Ontwikkeling van nieuwe moleculaire technieken en onderzoek naar de Gpr1 en calcineurine-afhankelijke regulatie van stressresistentie in Candida glabrata

SUMMARY Glucose is not only an energy source, but acts as a signaling molecule, as we have described in this study tightly regulating stress resistance. Characterization of the GPCR-cAMP-PKA signaling pathway has been well established in many fungal species, such as S. cerevisiae and C. albicans, however very little is known in C. glabrata. We show here a close correlation between environmental sugar content and GPCR Gpr1 in stress resistance in C. glabrata. By contrast, the calcineurin/Crz1 involved regulation of stress resistance and virulence in C. glabrata has been studied years ago. We have found that the calcineurin mediated regulation of thermotolerance, cell wall stress resistance and serum tolerance are highly dependent on the environmental glucose content and presence of the sugar receptor Gpr1. The subsequential stress tests of the GPA2 constitutively activated allele expressed strains and the pde2 mutant suggest that the glucose linked stress regulation is mediated via cAMP-PKA. On the other hand, we have also identified that upon heat stress calcineurin regulates trehalose mobilization via trehalase, which is a conventional downstream target of the cAMP-PKA pathway in many fungi. Trehalose can be widely found in fungal cells and acts as a storage carbohydrate and stress protectant. However, the research into the trehalose metabolism pathways in C. glabrata is almost non-existing up to date. We show that the neutral trehalase Nth1 (CAGL0M10439g) is involved in calcineurin linked regulation of thermotolerance, while similar to the baker’s yeast S. cerevisiae, the other homolog Nth2 (CAGL0C04323g) is a putative trehalase without clear enzymatic activity being detected. We also show that this trehalose mobilization regulation is Crz1 independent. Our results suggest a novel antagonistic regulation of the GPCR-cAMP-PKA and calcineurin signaling pathways to stresses. However, additional experiments need to be done to uncover the mechanism how calcineurin regulates trehalase activity. Phosphorylation regulation of both PKA and trehalase is crucial for their activities. It would be helpful to screen some components, such as Bcy1 and trehalase, of cAMP-PKA signaling pathway with the yeast two hybrid system, which will then be necessarily followed by an in vitro co-immunoprecipitation and other in vivo site specific phosphorylation assays for further verification. In C. glabrata, calcineurin regulates resistance towards the most frequently used antifungals such as echinocandins, azoles and polyene. Azoles and polyenes are known to be antagonistic in treatment. We have found that the gpr1, cnb1 and crz1 mutants response oppositely towards fluconazole and amB, which could be due to altered ergosterol levels in these strains. The explanation is that high ergosterol levels may antagonizes fluconazole, and synergistically provide an additional amount of target for amB, and vice versa. We also observed that the nth1 mutant exhibits a decreased susceptibility to amB by not increasing the sensitivity to fluconazole. Deletion of NTH1 in the cnb1∆ strain even slightly decreases the sensitivity to fluconazole, suggesting that increased trehalose may confer a robust membrane and enhance the cell fitness in general. However, the MIC of the two antifungals is not altered in all of the tested mutants, except the crz1 strain, which shows an increased MIC of fluconazole. These results indicate that instead of the cell resistance, the altered antifungal sensitivity we have observed in the spot assays might be due to the cell tolerance. It will be interesting in the future to screen the deletion collection for their susceptibility to the two types of antifungals, as such to systemically figure out the genes possibly involved in ergosterol metabolism and understand their role in regulation of drug susceptibility better. Genetic modification of C. glabrata is still a challenge due to its very low HR and high NHEJ efficiencies. In this study we have also developed new genetic modification tools for this organism. We have deleted the DNA ligase IV (Lig4) and greatly increased the rate of obtaining successful transformants. We subsequently showed that the lig4 mutants behave exactly as the wild type strain for all conditions tested, even when challenged with DNA damage agents. On the other hand, the previously commonly used yku80 mutants exhibit altered sensitivity to caspofungin, fluconazole, SDS and calcofluor white in comparison to the wild type strain. Beside this, the KU genes are also known to be crucial in maintaining the subtelomeric region length. All these indicates that inhibition of the KU genes will have risk to interfere the research work in C. glabrata . The lig4 mutant is thus suggested to be used in the future and is now available for the candida research community. CRISPR-Cas is a revolutionary technique that has been developed fast very recently. It without doubt dramatically increased the efficiency in genetic manipulation from mammals to plants. Its feature of genome wide applicability and marker free editing make it favorable to the scientists these years. We have applied the CRISPR-Cas9 system for the first time in C. glabrata by employing two plasmids to express the Cas9 protein and guide RNA. Due to the strong NHEJ efficiency, we show the risk of mutagenesis by applying this technique in the WT strain, while that can be avoided by using our lig4 strain. The highest HR frequency reached 94%, demonstrating the high efficiency of the Cas9 protein in C. glabrata. Moreover, we have showed that the HR efficiency is increased by at least 10 folds when using the CRISPR-Cas9 system compared to the traditional method. The plasmids of the CRISPR system for C. glabrata are now available for the candida research community.TABLE OF CONTENTS I LIST OF ABBREVIATIONS VI LIST OF GENES VIII NOMENCLATURE XI SAMENVATTING XII SUMMARY XIV INTRODUCTION 1 1.1 Candida and candidiasis 2 1.1.1 Candida 2 1.1.2 Candida albicans 2 1.1.3 Candida glabrata 3 1.1.4 Candidiasis 4 1.2 Identification and diagnosis 6 1.3 Antifungals 8 1.3.1 Azoles 9 1.3.2 Polyenes 10 1.3.3 Echinocandins 10 1.3.4 Antifungal resistance 10 1.4 Host immune response 12 1.5 Virulence factors 13 1.5.1 Filamentation 13 1.5.2 Adherence and biofilm 14 1.5.3 Evasion of the host immune response 18 1.6 GPCR-cAMP-PKA pathway 19 1.6.1 Gpr1-Gpa2 21 1.6.2 cAMP 21 1.6.3 PKA 21 1.6.4 Trehalose and glycogen accumulation 24 1.6.5 Trehalase 26 1.6.6 Msn2/4 27 1.7 Calcineurin-Crz1 pathway 28 1.7.1 Calcineurin in mammalian cells 29 1.7.2 Calcineurin in fungi 30 1.7.3 Crz1 31 1.7.4 Rcn1-Rcn2 33 1.7.5 Calcineurin-Crz1 involved virulence and drug resistance 34 1.8 Genetic manipulation in C. glabrata 34 1.9 Non-Homologous-End-Joining 35 1.10 CRISPR-CAS 37 AIMS 39 MATERIALS AND METHODS 43 2.1 Yeast strains, primers, and media 44 2.2 Deletion strains and plasmids construction 53 2.2.1 Deletion of GPR1 53 2.2.2 Construction of the GPA2 constitutively active allele GPA2Q299L 53 2.2.3 Construction of GFP tagged CRZ1 expression cassette 53 2.2.4 Deletion of NTH1 and NTH2 54 2.2.5 Construction of the NTH1 expression plasmid 54 2.2.6 Construction of the LIG4 deletion strain and the KU80 deletion strain 54 2.2.7 Deletion of ADE2, ARG8 and CNA1 55 2.2.8 Generation of the LIG4 re-integration construct 55 2.2.9 Construction of Cas9 and guide RNA plasmids and donor DNA fragments 56 2.3 Southern blot 57 2.4 Real Time PCR analysis 58 2.5 Ergosterol measurement 59 2.6 Trehalose quantification 59 2.7 Trehalase activity assay 59 2.8 In vitro PKA activity assay 60 2.9 Growth and stress experiments (lig4 strain linked assays) 60 2.10 Minimal inhibitory concentrations (MIC) using the broth dilution assay and E-test strips 61 2.11 Transformation and colony screening (for CRISPR work) 61 2.12 Western Blot 62 RESULTS AND DISCUSSION 63 3 The GPCR signaling pathway antagonizes calcineurin/Crz1 dependent regulation of thermotolerance and cell wall stress resistance in C. glabrata 64 3.1 Introduction 64 3.2 Results 65 3.2.1 The glucose concentration plays an important role in the level of thermotolerance of the cnb1∆ strain in C. glabrata. 65 3.2.2 Glucose linked heat sensitivity of cnb1 and crz1 mutants is mediated by Gpr1. 68 3.2.3 No antagonistic impact of Gpr1 on the calcineurin/Crz1 linked regulation of osmotic stress, membrane stress, ER stress and acidic/alkaline pH stress was observed. 69 3.2.4 Gpr1 antagonizes the calcineurin/Crz1 pathway not only in the regulation of thermotolerance, but also for cell wall stress resistance. 70 3.2.5 Constitutive activation of Gpa2 increases the susceptibility of the cnb1∆ gpr1∆ strain to both thermotolerance and cell wall stress. 72 3.2.6 Deletion of GPR1 restores the optimal growth of the cnb1∆ strain in serum, while constitutive activation of Gpa2 increases serum sensitivity of the strain. 74 3.2.7 The pde2 mutant shows an increased susceptibility to both thermal and cell wall stress. 76 3.2.8 Trehalose level is lower in the cnb1 mutant than in its background strain after being applied with heat stress for 4 hours. 77 3.2.9 The trehalase activity is higher in the cnb1 mutant than in the wild type and crz1 strains under heat stress 78 3.2.10 Real-Time PCR shows that the expression of the neutral trehalase is increased in the cnb1 mutant compared to the wild type strain. 80 3.2.11 Deletion of NTH1 in the cnb1∆ strain restores thermotolerance. 82 3.2.12 In vitro PKA activity assay shows that the PKA activity of the cnb1 mutant was not higher than that of the wild type strain. 83 3.2.13 The cnb1∆ gpr1∆ strain accumulates more trehalose than the cnb1∆ strain. 84 3.3 Discussion 85 4 Opposite sensitivities of gpr1, cnb1 and crz1 mutants to the two ergosterol linked antifungals, fluconazole and amphotericinB 88 4.1 Introduction 88 4.2 Results 89 4.2.1 Gpr1, calcineurin and Crz1 play opposite roles in the response to amphotericin B and fluconazole. 89 4.2.2 Deletion of NTH1 decreases C. glabrata amB susceptibility. 91 4.2.3 The MIC values of gpr1, cnb1 and nth1 mutants to the two antifungals are not different in comparison to the WT strain. 92 4.3 Discussion 92 5 Deletion of the DNA ligase IV gene in Candida glabrata significantly increases gene-targeting efficiency 94 5.1 Introduction 94 5.2 Results and discussion 95 5.2.1 The C. glabrata lig4 mutants behave exactly as the wild type for all conditions we have tested. 95 5.2.2 The C. glabrata Ku80, not the Lig4, is involved in cell membrane stress and cell wall stress regulations. 100 5.2.3 The gene-targeting efficiency is strongly increased in the lig4 mutant. 103 6 Genome engineering in Candida glabrata using the CRISPR-Cas system 110 6.1 Introduction 110 6.2 Results 111 6.2.1 Construction of the plasmids for use of the CRISPR-Cas9 system in C. glabrata. 111 6.2.2 Using the CRISPR-Cas9 system in the C. glabrata lig4 mutant strongly increases the accuracy in genome engineering compared to the ATCC2001 wild type strain. 113 6.2.3 The correct-transformant rate is highly dependent on the locus of insertion. 115 6.3 Discussion 118 GENERAL DISCUSSION 121 7 General Discussion 122 7.1 Gpr1 antagonizes calcineruin in regulation of stress resistance 122 7.2 Calcineurin regulates trehalase activity and trehalose level upon heat stress. 122 7.3 Sensitivity of gpr1, cnb1 and crz1 mutants is opposite to amB and fluconazole. 125 7.4 Development of molecular tools in C. glabrata. 125 7.5 Deletion of the LIG4 increases the gene targeting efficiency. 125 7.6 Application of CRISPR-Cas system in C. glabrata. 126 REFERENCE 127nrpages: 155status: publishe