The authors have declared that no competing interests exist.
Conceived and designed the experiments: EP PBA PV. Performed the experiments: GL EP. Analyzed the data: GL EP PV PBA. Contributed reagents/materials/analysis tools: EP PBA PV GL. Wrote the paper: GL.
In the last few decades, fungal infections, particularly nosocomial, increased all around the world. This increment stimulated the search for new antifungal agents, especially those derived from nature. Among natural products, those from marine sources have gained prominence in the last years. Purified phlorotannins extracts from three brown seaweeds (
Resistance to antifungal agents has significantly increased over the past few decades.
The cell membrane and cell wall of fungi are the most important targets for antifungal drugs. These physical and chemical barriers are responsible for the communication with the environment and, therefore, have a key role in metabolic processes
Antifungals can also affect the germ tube formation and adhesion of yeasts, and interact with the respiratory chain processes in mitochondria
Mitochondria are present in most eukaryotic cells and comprise the respiratory chain. These organelles play several important roles, including generation and regulation of reactive oxygen species (ROS), calcium (Ca2+) homeostasis, regulation of apoptosis and metabolic processes, also being responsible for more than 90% of cellular ATP production
While the medicinal properties of herbs have been recognized since ancient times, there has been a resurgence of interest in the antimicrobial properties of marine organisms. Seaweeds are particularly attractive, not only for the abundance of substances with industrial interest, but also for the diversity on secondary metabolites with interesting pharmaceutical properties
Dimethyl sulfoxide (DMSO), trypan blue solution (0.4%), sodium chloride (NaCl), 3-(N-morpholino) propanesulfonic acid (MOPS), N-acetylglucosamine, D-(+)-glucosamine hydrochloride, ergosterol (75%), thiazolyl blue tetrazolium bromide (MTT), rhodamine 123 (RHO), 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate, ammonium sulphamate (NH4 sulphamate), ferric chloride (FeCl3) and curdlan from
Brown seaweeds used in this work were randomly collected in the coast of Peniche (West Portugal) and taxonomically identified
Cultures were obtained from the Laboratory of Microbiology, Faculty of Pharmacy, Porto University (Portugal). The antifungal activity of the purified phlorotannins extracts was evaluated against
All microorganisms were stored in Sabouraud broth medium with 20% glycerol at −70°C and sub-cultured in SDA before each test, to ensure optimal growth conditions and purity.
Broth microdilution methods based on the Clinical and Laboratory Standard Institute (CLSI) reference documents M27A-3 and M38-A2 for yeast and filamentous fungi, respectively, with minor modifications, were used to determine minimum inhibitory concentrations (MIC)
The minimum lethal concentrations (MLC) of purified phlorotannins extracts was determined after 48 h (for
Germ tube inhibition assay was performed according to Pinto and co-workers
Cell separation and adherence assays were modified from those proposed by Lima-Neto and co-workers
Adherence assays were developed by mixing 1 mL of each suspension in a test tube, followed by incubation in the presence of the test compounds, at 35°C under gentle stirring for 2 h. A control without test compound and a control with epithelial cells pre-treated with the test compound were performed along with the samples. After incubation, two drops of trypan blue solution (0.4%) were added to each tube and the mixture was gently shaken. Ten microliters of the stained suspension were transferred to a Neubauer chamber and examined under light microscopy.
Fungi growth conditions for sterol extraction were performed according to Pinto and co-workers
After the incubation period with purified phlorotannins extracts, total intracellular sterols were extracted by saponification
Cell wall 1,3-β-D-glucans content was determined using the aniline blue assay
Results (mean ± standard deviation) are expressed as g (curdlan)/100 g (dry microorganism) of three independent assays performed in duplicate.
The methodology used for cell wall chitin quantification was based on the protocol proposed by Fortwendel and co-workers
Final results represent the average (± standard deviation) of three independent experiments performed in duplicate.
Mitochondrial dehydrogenase activity was evaluated by the MTT assay
After the exposure time, cell suspensions were centrifuged, the supernatant was removed, and 500 µL of MTT solution (0.5 mg/mL in RPMI, 35°C) were added to the cell pellets and left incubating for 30 minutes at 35°C (for
The mitochondrial membrane potential was evaluated by the incorporation of the fluorescent dye RHO
Fluorescence intensity was determined in a fluorescence microplate reader (Synergy HT, BioTek Instruments, Winooski, USA) equipped with Gen5 software, with excitation wavelength 485/20 nm and emission wavelength 528/20 nm. Results are expressed in% of fluorescence relative to control, for three independent assays performed in duplicate. Sodium azide (an inhibitor of the mitochondrial respiratory chain) at a final concentration of 20 mM was used for all the experiments as control (data not shown).
Data were analyzed by using GraphPad PRISM software (GraphPad software, San Diego, CA, USA) (version 5.02 for Windows). One-way analysis of variance (ANOVA), using the Dunnett Multiple Comparison test, was carried out on data obtained from three independent assays performed in duplicate for each sample. Levels of statistical significance at
The antifungal activity of purified phlorotannins extracts from the studied species is presented in
Seaweed | |||||||||
Strains | MIC |
MIC50 |
MLC |
MIC |
MIC50 |
MLC |
MIC |
MIC50 |
MLC |
15.6 | – | >62.5 | 31.3 | – | >62.5 | 31.3 | – | >62.5 | |
31.3 | – | >62.5 | 31.3 | – | >62.5 | >62.5 | – | – | |
>62.5 | – | – | >62.5 | – | – | >62.5 | – | – | |
62.5 | – | >62.5 | 62.5 | – | >62.5 | >62.5 | – | – | |
62.5 | – | >62.5 | >62.5 | 62.5 | – | >62.5 | – | – | |
62.5 | 31.3 | >62.5 | >62.5 | 62.5 | >62.5 | >62.5 | – | – | |
>62.5 | 2.0 | – | >62.5 | – | – | >62.5 | – | – | |
62.5 | 31.3 | >62.5 | 62.5 | – | >62.5 | >62.5 | – | – | |
3.9 | – | 7.8 | 15.6 | – | 15.6 | 7.8 | – | 7.8 | |
3.9 | – | 7.8 | 7.8 | – | 31.3 | 3.9 | – | 31.3 | |
7.8 | – | 7.8 | 31.3 | – | 31.3 | 15.6 | – | 15.6 | |
31.3 | – | 31.3 | 31.3 | – | >62.5 | 15.6 | – | 15.6 | |
31.3 | – | >62.5 | >62.5 | – | – | 31.3 | – | >62.5 | |
>62.5 | – | – | >62.5 | – | – | >62.5 | – | – | |
>62.5 | – | – | >62.5 | – | – | >62.5 | – | – | |
>62.5 | – | – | >62.5 | – | – | >62.5 | – | – |
MIC, MIC50 and MLC were determined by a microdilution method and expressed in mg/mL (dry matter).
“–”Not determined.
Of the genus
Arrows show a constriction resulting from an incomplete budding, where the bud remains attached to the mother cell, originating pseudohyphae. Levels of magnification are as shown: Bars, 50 µm (A1 and B1) and 250 µm (A2 and B2). Results are expressed as mean (±SD) of three independent assays. *
According to these results, and in order to clarify if pseudohyphae formation could affect the adherence of
The effect of purified phlorotannins extracts on fungal membrane composition was evaluated by determining ergosterol by HPLC-DAD, after fungal treatment with sub-inhibitory extracts concentrations (1/2 to 1/8 of the MIC) (
Results are expressed as mean (±SD) of three independent assays. *
The effect of purified phlorotannins extracts on fungal cell wall composition was evaluated by measuring the amount of 1,3-β-D-glucans and chitin. 1,3-β-D-glucans levels were quantified by a fluorescence assay after microorganisms treatment with sub-inhibitory concentrations of purified phlorotannins extracts (1/2 to 1/16 of the MIC). Caspofungin, an inhibitor of glucans synthesis, was used as control. 1,3-β-D-glucans levels on cells treated with purified phlorotannins extracts were compared with those of untreated cells. None of the samples significantly affected the glucans composition of the studied microorganisms.
Chitin levels in fungal cell wall were determined spectrofotometrically. Purified phlorotannins extracts had no effect on the chitin levels of yeast cells. Regarding dermatophyte, only
In order to check whether the phlorotannins could affect the mitochondrial function, the MTT reduction assay was performed with purified phlorotannins extracts concentrations ranging from MIC to MIC/1024 (
Results are expressed as the percent change of MTT reduction using the nontreated cells as control (mean (±SD) of three independent assays performed in duplicate). For concentrations lower than MIC/1024 the mitochondrial activity was similar to the untreated cells. Arrows show the formazan salts produced by
Along with the MTT reduction assay, the mitochondrial membrane potential was evaluated by measuring the incorporation of the fluorescent probe RHO by
For concentrations lower than MIC/1024 the percentage of fluorescence was similar to the untreated cells. Results are expressed as mean (±SD) of three independent assays performed in duplicate. *
According to the displayed antifungal activity, the purified phlorotannins extracts were object of further investigation, in an attempt to elucidate the mechanism underlying the antifungal action of these compounds. Thereby, the effect on the dimorphic transition and adherence (in
The ability to produce a germ tube is characteristic of
Our results demonstrate that phlorotannins of
Ergosterol is an important component of fungal cells, responsible for the maintenance of cell membrane structure and functions. Its synthesis is inhibited by azoles, which are fungistatic drugs commonly used in treatment and prevention of candidiasis
Unlike vertebrates, yeast respiratory chain involves more complex and flexible pathways not fully elucidated.
A large proportion of cellular dehydrogenases, namely succinate, NADH, glycerol 3P-dehydrogenase and lactate dehydrogenases, which are part of the CRC, are responsible for the reduction of MTT to formazan salts
Face to the results (
During normal respiration, small amounts of toxic intermediate species are generated by partial reduction of oxygen, including superoxide, hydrogen peroxide and hydroxyl radicals. Although cells have several enzymatic (superoxide dismutase, glutathione peroxidase and catalase) and non-enzymatic (endogenous and exogenous antioxidants) systems contributing for free radicals inactivation, these molecules can accumulate and cause cell damage
Along with the mitochondrial dehydrogenases function, the mitochondrial membrane potential is a key indicator of cellular viability. It reflects the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation. RHO is a fluorescent probe commonly used for the evaluation of mitochondrial membrane potential
According to the results, it appears that purified phlorotannins extracts have a dual mechanism for regulating the mitochondrial membrane potential (
Cell systems posses several proteins which are responsible for regulating programmed cell death and mitochondria integrity. In cases of severe cellular injury, anti-apoptotic proteins can block cell death by reducing the oxidative stress. The expression of these proteins is capable stabilizing the mitochondrial membrane potential
According to this, it could be hypothesised that at the highest concentration (MIC) there is increased expression of anti-apoptotic proteins by the cell, in a tentative to resist apoptosis, and therefore a quicker stabilization of the membrane potential takes place. As the concentration of phlorotannins decreases (from MIC to MIC/8), the cellular injury and the expression of anti-apoptotic proteins lowers, so that the membrane potential is not established as soon.
The second mechanism by which phlorotannins seem to affect the membrane potential is not probably related to anti-apoptotic defences. The accumulation of ROS inside cells can lead to alterations in cell membranes permeability, leading to the increase of cytosolic Ca2+ concentrations, both by the release of Ca2+ from the intracellular stores and by the increased influx over the cytoplasm membrane
Taking into account such hypotheses, there seem to be two possible mechanisms to explain the behaviour of mitochondria in
In conclusion, this work evidences the antifungal capacity of phlorotannins against a wide range of yeast and filamentous fungi. In a general way,
The hyperactivity of mitochondrial dehydrogenases caused by phlorotannins can lead to the accumulation of ROS and Ca2+, which in turn may result in membrane hyperpolarization, incompatible with normal cell metabolism. Nevertheless, the mechanism of action of these compounds needs further investigation, namely in what concerns to their action over some important cell enzymes like proteases, lipases and α-glucosidase, which play an important role in cell metabolism
The increment in combining antifungal medications with different mechanisms of action can lead to better therapeutic responses. Contrary to what happens with some commercially available antifungal drugs, the effect on yeast mitochondria activity can be the primary mechanism of action of phlorotannins. Thus, the challenge remains to associate compounds from natural matrices with existing antifungal drugs for which there is some resistance. There are some data indicating that phlorotannins do not act similarly in the mitochondria of mammalian and yeast