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hyphae-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hyphae-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans.ImportanceRegulation of Ace2 and Morphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis; suppression of the hyphae-to-yeast transition. Very few regulators of this transition have been described and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.

scholarly journals Supplier-initiating risk management behaviour and supply-side resilience: the effects of interpersonal relationships and dependence asymmetry in buyer-supplier relationships

2020 ◽  
Vol 40 (7/8) ◽  
pp. 971-995
Author(s):  
Yiyi Fan ◽  
Mark Stevenson ◽  
Fang Li
Keyword(s):  
Risk Management ◽  
Interpersonal Relationships ◽  
Supply Side ◽  
Large Set ◽  
Supplier Relationships ◽  
Content Type ◽  
Management Behaviour ◽  
Wide Range ◽  
Dependence Asymmetry ◽  
The Relationship

PurposeThe aim of the study is to explore how two dimensions of interpersonal relationships (i.e. size and range of relationships) affect supplier-initiating risk management behaviours (SIRMB) and supply-side resilience. Further, the study aims to explore the moderating role of dependence asymmetry.Design/methodology/approachNine hypotheses are tested based on a moderated mediation analysis of survey data from 247 manufacturing firms in China. The data are validated using a subset of 57 attentive secondary respondents and archival data.FindingsSIRMB positively relates to supply-side resilience. Further, SIRMB mediates the positive relationship between range and supply-side resilience, and this relationship is stronger at lower levels of dependence asymmetry. Yet, although dependence asymmetry positively moderates the relationship between range and SIRMB, it negatively moderates the relationship between size and SIRMB. We did not, however, find evidence that size has a conditional indirect effect on supply-side resilience through SIRMB.Practical implicationsManagers in buying firms can incentivise SIRMB to enhance supply-side resilience by developing a diverse rather than a large set of interpersonal relationships with a supplier. This might include allocating particular employees with a wide range of contacts within a supplier to that relationship, while it may be necessary to adopt different networking strategies for different supplier relationships. Firms in a highly asymmetrical relationship may seek to raise supplier expectations about the necessity to initiate risk management behaviour or look to change the dynamic of the relationship by managing contracts for fairness.Originality/valueNew knowledge on SIRMB as a mediating variable underpinning the relationship between interpersonal relationships and supply-side resilience is provided; and empirical evidence on the opposing moderation effect of dependence asymmetry is presented.

scholarly journals Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

2012 ◽  
Vol 11 (10) ◽  
pp. 1289-1299 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Katherine S. Barker ◽  
Elizabeth L. Berkow ◽  
Geoffrey Toner ◽  
Sean G. Chadwick ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Amino Acid Substitutions ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increasedERG11expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

scholarly journals The Three Clades of the Telomere-AssociatedTLOGene Family of Candida albicans Have Different Splicing, Localization, and Expression Features

2012 ◽  
Vol 11 (10) ◽  
pp. 1268-1275 ◽  
Author(s):  
Matthew Z. Anderson ◽  
Joshua A. Baller ◽  
Keely Dulmage ◽  
Lauren Wigen ◽  
Judith Berman
Keyword(s):  
Candida Albicans ◽  
Gene Family ◽  
Transcription Regulation ◽  
Family Members ◽  
Gene Products ◽  
Content Type ◽  
Human Host ◽  
Wide Range ◽  
N Terminus

ABSTRACTCandida albicansgrows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associatedTLOgene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies inC. albicans. This correlates with increased virulence and clinical prevalence relative to those of otherCandidaclade species. The 14 expressedTLOgene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade β have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally,TLOα genes are generally expressed at much higher levels than areTLOγ genes. We propose that expansion of theTLOgene family and the predicted role of Tlo proteins in transcription regulation provideC. albicanswith the ability to adapt rapidly to the broad range of different environmental niches within the human host.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Disruption of the Transcriptional Regulator Cas5 Results in Enhanced Killing of Candida albicans by Fluconazole

2014 ◽  
Vol 58 (11) ◽  
pp. 6807-6818 ◽  
Author(s):  
Erin M. Vasicek ◽  
Elizabeth L. Berkow ◽  
Vincent M. Bruno ◽  
Aaron P. Mitchell ◽  
Nathan P. Wiederhold ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Resistance Mechanisms ◽  
Transcriptional Network ◽  
Transcriptional Analysis ◽  
Resistance Mutations ◽  
Content Type ◽  
Minimum Fungicidal Concentration ◽  
Fungistatic Activity ◽  
Yeast Extract Peptone Dextrose ◽  
Genes Encoding

ABSTRACTAzole antifungal agents such as fluconazole exhibit fungistatic activity againstCandida albicans. Strategies to enhance azole antifungal activity would be therapeutically appealing. In an effort to identify transcriptional pathways that influence the killing activity of fluconazole, we sought to identify transcription factors (TFs) involved in this process. From a collection ofC. albicansstrains disrupted for genes encoding TFs (O. R. Homann, J. Dea, S. M. Noble, and A. D. Johnson, PLoS Genet. 5:e1000783, 2009,http://dx.doi.org/10.1371/journal.pgen.1000783), four strains exhibited marked reductions in minimum fungicidal concentration (MFCs) in both RPMI and yeast extract-peptone-dextrose (YPD) media. One of these genes,UPC2, was previously characterized with regard to its role in azole susceptibility. Of mutants representing the three remaining TF genes of interest, one (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 μg/ml after 72 h in YPD medium. This mutant also showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid medium containing 10 μg/ml fluconazole, and exhibited increased susceptibility by time-kill analysis.CAS5disruption in highly azole-resistant clinical isolates exhibiting multiple resistance mechanisms did not alter susceptibility. However,CAS5disruption in strains with specific resistance mutations resulted in moderate reductions in MICs and MFCs. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role ofCAS5in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences the response ofC. albicansto fluconazole. Further delineation of this transcriptional network may identify targets for potential cotherapeutic strategies to enhance the activity of the azole class of antifungals.

scholarly journals Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans

2021 ◽  
Vol 17 (8) ◽  
pp. e1009861
Author(s):  
Kyunghun Min ◽  
Thomas F. Jannace ◽  
Haoyu Si ◽  
Krishna R. Veeramah ◽  
John D. Haley ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Regulatory Subunit ◽  
Chromosome 2 ◽  
Casein Kinase 1 ◽  
Hyphal Morphogenesis ◽  
Wide Range ◽  
Kinase A

Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase A (PKA) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

scholarly journals Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

2012 ◽  
Vol 78 (15) ◽  
pp. 5093-5103 ◽  
Author(s):  
Dandan Chen ◽  
Qi Zhang ◽  
Qinglin Zhang ◽  
Peilin Cen ◽  
Zhinan Xu ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Acyl Carrier Protein ◽  
Building Blocks ◽  
Transcriptional Analysis ◽  
Genetic Enhancement ◽  
Side Chain ◽  
Original Strain ◽  
Content Type ◽  
Streptomyces Tsukubaensis ◽  
Wide Range

ABSTRACTFK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strainStreptomyces tsukubaensisby the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact thatS. tsukubaensisis amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.

scholarly journals Sch9 Kinase Integrates Hypoxia and CO2Sensing To Suppress Hyphal Morphogenesis in Candida albicans

2011 ◽  
Vol 10 (4) ◽  
pp. 502-511 ◽  
Author(s):  
Catrin Stichternoth ◽  
Alida Fraund ◽  
Eleonora Setiadi ◽  
Luc Giasson ◽  
Anna Vecchiarelli ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Wild Type Strain ◽  
Cell Wall Proteins ◽  
Content Type ◽  
Chronological Aging ◽  
Hyphal Morphogenesis ◽  
Hypha Formation ◽  
Virulence Trait ◽  
Genes Encoding ◽  
A Minor

ABSTRACTThe yeast-hypha transition is an important virulence trait ofCandida albicans. We report that the AGC kinase Sch9 prevents hypha formation specifically under hypoxia at high CO2levels.sch9mutants showed no major defects in growth and stress resistance but a striking hyperfilamentous phenotype under hypoxia (<10% O2), although only in the presence of elevated CO2levels (>1%) and at temperatures of <37°C during surface growth. Thesch9hyperfilamentous phenotype was independent of Rim15 kinase and was recreated by inhibition of Tor1 kinase by rapamycin or caffeine in a wild-type strain, suggesting that Sch9 suppression requires Tor1. Caffeine inhibition also revealed that both protein kinase A isoforms, as well as transcription factors Czf1 and Ace2, are required to generate thesch9mutant phenotype. Transcriptomal analyses showed that Sch9 regulates most genes solely under hypoxia and in the presence of elevated CO2. In this environment, Sch9 downregulates genes encoding cell wall proteins and nutrient transporters, while under normoxia Sch9 and Tor1 coregulate a minor fraction of Sch9-regulated genes, e.g., by inducing glycolytic genes. Other than inSaccharomyces cerevisiae, bothsch9andrim15mutants showed decreased chronological aging under normoxia but not under hypoxia, indicating significant rewiring of the Tor1-Sch9-Rim15 pathway inC. albicans. The results stress the importance of environmental conditions on Sch9 function and establish a novel response circuitry to both hypoxia and CO2inC. albicans, which suppresses hypha formation but also allows efficient nutrient uptake, metabolism, and virulence.

scholarly journals The 5′ Untranslated Region of theEFG1Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis inCandida albicans

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Prashant R. Desai ◽  
Klaus Lengeler ◽  
Mario Kapitan ◽  
Silas Matthias Janßen ◽  
Paula Alepuz ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Untranslated Regions ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Content Type ◽  
Reading Frame ◽  
Human Fungal Pathogen ◽  
Hyphal Morphogenesis ◽  
Incandida Albicans

ABSTRACTExtensive 5′ untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis inCandida albicans. The major transcripts of theEFG1gene, which are responsible for cellular morphogenesis and metabolism, contain a 5′ UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5′ UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of theEFG1transcript. Polysomal analyses revealed that the 218-nt 5′ UTR sequence is required for efficient translation of the Efg1 protein. Replacement of theEFG1open reading frame (ORF) by the heterologous reporter geneCaCBGlucconfirmed the positive regulatory importance of the identified 5′ UTR sequence. In contrast to other reported transcripts containing extensive 5′ UTR sequences, these results indicate the positive translational function of the 5′ UTR sequence in theEFG1transcript, which is observed in the context of the nativeEFG1promoter. It is proposed that the 5′ UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of theEFG1transcript.IMPORTANCEMany of the virulence traits that makeCandida albicansan important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5′ untranslated regulatory sequence (5′ UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5′ UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5′ UTR sequences, it appears that the virulence ofC. albicansdepends on the combination of transcriptional and translational regulatory mechanisms.

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