Apoptosis-inducing antifungal peptides and proteins

Abstract Despite the availability of various classes of antimycotics, the treatment of patients with systemic fungal infections is challenging. Therefore the development of new antifungals is urgently required. Promising new antifungal candidates are antimicrobial peptides. In the present review, we provide an overview of antifungal peptides isolated from plants, insects, amphibians and mammals that induce apoptosis. Their antifungal spectrum, mode of action and toxicity are discussed in more detail

Structure-activity relationship study of the plant-derived decapeptide OSIP108 inhibiting Candida albicans biofilm formation

We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity

Stimulation of superoxide production increases fungicidal action of miconazole against Candida albicans biofilms

We performed a whole-transcriptome analysis of miconazole-treated Candida albicans biofilms, using RNA-sequencing. Our aim was to identify molecular pathways employed by biofilm cells of this pathogen to resist action of the commonly used antifungal miconazole. As expected, genes involved in sterol biosynthesis and genes encoding drug efflux pumps were highly induced in biofilm cells upon miconazole treatment. Other processes were affected as well, including the electron transport chain (ETC), of which eight components were transcriptionally downregulated. Within a diverse set of 17 inhibitors/inducers of the transcriptionally affected pathways, the ETC inhibitors acted most synergistically with miconazole against C. albicans biofilm cells. Synergy was not observed for planktonically growing C. albicans cultures or when biofilms were treated in oxygen-deprived conditions, pointing to a biofilm-specific oxygen-dependent tolerance mechanism. In line, a correlation between miconazole's fungicidal action against C. albicans biofilm cells and the levels of superoxide radicals was observed, and confirmed both genetically and pharmacologically using a triple superoxide dismutase mutant and a superoxide dismutase inhibitor N-N'-diethyldithiocarbamate, respectively. Consequently, ETC inhibitors that result in mitochondrial dysfunction and affect production of reactive oxygen species can increase miconazole's fungicidal activity against C. albicans biofilm cells

Potentiation of Antibiofilm Activity of Amphotericin B by Superoxide Dismutase Inhibition

This study demonstrates a role for superoxide dismutases (Sods) in governing tolerance of Candida albicans biofilms to amphotericin B (AmB). Coincubation of C. albicans biofilms with AmB and the Sod inhibitors N,N′-diethyldithiocarbamate (DDC) or ammonium tetrathiomolybdate (ATM) resulted in reduced viable biofilm cells and increased intracellular reactive oxygen species levels as compared to incubation of biofilm cells with AmB, DDC, or ATM alone. Hence, Sod inhibitors can be used to potentiate the activity of AmB against C. albicans biofilms

The plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability

We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Characterization of the cAMP-PKA pathway in Candida glabrata.

Nutrients are not only building blocks and energy providers, but can also function as signaling molecules that transmit important environmental information to cells. This informs, for example, yeast cells if they can grow or if they have to adapt to nutrient starvation. The cAMP-PKA pathway mediates the glucose- response in Saccharomyces cerevisiae and has been well established in this model organism. This pathway senses glucose and sucrose through a G protein-coupled receptor and regulates various downstream effects, including stress response, growth, trehalose levels and pseudohyphal growth.Several of these downstream targets of the cAMP-PKA pathway, including growth and stress resistance, are also important for pathogens. Candida glabrata, a human pathogen, contains homologs of almost all components of the cAMP-PKA pathway, as described in S. cerevisiae. Here, we characterized the cAMP-PKA pathway of C. glabrata and its role in glucose-responsive characteristics, including trehalose mobilization and heat stress resistance.We provide evidence that C. glabrata cells are able to respond to glucose and sucrose, but also to fructose, by inducing cAMP signaling. This response requires the G protein-coupled receptor Gpr1 and the Ga subunit Gpa2, suggesting a role for the GPCR complex as a sugar sensing module. Complementation experiments suggest that the three sugars do not activate the receptor similarly, as a cAMP signal in a Scgpr1D strain expressing CgGPR1 could only be induced in response to glucose, but not to sucrose or fructose. Deletion of 2 other components of the cAMP-PKA pathway, PDE1 or TPK2, results in an increased and longer-lasting cAMP signal, indicating that the corresponding proteins control the cAMP signal. Accumulation of trehalose, which starts when glucose is depleted, is reduced when PDE2 or the KRH genes are absent. On the other hand, deletion of GPR1 or GPA2 results in a delay in trehalose degradation, whereas deletion of one or both of the KRH genes results in a faster trehalose mobilization. In addition, heat stress experiments demonstrated that gpr1D and gpa2D strains have an opposite phenotype as the krh1D krh2D strain. These results suggest that, like in the model organism S. cerevisiae, the GPCR complex acts positively on the downstream cAMP-PKA pathway, while the Krh and Pde proteins seem to act as negative regulators of the pathway.Adhesion to human cells or inert surfaces is critical for the virulence of a fungal pathogen. In this study we demonstrated that addition of a cAMP-inducing sugar reduces adherence to polystyrene in a Gpr1-dependent manner. However, none of the tested deletion strains displayed a defect in survival in the fly model of C. glabrata candidiasis.Furthermore, addition of a cAMP-inducing sugar induces a fast and transient burst of expression of the adhesin-encoding genes EPA1 and EPA3, in a concentration-dependent manner. This is the first time that EPA3 expression is experimentally induced in response to environmental conditions, as this gene is normally strongly silenced. Deletion of GPR1 decreases the induction of expression of EPA1 and EPA3, which seems counterintuitive as deletion of GPR1 also results in increased adherence. Whether the increased expression of EPA1 and EPA3 can be linked to the reduced adhesion of C. glabrata to plastic, in the presence of cAMP-inducing sugars, remains to be established. Finally, we also demonstrated that, when cell culture medium (RPMI) is used, the Krh and Pde proteins are involved in adhesion and biofilm development on polystyrene.Together these data indicate that the C. glabrata proteins, homologous to those of the S. cerevisiae cAMP-PKA pathway have, in general, retained their function. In addition, the sugar-inducible expression of EPA1 and EPA3 was identified as a new specific readout to measure the activity of the Gpr1-Gpa2 signaling pathway in C. glabrata.TABLE OF CONTENTS I LIST OF ABBREVIATIONS III LIST OF GENES V NOMENCLATURE VIII INTRODUCTION AND AIMS 1 1. LITERATURE OVERVIEW 3 1.1. CANDIDA GLABRATA 5 1.1.1. INTRODUCTION 5 1.1.2. OVERVIEW OF THE C. GLABRATA GENOME 5 1.1.3. TAXONOMY OF C. GLABRATA 6 1.1.4. MATING 6 1.1.5. EPIDEMIOLOGY, HOST DEFENSE MECHANISMS AND SUPERFICIAL AND SYSTEMIC INFECTIONS 6 1.1.5.1. INTRODUCTION 6 1.1.5.2. THE HOST IMMUNE SYSTEM 7 1.1.5.3. CANDIDA INFECTIONS 8 1.1.5.3.1. SUPERFICIAL INFECTIONS 8 1.1.5.3.2. SYSTEMIC INFECTIONS 9 1.1.6. IDENTIFICATION OF C. GLABRATA 10 1.1.7. VIRULENCE FACTORS IN C. ALBICANS AND C. GLABRATA 11 1.1.7.1. ADHESINS 11 1.1.7.2. FILAMENTATION 15 1.1.7.3. INVASION THROUGH THE EPITHELIAL BARRIER 17 1.1.7.4. PHENOTYPIC SWITCHING 18 1.1.7.5. BIOFILM 20 1.1.8. ANTIFUNGAL DRUGS AND RESISTANCE 22 1.1.8.1. CLASSIFICATION OF ANTIMYCOTICS 22 1.1.8.2. RESISTANCE TO ANTIFUNGAL DRUGS 23 1.1.8.2.1. RESISTANCE TO DRUGS IN CLINICAL ISOLATES 23 1.1.8.2.2. ANTIFUNGAL RESISTANCE IN BIOFILMS 23 1.2. THE CAMP-PKA PATHWAY IN FUNGI 24 1.2.1. INTRODUCTION 24 1.2.2. GLUCOSE-RESPONSE SIGNALING CASCADES IN S. CEREVISIAE 25 1.2.2.1. THE GLUCOSE REPRESSION PATHWAY 25 1.2.2.2. THE SNF3/RGT2 GLUCOSE-SENSING PATHWAY 27 1.2.2.3. THE CAMP-PKA PATHWAY 29 1.2.2.3.1. ADENYLATE CYCLASE SYNTHESIZES CAMP 30 1.2.2.3.2. UPSTREAM ACTIVATORS OF ADENYLATE CYCLASE 30 1.2.2.3.3. THE G PROTEIN-COUPLED RECEPTOR GPR1 31 1.2.2.3.4. PROTEIN KINASE A 33 1.2.2.3.5. CAMP DEGRADATION BY PDE1 AND PDE2 34 1.2.2.3.6. A BYPASS OF ADENYLATE CYCLASE BY KRH1 AND KRH2 35 1.2.2.3.7. DOWNSTREAM TARGETS OF PKA 36 1.2.2.3.8. FEEDBACK INHIBITION 41 1.2.3. THE FGM PATHWAY 41 1.2.4. THE CAMP-PKA PATHWAY IN PATHOGENIC FUNGI 42 1.2.4.1. GPCR SYSTEMS IN FUNGI 42 1.2.4.2. COMPONENTS OF THE CAMP-PKA PATHWAY IN C. ALBICANS 43 1.2.4.3. ROLE OF PKA IN VIRULENCE IN OTHER FUNGI 45 2. RESULTS AND DISCUSSION 47 2.1. C. GLABRATA POSSESSES HOMOLOGS OF ALL COMPONENTS OF THE S. CEREVISIAE PKA MACHINERY 49 2.1.1. CONSTRUCTION OF MUTANTS INVOLVED IN THE CAMP-PKA PATHWAY IN C. GLABRATA 49 2.1.2. CYR1 AND RAS1 ARE ESSENTIAL FOR NORMAL GROWTH IN C. GLABRATA 50 2.2. DIFFERENT SUGARS INDUCE A GPR1- AND GPA2-DEPENDENT CAMP SIGNAL 52 2.2.1. INTRACELLULAR CAMP LEVEL RISES IN RESPONSE TO SUGARS BUT NOT TO SERUM IN C. GLABRATA 52 2.2.2. THE C. GLABRATA GPCR COMPLEX IS REQUIRED FOR SUGAR SENSING 55 2.2.3. CGGPR1 RESTORES GLUCOSE-INDUCED CAMP SIGNALING AND HSP12 REPRESSION IN A S. CEREVISIAE GPR1D STRAIN 58 2.2.4. GPR1 AND GPA2 MODULATE TREHALOSE MOBILIZATION 63 2.2.5. GPR1 AND GPA2 CONTRIBUTE TO HEAT STRESS SUSCEPTIBILITY OF CELLS GROWING ON FERMENTABLE CARBON SOURCES 65 2.3. CHARACTERIZATION OF OTHER COMPONENTS OF THE CAMP-PKA PATHWAY IN C. GLABRATA 68 2.3.1. PDE1 AND TPK2 REGULATE THE GLUCOSE-INDUCED CAMP SIGNAL 68 2.3.2. THE KRH PROTEINS, PDE2 AND TPK2 REGULATE TREHALOSE LEVELS 71 2.3.2.1. THE KELCH REPEAT HOMOLOGS REGULATE TREHALOSE MOBILIZATION AND ACCUMULATION 71 2.3.2.2. PHOSPHODIESTERASE PDE2 IS INVOLVED IN TREHALOSE ACCUMULATION. 75 2.3.2.3. DELETION OF TPK2 RESULTS IN AN INCREASED MOBILIZATION OF TREHALOSE 77 2.3.3. THE KRH PROTEINS ARE INVOLVED IN HEAT STRESS RESISTANCE 78 2.4. THE CAMP-PKA PATHWAY OF C. GLABRATA AND VIRULENCE-RELATED CHARACTERISTICS 80 2.4.1. C. GLABRATA AND ADHESION, BIOFILM AND EPA-EXPRESSION 80 2.4.1.1. CAMP-INDUCING SUGARS INHIBIT FUNGAL ADHERENCE IN A GPR1-DEPENDENT MANNER 80 2.4.1.2. GLUCOSE, FRUCTOSE AND SUCROSE INDUCE THE EXPRESSION OF EPA1 AND EPA3 82 2.4.1.2.1. SUGAR-INDUCED EPA1 AND EPA3 EXPRESSION IS GPR1-DEPENDENT 82 2.4.1.2.2. EXPRESSION OF EPA1 AND EPA3 IS GLUCOSE-CONCENTRATION DEPENDENT AND TRANSIENT 83 2.4.1.3. KRH AND PDE PROTEINS ARE INVOLVED IN ADHESION AND BIOFILM DEVELOPMENT IN RPMI 85 2.4.2. THE CAMP-PKA PATHWAY AND VIRULENCE 87 2.4.2.1. DELETION OF GPR1 OR THE KRH GENES DOES NOT ALTER VIRULENCE IN A FLY MODEL OF C. GLABRATA CANDIDIASIS 87 2.4.3. THE CAMP-PKA PATHWAY AND RESISTANCE TO STRESS AND ANTIFUNGALS 90 2.4.3.1. DELETION OF PDE1 AND/OR PDE2 RENDERS THE CELL MORE SENSITIVE TO STRESS AND/OR FLUCONAZOLE 90 3. GENERAL DISCUSSION 93 4. MATERIALS AND METHODS 105 4.1. STRAINS AND GROWTH CONDITIONS 107 4.2. CONSTRUCTION OF STRAINS 107 4.2.1. GENE DELETION IN CANDIDA GLABRATA 107 4.2.2. GENE COMPLEMENTATION IN S. CEREVISIAE 109 4.3. METHODS 113 4.3.1. DETERMINATION OF CAMP LEVELS 113 4.3.2. TREHALOSE DETERMINATION 114 4.3.3. HEAT STRESS EXPERIMENTS 114 4.3.4. PROTEIN-PROTEIN INTERACTION ASSAY USING THE SPLIT-UBIQUITIN SYSTEM 114 4.3.5. ADHESION AND XTT QUANTIFICATION ASSAY 115 4.3.6. BIOFILM ASSAY IN 96-WELL POLYSTYRENE PLATES 115 4.3.7. BIOFILM ASSAY DEVELOPED IN POLYURETHANE CATHETERS 115 4.3.8. RNA EXTRACTION, CDNA SYNTHESIS AND REAL TIME PCR 116 4.3.9. DROSOPHILA MELANOGASTER SURVIVAL EXPERIMENTS 116 4.3.10. MINIMAL INHIBITORY CONCENTRATION (MIC) OF FLUCONAZOLE 117 4.3.11. REPRODUCIBILITY OF THE RESULTS 117 SUMMARY 119 SAMENVATTING 121 REFERENCES 123nrpages: 156status: publishe

Structure-activity relationship study of the plant-derived decapeptide OSIP108 inhibiting Candida albicans biofilm formation

We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity.status: publishe