Interplay between Candida albicans and the Antimicrobial Peptide Armory

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and C. albicans resistance mechanisms and current developments in the use of AMPs as antifungal agents.     

Mechanism of action of novel synthetic dodecapeptides against Candida albicans

Background

Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,β-didehydrophenylalanine (?Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated.

Methods

The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy.

Results and conclusions

The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250 μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans.

General significance

The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.     

Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species

In this study we investigated the effects of Candida albicans, Candida krusei, Candida tropicalis and Candida parapsilosis on human beta-defensin 2 (HBD-2) production in Caco-2 intestinal cell line, and the production of alpha-defensins (human neutrophil peptides, HNP 1–3) in peripheral blood. Opportunistic pathogen yeasts can modulate the host immune function by inducing defensins, the natural antimicrobial peptides. Here we show that Candida spp. stimulated HBD-2 expression in and release from Caco-2 cells, with C. albicans inducing the highest levels of HBD-2. Similarly, HNP 1–3 secretion was significantly increased in whole blood after exposure to Candida yeast cells, with C. albicans producing the greatest effect. Our investigations underscore the important role of beta and alpha defensins produced by intestinal epithelial cells locally and neutrophils systemically in the antifungal defense against Candida.     

Backbone 1H, 15N, 13C and Ile, Leu, Val methyl chemical shift assignments for the 33.5 kDa N-terminal domain of Candida albicans ALS1

The agglutinin-like-sequence (ALS) family of adhesion proteins are a key virulence factor for C. albicans. These proteins have been implicated in several functions, notably adhesion and invasion of different cell types, as well as binding to peptides and proteins in the cell surface and extracellular matrix. In order to understand their binding mechanism and en route to a full structural determination by NMR, here we report the resonance assignments of backbone atoms plus Ile, Leu and Val methyls for residues 18–329 of ALS1, which comprises the 33.5 kDa binding domain.     

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46–100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25–100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.     

Plagiochin E,an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans

Background

Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied.

Methods

We assayed the mitochondrial membrane potential (mtΔψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F₀F₁-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated.

Results

Exposure to PLE resulted in an elevation of mtΔψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F₀F₁-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE.

Conclusions

PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.

General significance

The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.     

The Peptide-binding Cavity Is Essential for Als3-mediated Adhesion of Candida albicans to Human Cells

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.     

Antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from Psacothea hilaris

Psacotheasin is a 34-mer knottin-type peptide that is derived from Psacothea hilaris larvae. In this study, the antifungal activity and mechanism(s) by which psacotheasin affects human fungal pathogens were investigated. Psacotheasin shows remarkable antifungal properties without hemolytic activity against human erythrocytes. To understand the antifungal mechanism(s) of psacotheasin in Candida albicans, flow cytometric analysis with DiBAC₄(3) and PI was conducted. The results showed that psacotheasin depolarized and perturbed the plasma membrane of the C. albicans. Three-dimensional (3D)-flow cytometric contour-plot analysis, accompanied by decreased forward scatter (FS), which indicates cell size, confirmed that psacotheasin exerted antifungal effects via membrane permeabilization. The membrane studies, using a single GUV and FITC-dextran (FD) loaded liposomes, indicate that psacotheasin acts as a pore-forming peptide in the model membrane of C. albicans and the radius of pores were presumed to be anywhere from 2.3 to 3.3 nm. Therefore, the current study suggests that the mechanism(s) of psacotheasin’s antifungal properties function within the membrane.     

Anticandidal activity of synthetic peptides: Mechanism of action revealed by scanning electron and fluorescence microscopies and synergism effect with nystatin

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo‐CBP₃‐PepI (CPAIQRCC) and Mo‐CBP₃‐PepII (NIQPPCRCC), are described, bioinspired by Mo‐CBP₃, which is an antifungal chitin‐binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50‐fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.     

Cooperative Anti-Candida Effects of Lactoferrin or Its Peptides in Combination with Azole Antifungal Agents

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide Lactoferricin® B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/41/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.     

Chemical synthesis,structure–activity relationship,and properties of shepherin I: a fungicidal peptide enriched in glycine-glycine-histidine motifs

Although glycine-rich antimicrobial peptides (AMPs) are found in animals and plants, very little has been reported on their chemistry, structure activity-relationship, and properties. We investigated those topics for Shepherin I (Shep I), a glycine-rich AMP with the unique amino acid sequence G¹YGGHGGHGGHGGHGGHGGHGHGGGGHG²⁸. Shep I and analogues were synthesized by the solid-phase method at 60 °C using conventional heating. Purification followed by chemical characterization confirmed the products' identities and high purity. Amino acid analysis provided their peptide contents. All peptides were active against the clinically important Candida species, but ineffective against bacteria and mycelia fungi. Truncation of the N- or C-terminal portion reduced Shep I antifungal activity, the latter being more pronounced. Carboxyamidation of Shep I did not affect the activity against C. albicans or C. tropicalis, but increased activity against S. cerevisiae. Carboxyamidated analogues Shep I (3-28)a and Shep I (6-28)a were equipotent to Shep I and Shep Ia against Candida species. As with most cationic AMPs, all peptides had their activity significantly reduced in high-salt concentrations, a disadvantage that is defeated if 10 µM ZnCl₂ is present. At 100 µM, the peptides were practically not hemolytic. Shep Ia also killed C. albicans MDM8 and ATCC 90028 cells. Fluo-Shep Ia, an analogue labeled with 5(6)-carboxyfluorescein, was rapidly internalized by C. albicans MDM8 cells, a salt-sensitive process dependent on metabolic energy and temperature. Altogether, such results shed light on the chemistry, structural requirements for activity, and other properties of candidacidal glycine-rich peptides. Furthermore, they show that Shep Ia may have strong potential for use in topical application.     

The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.     

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC₈₀ values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC₈₀ of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.     

Effect of Tetrandrine against Candida albicans Biofilms

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.     

The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.     

Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A

Background

Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms.

Methods

Combinations of five antifungal drugs- fluconazole (FLC), voriconazole (VOR), caspofungin (CSP), amphotericin B (AmB) and nystatin (NYT) with cyclosporine A (CSA) were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI) of combination effects. Biofilm growth was analyzed using XTT-metabolic assay.

Results

MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively.

Conclusions

The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.