Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity.

With the appearance of the antifungal resistance, novel antifungal agents need to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of aryltetrazole with 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole or 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole and Michael-type addition of aryltetrazole to phenyl vinyl sulfone. The chemical structures of the synthesized compounds were confirmed by means of 1H NMR, 13C NMR, IR and HRMS spectral data. The compounds were tested against the moulds: Fusarium sambucinum, Fusarium oxysporum, Colletotrichum coccodes, Aspergillus niger, and the yeast Candida albicans. The results showed that among the moulds only C. coccodes was significantly sensitive to all the structures examined. All the tetrazole derivatives acted at the same level against C. albicans and demonstrated a high cell growth inhibition (97-99%) at the concentrations ranging from 16 to 0.0313µg/mL. The mode of action of 2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c) and 2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d) was established by verifying fungal growth in the presence of osmotic protector-sorbitol. The effect of compound 5c or 5d combined with Fluconazole was determined using the checkerboard method. The calculated fractional inhibitory concentration index (FIC) indicated antagonism (FIC >1). Additionally, survival experiments with lepidopteran Galleria mellonella treated with compounds 5c and 5d were performed and demonstrated the lack of toxicity of these compounds.

Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives.

A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 µg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100).

Tetrazole activity against Candida albicans. The role of KEX2 mutations in the sensitivity to (±)-1-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propan-2-yl acetate.

Series of 4-(5-aryl-2H-tetrazol-2-yl)butan-2-ol, 1-(5-aryl-2H-tetrazol-2-yl)propan-2-ol and their acetates have been screened against Candida albicans. Among the tested compounds, (±)-1-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propan-2-yl acetate (E5) proved to be the most effective inhibitor of fungal growth and was further evaluated against young (adhesion phase) and mature biofilm in vitro. The activity exhibited by the tested tetrazole derivatives against C. albicans associated with minor cytotoxicity towards Vero epithelial cells make us suggest that E5 could be a promising structure in the development of new antifungals. Serine protease Kex2 appeared essential for the resistance mechanism. Further investigations of in vivo activity, drug interactions, and E5 structure optimization are needed.

New synthetic sulfone derivatives inhibit growth, adhesion and the leucine arylamidase APE2 gene expression of Candida albicans in vitro.

The successful preventing and effective treatment of invasive Candida albicans infections required research focused on synthesis of new classes of agents and antifungal activity studies. Bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named compound 6); dichloromethyl-4-chloro-3-nitrophenyl sulfone (named 7); and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (named 11) on inhibition of planktonic cells' growth, leucine arylamidase APE2 gene expression, and adhesion to epithelial cells were investigated. In vitro anti-Candida activities were determined against wild-types, and the morphogenesis mutants: Δefg1 and Δcph1. MICs of compounds 6, 7 and 11 (concentrated at 0.25-16µg/ml) were determined using the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document). APE2 expression was analyzed using RT-PCR; relative quantification was normalized against ACT1 in cells growth in YEPD and on Caco-2 cell line. Adherence assay of C. albicans to Caco-2 was performed in 24-well-plate. The structure activity relationship suggested that sulfone containing hydrazine function at C-1 (compound 11) showed higher antifungal activity (cell inhibition%=100 at 1-16µg/ml) than the remaining sulfones with chlorine at C-1. Δcph1/Δefg1 was highly sensitive to compound 11, while the sensitivity was reduced in Δcph1/Δefg1::EFG1 (%=100 at 16-fold higher concentration). Compound 11 significantly affected adherence to epithelium (P ⩽0.05) and hyphae formation. The APE2 up-regulation plays role in sulfones' resistance on MAP kinase pathway. Either CPH1 or EFG1 play a role in the resistance mechanism in sulfones. The strain-dependent phenomenon is a factor in the sulfone resistance mechanism. Sulfones' mode of action was attributed to reduced virulence arsenal in terms of adhesiveness and pathogenic potential related to the APE2 expression and morphogenesis.

Susceptibility of Candida albicans to new synthetic sulfone derivatives.

The influence of halogenated methyl sulfones, i.e. bromodichloromethyl-4-chloro-3-nitrophenyl sulfone (named halogenated methyl sulfone 1), dichloromethyl-4-chloro-3-nitrophenyl sulfone (halogenated methyl sulfone 2), and chlorodibromomethyl-4-hydrazino-3-nitrophenyl sulfone (halogenated methyl sulfone 3), on cell growth inhibition, aspartic protease gene (SAP4-6) expression, adhesion to epithelium, and filamentation was investigated. Antifungal susceptibility of the halogenated methyl sulfones was determined with the M27-A3 protocol in the range of 16-0.0313 µg/mL. Adherence to Caco-2 cells was performed in 24-well plates; relative quantification was normalized against ACT1 in cells after 18 h of growth in YEPD and on Caco-2 cells. SAP4-6 expression was analyzed using RT-PCR. Structure-activity relationship studies suggested that halogenated methyl sulfone 1 containing bromodichloromethyl or dichloromethyl function at C-4 (halogenated methyl sulfone 2) of the phenyl ring showed the best activity (100% cell inhibition at 0.5 µg/mL), while hydrazine at C-1 (halogenated methyl sulfone 3) reduced the sulfone potential (100% = 4 µg/mL). SAP4-6 were up- or down-regulated depending on the strains' genetic background and the substitutions on the phenyl ring. Halogenated methyl sulfone 2 repressed germination and affected adherence to epithelium (P ≤ 0.05). The tested halogenated methyl sulfones interfered with the adhesion of Candida albicans cells to the epithelial tissues, without affecting their viability after 90 min of incubation. The mode of action of the halogenated methyl sulfones was attributed to the reduced virulence of C. albicans. SAP5 and SAP6 contribute to halogenated methyl sulfones resistance. Thus, halogenated methyl sulfones can inhibit biofilm formation due to their interference with adherence and with the yeast-to-hyphae transition.

Role of SAP7-10 and Morphological Regulators (EFG1, CPH1) in Candida albicans' Hypha Formation and Adhesion to Colorectal Carcinoma Caco-2.

Secreted aspartic proteases (Saps) are considered as key virulence factors of Candida albicans. Hopefully our outlook will widen the knowledge of SAP7's role in C. albicans pathogenesis. The goal of our study was to investigate SAP7 expression during C. albicans adhesion to intestinal human cells. Another objective was to study the role of SAP8-10 and transcriptional regulators: EFG1 and CPH1, using the mutants: Δsap, Δefg1, Δcph1 during growth on Caco-2 monolayer. SAP7 expression was analyzed using real time RT-PCR; relative quantification was normalized against ACT1 in cells after growth on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. The results proved that SAP7 can play a role during the initial adaptation of C. albicans to intestinal tract and decreases over time. Up-regulation of SAP7 occured in the absence of SAP8 and SAP10 (genetic alternations dependence). SAP7 can be regulated by the morphogensis' regulators during C. albicans growth on epithelium. Adhesion of the mutants was indistinguishable from SC5314. The lack of neither SAP8-10 nor EFG1/CPH1 influences the adhesive behaviour of C. albicans. Deletion of SAP8-10 resulted in no filamentation defects. The results help better understand the role of SAP7 during adhesion and morphogenesis in C. albicans.

Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum PQ-96.

The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

Quantitative expression of Candida albicans aspartyl proteinase genes SAP7, SAP8, SAP9, SAP10 in human serum in vitro.

The different members of the secreted aspartyl proteinase (Sap) family of the human pathogenic yeast Candida albicans are proposed to play different roles during infection and are differentially expressed at various body sites. In recent reports, expression analysis has focused on the genes SAP1-6, while the expression pattern of SAP7-10 was less well studied. We analyzed the SAP7-SAP10 expression profile of C. albicans under human serum influence that may be elucidated in the course of blood infection in humans and how this in vitro expression profile is associated with hyphal formation. The phenotypes of strains were examined under scanning electron microscopy. Quantitative RT-PCR (2-(deltadeltaC)T) was used to monitor SAP expression of C. albicans wild type cells and mutants lacking SAP9 and/or SAP10. Of the four analyzed SAP genes, only SAP7 was detectably induced in the double mutant and in the wild type strains in the model that mimics bloodstream infections. On the other hand, in the wild types (isolate 83 and CAF2-1), SAP7 was expressed 0.8- or 0.4-fold less than SAP10, respectively. Our findings suggest that Sap7 may respond to the challenge of the human blood environment. Furthermore, the results support the notion that compensatory upregulation of SAP7 and SAP8 in the deltasap9/deltasap10 mutant occurs in these conditions. SAP7-10 expression was strain-specific. Our findings point to a link between morphogenesis and expression of SAP9 in serum, where these conditions induce both hyphae and SAP9, but temporal gene expression patterns might be controlled by other factors.

Sulfone derivatives reduce growth, adhesion and aspartic protease SAP2 gene expression.

Fungal virulence factors represent a strategy for the design of new compounds with effective activities against Candida spp. Dichloromethyl-4-chloro-3-nitrophenylsulfone (named Compound 1) and chlorodibromomethyl-4-hydrazino-3-nitrophenylsulfone (Compound 2) versus Candida albicans virulence factors (SAP2 expression and adhesion to Caco-2 cell line) were investigated. Candida albicans SC5314 and its mutants: Δsap9, Δsap10, Δsap9/10 were used. MICs of the Compounds (concentrated at 0.0313-16 µg/ml) were determined using M27-A3. Percentage of cell inhibition was assessed spectrophotometrically (OD405) after 48 h at 35 °C. The SAP2 expression was analyzed with the use of RT-PCR; relative quantification was normalized against ACT1 in cells grown in YEPD and on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. Compound 1 showed higher activity (% = 100 at 4 µg/ml) than Compound 2 (MIC90 = 16 µg/ml). Dichloromethyl at the para position of the phenyl ring exerted anti-Candidal potential. Under Compound 1, SAP2 was down-regulated in all the strains (P ≤ 0.05). Conversely, SAP2 was over-expressed in Δsap9-10 (untreated cells) compared with the wild-type. The Compounds significantly affected adherence to epithelium (P ≤ 0.05). The tested sulfones interfered with the adhesion of C. albicans cells to the epithelial tissues without affecting their viability after 90-min of incubation. The Compounds' mode of action was attributed to the reduced adhesiveness and the lower SAP2 expression. Saps9-10 play a role in C. albicans adhesion and they can be involved in the sulfone resistance mechanisms.

Cell wall carbohydrates content of pathogenic Candida albicans strain morphological forms.

INTRODUCTION: The study evaluated the cell wall carbohydrates fraction in blastoconidia grown in YEPD medium at 30 degrees C and in the conglomerate of true hyphae grown in human serum at 37 degrees C. MATERIAL AND METHODS: The clinical isolate obtained from a child with widespread C. albicans infection was used in the study. The cells were broken with glass beads, centrifuged to harvest the cell wall followed by subjection to TFA hydrolysis and in the result of that released monosaccharides were detected by HPAEC-PAD. Both, serum and temperature conditions (37 degrees C) affected germination process influencing the cell wall carbohydrates content when incubation in serum was prolonged from 1 to 18 h. RESULTS: The mannan content of blastoconidia was almost twofold higher compared to filamentous forms (149.25 +/- 299.24 vs 77.26 +/- 122.07). The glucan content was threefold lower in blastoconidia compared to hyphae (251.86 +/- 243.44 vs 755.81 +/- 1299.30). The chitin level was fourfold lower in blastoconidia compared to filaments (23.86 +/- 54.09 vs 106.29 +/- 170.12). The reason for the differences in the carbohydrates content may be related to type of morphology induced in different environmental conditions. Among tested carbohydrates, glucan appeared to be present in appreciably larger amounts in both tested morphological fractions. The ultrastructure of the blastoconidial cell wall revealed striking differences compared to the hyphae indicating the carbohydrates content alterations for wall assembly during hyphal growth at alkaline pH and temp. 37 degrees C. CONCLUSIONS: The study provided evidence for the relationship between morphogenesis, cell-cell adhesion induced by serum and changes in the level of carbohydrates content.

Antifungal agents commonly used in the superficial and mucosal candidiasis treatment: mode of action and resistance development.

Recent progress in medical sciences and therapy resulted in an increased number of immunocompromised individuals. Candida albicans is the leading opportunistic fungal pathogen causing infections in humans, ranging from superficial mucosal lesions to disseminated or bloodstream candidiasis. Superficial candidiasis not always presents a risk to the life of the infected host, however it significantly lowers the quality of life. Superficial Candida infections are difficult to treat and their frequency of occurrence is currently rising. To implement successful treatment doctors should be up to date with better understanding of C. albicans resistance mechanisms. Despite high frequency of Candida infections there is a limited number of antimycotics available for therapy. This review focuses on current understanding of the mode of action and resistance mechanisms to conventional and emerging antifungal agents for treatment of superficial and mucosal candidiasis.

Ultrastructure of Candida albicans pleomorphic forms: phase-contrast microscopy, scanning and transmission electron microscopy.

A modified method of glutaraldeyde-osmium tetroxide fixation was adjusted to characterize the ultrastructure of Candida albicans pleomorphic forms, using phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy. The discovered morphological criteria defining the individual morphotypes are discussed in terms of mycological and histopathological diagnostics of candidiasis. The relations are discussed between fungal pleomorphism, virulence and susceptibility of different morphotypes to fungicides.

In vitro study of secreted aspartyl proteinases Sap1 to Sap3 and Sap4 to Sap6 expression in Candida albicans pleomorphic forms.

Transition from round budding cells to long hyphal forms and production of secreted aspartic proteases (Saps) are considered virulence-associated factors of Candida albicans. Although plenty of data dealing with Saps involvement in the infection process have been published, Saps expression by the different pleomorphic forms as well as the capacity of C. albicans filaments to express Sap1-6 under serum influence are poorly investigated. In this study, we used immunofluorescence and immunoelectron microscopy for the detection of Sap1-6 isoenzymes in C. albicans pleomorphic cells (blastoconidia, germ tubes, pseudohyphae, true hyphae) grown in Sap-inductive human serum and Sap non-inductive medium - yeast extract-peptone-glucose (YEPD). Isoenzymes were below the detection level in all blastoconidial cells grown in YEPD for 18 h. Sap1-6 expression was hardly detected in C. albicans cells cultivated in serum for 20 min. Increasing level of Sap1-6 expression was observed when C. albicans was incubated for 2, 6 and 18 h in serum corresponding to the development of germ tubes, pseudohyphae and true hyphae. The expression of Sap1-3 in pseudohyphae and true hyphae was more intensive compared to Sap4-6. Thus, we could show that human serum induced hyphae formation and the expression of Sap1-6 were co-regulated.

[Virulence factors of Candida albicans]. / Czynniki zjadliwosci Candida albicans.

Candida albicans is the most common etiological factor of opportunistic human fungal infections. In this review, we focus on the major virulence factors that mediate the pathogenesis of C. albicans. Among these virulence factors, secreted aspartyl proteases, adherence, pleomorphism are the most important features of C. albicans infections. Ability to exist as different pleomorphic forms is defined as pleomorphism. A number of quorum sensing (QS) molecules have been described which affect morphogenesis process in C. albicans. Furthermore, the morphological transition of C. albicans in response to changing environmental conditions represent a means by which the strain adapts to different biological niches. Furthermore, every morphotype has own virulence profile and each pleomorphic form provide critical functions required for pathogenesis. Candida albicans is a producer of extracellular hydrolytic enzymes. Among them lipases, phospholipases and secreted aspartyl proteinases (Sap) are most significant in virulence. Sap proteins contribute to pathogenesis by digestion of host cell membranes and molecules of the host immune system to avoid antimicrobial attack by the host. One of the key features in the development of candidiasis is adhesion ofC. albicans to buccal and vaginal epithelial cells. The adhesion to host cells represents the first step in the internalization process which involves adhesins. Knowledge of the role of the various C. albicans' virulence factors during in vivo infections is still incomplete, therefore further studies including quantification of genes expression and histopathological examination of tissues damage are required to fully understand pathogenesis of this opportunistic pathogen.

Role of Virulence Determinants in Candida albicans' Resistance to Novel 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone.

We investigated the role of KEX2, SAP4-6, EFG1, and CPH1 in the virulence of Candida under a novel compound 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone (Compound 4). We examined whether the exposure of C. albicans cells to Compound 4, non-cytotoxic to mammalian cells, reduces their adhesion to the human epithelium. We next assessed whether the exposure of C. albicans cells to Compound 4 modulates the anti-inflammatory response (IL-10) and induces human macrophages to respond to the Candida cells. There was a marked reduction in the growth of the sap4Δsap5Δsap6Δ mutant cells when incubated with Compound 4. Under Compound 4 (minimal fungicidal concentration MFC = 0.5-16 µg/mL): (1) wild type strain SC5314 showed a resistant phenotype with down-regulation of the KEX2 expression; (2) the following mutants of C.albicans: sap4Δ, sap5Δ, sap6Δ, and cph1Δ displayed decreased susceptibility with the paradoxical effect and up-regulation of the KEX2 expression compared to SC5314; (3) the immune recognition of C. albicans by macrophages and (4) the stimulation of IL-10 were not blocked ex vivo. The effect of deleting KEX2 in C. albicans had a minor impact on the direct activation of Compound 4's antifungal activity. The adhesion of kex2Δ is lower than that of the wild parental strain SC5314, and tends to decrease if grown in the presence of a sub-endpoint concentration of Compound 4. Our results provide evidence that SAP4-6 play a role as regulators of the anti-Candida resistance to Compound 4. Compound 4 constitutes a suitable core to be further exploited for lead optimization to develop potent antimycotics.

Natural Antimicrobial Peptides as Inspiration for Design of a New Generation Antifungal Compounds.

Invasive fungal infections are associated with high mortality rates, despite appropriate antifungal therapy. Limited therapeutic options, resistance development and the high mortality of invasive fungal infections brought about more concern triggering the search for new compounds capable of interfering with fungal viability and virulence. In this context, peptides gained attention as promising candidates for the antimycotics development. Variety of structural and functional characteristics identified for various natural antifungal peptides makes them excellent starting points for design novel drug candidates. Current review provides a brief overview of natural and synthetic antifungal peptides.

Effect of serine protease KEX2 on Candida albicans virulence under halogenated methyl sulfones.

AIM: The effect of KEX2 mutations on C. albicans virulence and resistance to halogenated methyl sulfones was assessed. MATERIALS & METHODS: The mechanism of action of sulfones was studied using flow cytometry and microscopy. Expression of KEX2 and SAP5 was assessed using quantitative Real-Time-PCR. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and lactate dehydrogenase assays were elaborated to study, respectively, metabolism of Candida treated with sulfones and their cytotoxicity against tissues. Inflammatory response was detected by ELISA. RESULTS: Lysosome permeabilization and dose-dependent programmed cell death under sulfones were noted. KEX2 induction depended on halogenomethylsulfonyl groups, which affected cell wall biosynthesis and adhesion. CONCLUSION: Sulfones treatment reduced Candida pathogenicity in Galleria mellonella. Sulfones are an alternative for antifungal therapies due to their safety profile and antibiofilm activity.

Antifungal Effect of Novel 2-Bromo-2-Chloro-2-(4-Chlorophenylsulfonyl)-1-Phenylethanone against Candida Strains.

We investigated the antifungal activity of novel a 2-bromo-2-chloro-2-(4-chlorophenylsulfonyl)-1-phenylethanone (compound 4). The synthesis of compound 4 was commenced from sodium 4-chlorobenzene sulfinate and the final product was obtained by treatment of α-chloro-ß-keto-sulfone with sodium hypobromite. The sensitivity of 63 clinical isolates belonging to the most relevant Candida species toward compound 4 using the method M27-A3 was evaluated. We observed among most of the clinical strains of C. albicans MIC ranging from 0.00195 to 0.0078 µg/mL. Compound 4 at 32 µg/mL exhibited fungicidal activity against nine Candida strains tested using the MFC assay. Compound 4 displayed anti-Candida activity (with clear endpoint) against 22% of clinical strains of Candida. Under compound 4, Candida susceptibility and tolerance, namely paradoxical effect (PG), was found for only two clinical isolates (C. glabrata and C. parapsilosis) and reference strain 14053 using both M27-A3 and MFC method. We found that compound 4 does not induce toxicity in vivo against larvae of Galleria mellonella (≥97% survival) and it displays reduced toxicity on mammalian cells in vitro (< CC20 at 64 µg/mL). Furthermore, XTT assay denoted clear metabolic activity of sessile cells in the presence of compound 4. Thus, the effect of compound 4 on formed C. albicans biofilms was minimal. Moreover, strain 90028 exhibited no defects in hyphal growth on Caco-2 monolayer under compound 4 influence at MIC = 16 µg/mL. The MIC values of compound 4 against C. albicans 90028, in medium with sorbitol did not suggest that compound 4 acts by inhibiting fungal cell wall synthesis. Our findings with compound 4 suggest a general strategy for antifungal agent development that might be useful in limiting the emergence of resistance in Candida strains.

Design and studies of multiple mechanism of anti-Candida activity of a new potent Trp-rich peptide dendrimers.

PURPOSE: Eight peptide dendrimers were designed as structural mimics of natural cationic amphiphilic peptides with antifungal activity and evaluated for their anti-Candida potential against the wild type strains and mutants. METHODS: Dendrimer 14 containing four Trp residues and dodecyl tail and a slightly smaller dendrimer 9 decorated with four N-methylated Trp that displayed 100 and 99.7% of growth inhibition at 16 µg/mL respectively, were selected for evaluation against the Candida albicans mutants with disabled biosynthesis of aspartic proteases responsible for host tissue colonization and morphogenesis during biofilm formation (sessile model). Flow cytometry method was employed to detect apoptotic cells with membrane alterations (phosphatidylserine translocation), and differentiation of apoptotic from necrotic cells was also performed. Simultaneous staining of cell surface phosphatidylserine with Annexin-V-Fluorescein and necrotic cells with propidium iodide was conducted. RESULTS: 14 at 16 µg/mL caused C. albicans cells to undergo cellular apoptosis but its increasing concentrations induced necrosis. 14 influenced C. albicans biofilm viability as well as hyphal and cell wall morphology. Confocal microscopy and cell wall staining with calcofluor white revealed that in epithelial model the cell surface structure was perturbed at MIC of peptide dendrimer. It appears that tryptophan or 1-methyltryptophan groups displayed at the surface and positive charges hidden in the dendrimer tree along with hydrocarbon tail located at C-terminus are important for the anti-Candida activity since dendrimers containing tryptamine at C-terminus showed only a moderate activity. CONCLUSIONS: Our results suggest that membranolytic dendrimer 14, targeting cellular apoptotic pathway and impairing the cell wall formation in mature biofilm, may be a potential multifunctional antifungal lead compound for the control of C. albicans infections.

The in vitro effects of new D186 dendrimer on virulence factors of Candida albicans.

The emergence of drug-resistant Candida albicans strains necessitates identifying new antimycotics along with studying their modes of action. The influence of a new rationally designed dendrimer D186 containing N,N-dioctyl tail and four tryptophane residues on inhibition of planktonic cells, aspartic protease SAP5 expression and adhesion to epithelial cells was investigated. In vitro anti-Candida activities were determined against wild types, Δsap mutants and morphogenesis mutants: Δefg1, Δcph1 and Δcph1/Δefg1. MICs of D186 determined with M27-A3 protocol were in the range 2-16 µg ml(-1). Adherence assay of C. albicans to Caco-2 was performed in 24-well plate. Group I (MIC=8 µg ml(-1), inhibition=82.05-100%) was the most frequent followed by Group II (MIC=4 µg ml(-1), inhibition=99.64-100%) and Group III (MIC=2 µg ml(-1), inhibition=96.47-96.98%). SAP5 expression was analyzed using RT-PCR; relative quantification was normalized against ACT1 in cells after 18-h growth on Caco-2 cell line. D186 exhibited more potent inhibition activity (statistically significant P⩽0.05) against Δsap10 and Δsap9/Δsap10 (MIC=2 µg ml(-1)) than the remaining strains tested. Pretreating cells with D186 significantly inhibited adhesion of all Candida strains compared with their non-treated counterparts (P⩽0.05). D186 affected SAP5 expression of all strains suggesting that this gene is controlled by environmental conditions. A hypothesis can be formulated that the hydrophobicity of D186 and presence of four Trp residues favors its accumulation in the membrane causing membrane disruption, especially facilitated in mutants perturbed in the cell wall compositions. The D186 mode of action was attributed to reduced virulence in terms of adhesiveness and pathogenic potential related to SAP5 expression and morphogenesis.