Synergistic Antifungal Activity of Berberine Derivative B-7b and Fluconazole

Our previous study demonstrated berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. We also suggested BBR played a major antifungal role in the synergism of FLC and BBR, while FLC increased intracellular BBR concentrations. Our following systematic structural modification and reconstruction of BBR core identified the novel scaffold of N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(substituted phenyl)acet-amide derivatives 7a-i, including B-7b and B-7d exhibiting remarkable synergistic antifungal activity and low cytotoxicity. Here, the study mainly investigated the synergistic activity of FLC and B-7b and the underlying mechanism. In vitro interaction of FLC and B-7b was investigated against 30 FLC-resistant clinical isolates of C. albicans and non-C. albicans species, including Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The potent synergistic activity of B-7b in combination with FLC against FLC-resistant C. albicans was found through the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed its better synergism with FLC. And as expected, B-7b exhibited much lower cytotoxicity than BBR to human umbilical vein endothelial cells. In contrast to BBR, we found that endogenous ROS augmentation was not involved in the synergism of FLC and B-7b. According to the results from our present comparative proteomic study, it seemed that the disruption of protein folding and processing and the weakening of cells’ self-defensive ability contributed to the synergism of FLC and B-7b. Together, these results suggested novel scaffold BBR derivative B-7b could be a promising synergist in combination with FLC for the treatment of invasive fungal infections.     

Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans

Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100 μM silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K⁺ and accumulation of cytoplasmic and mitochondrial Ca²⁺. Among these phenomena, mitochondrial Ca²⁺ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca²⁺ in apoptosis by employing 20 mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca²⁺ signaling in C. albicans.     

β-amyrin-induced apoptosis in Candida albicans triggered by calcium

The emergence of drug-resistant pathogens has urged researchers to discover alternatives for traditional antibiotics. β-amyrin, which is included in the category of triterpenoids extracted from plants, is known for its antimicrobial activity, although the underlying mechanism has not yet been revealed. This study was conducted to elucidate the antifungal mode of action of β-amyrin against Candida albicans. Based on the relevance between triterpenoids and oxidative molecules, reactive oxygen species (ROS) concentrations were detected, which showed a noticeable increment. Disruption of Ca²⁺ homeostasis in the cytosol was additionally analyzed, which was supported by interactions between two. Subsequently, decrease in mitochondrial membrane potential, increment of mitochondrial Ca²⁺, and ROS concentration were monitored, which suggested mitochondrial dysfunction modulated by Ca²⁺. Further investigation confirmed oxidative damage through glutathione reduction and DNA fragmentation. Accumulation of lethal damages resulted in the activation of caspases and externalization of phosphatidylserine, indicating the induction of yeast apoptosis by β-amyrin in C. albicans.     

A recyclable and light-triggered nanofibrous membrane against the emerging fungal pathogen Candida auris

The emerging "super fungus" Candida auris has become an important threat to human health due to its pandrug resistance and high lethality. Therefore, the development of novel antimicrobial strategy is essential. Antimicrobial photodynamic therapy (aPDT) has excellent performance in clinical applications. However, the relevant study on antifungal activity and the mechanism involved against C. auris remains scarce. Herein, a recyclable and biodegradable polylactic acid-hypocrellin A (PLA-HA) nanofibrous membrane is newly developed. In vitro PLA-HA-aPDT could significantly reduce the survival rate of C. auris plankton and its biofilms, and the fungicidal effect of the membrane is still significant after four repeated uses. Simultaneously, PLA-HA exhibits good biocompatibility and low hemolysis. In vivo experiments show that PLA-HA-aPDT can promote C. auris-infected wound healing, reduce inflammatory response, and without obvious toxic side-effects. Further results reveal that PLA-HA-aPDT could increase endogenous reactive oxygen species (ROS) levels, leading to mitochondrial dysfunction, release of cytochrome C, activation of metacaspase, and nuclear fragmentation, thereby triggering apoptosis of C. auris. Compared with HA, PLA-HA shows stronger controllability and reusability, which can greatly improve the utilization efficiency of HA alone. Taken together, the efficacy, safety and antifungal activity make PLA-HA-aPDT a highly promising antifungal candidate for skin or mucous membrane C. auris infection.     

ClTI,a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds,exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.     

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.     

Diorcinol D Exerts Fungicidal Action against Candida albicans through Cytoplasm Membrane Destruction and ROS Accumulation

Candida albicans, which is the most common human fungal pathogen, causes high mortality among immunocompromised patients. Antifungal drug resistance becomes a major challenge for the management of Candida infection. Diorcinol D (DD), a diphenyl ether derivative isolated from an endolichenic fungus, exerted fungicidal action against Candida species. In this study, we investigated the possible mechanism of its antifungal activity. The change of membrane dynamics and permeability suggested that the cell membrane was disrupted by the treatment of DD. This was further supported by the evidences of intracellular glycerol accumulation, alteration of cell ultrastructure, and down-regulation of genes involved in cell membrane synthesis. In addition, the treatment of C. albicans with DD resulted in the elevation of reactive oxygen species (ROS), which caused the dysfunction of mitochondria. These altogether suggested that DD exerted its antifungal activity through cytoplasmic membrane destruction and ROS accumulation. This finding is helpful to uncover the underlying mechanisms for the diphenyl ether derivatives and provides a potential application in fighting clinical fungal infections.     

Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4 mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca²⁺) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca²⁺ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca²⁺ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.     

Epidemiology and Microbiologic Characterization of Nosocomial Candidemia from a Brazilian National Surveillance Program

Candidemia is a growing problem in hospitals all over the world. Despite advances in the medical support of critically ill patients, candidiasis leads to prolonged hospitalization, and has a crude mortality rate around 50%. We conducted a multicenter surveillance study in 16 hospitals distributed across five regions of Brazil to assess the incidence, species distribution, antifungal susceptibility, and risk factors for bloodstream infections due to Candida species. From June 2007 to March 2010, we studied a total of 2,563 nosocomial bloodstream infection (nBSI) episodes. Candida spp. was the 7^th most prevalent agent. Most of the patients were male, with a median age of 56 years. A total of 64 patients (46.7%) were in the ICU when candidemia occurred. Malignancies were the most common underlying condition (32%). The crude mortality rate of candidemia during the hospital admission was 72.2%. Non-albicans species of Candida accounted for 65.7% of the 137 yeast isolates. C. albicans (34.3%), Candida parapsilosis (24.1%), Candida tropicalis (15.3%) and Candida glabrata (10.2%) were the most prevalent species. Only 47 out of 137 Candida isolates were sent to the reference laboratory for antifungal susceptibility testing. All C. albicans, C. tropicalis and C. parapsilosis isolates were susceptible to the 5 antifungal drugs tested. Among 11 C. glabrata isolates, 36% were resistant to fluconazole, and 64% SDD. All of them were susceptible to anidulafungin and amphotericin B. We observed that C. glabrata is emerging as a major player among non-albicans Candida spp. and fluconazole resistance was primarily confined to C. glabrata and C. krusei strains. Candida resistance to echinocandins and amphotericin B remains rare in Brazil.Mortality rates remain increasingly higher than that observed in the Northern Hemisphere countries, emphasizing the need for improving local practices of clinical management of candidemia, including early diagnosis, source control and precise antifungal therapy.     

Calcium and oxidative stress mediate perillaldehyde-induced apoptosis in Candida albicans

New anti-Candida albicans drugs are needed due to the emergence of resistant cases in recent years. Perillaldehyde (PAE) is a natural monoterpenoid compound derived from Perilla frutescens. The minimum inhibitory concentration of PAE against C. albicans was 0.4 μL/mL. We aimed to elucidate the antifungal mode of action of PAE against C. albicans. The antifungal activity of PAE against C. albicans was found to correlate with an elevation in intracellular Ca²⁺ and accumulation of ROS. Several downstream apoptosis events such as the disruption of mitochondrial membrane potential, phosphatidylserine externalization, cytochrome c release, and metacaspase activation were observed in PAE-treated cells. DNA damage and nuclear fragmentation assays also revealed apoptosis of C. albicans cells. In summary, by means of fluorescent microscopy, flow cytometer analysis, and Western blot, our data uncovered that PAE exerts its antifungal activity through Ca²⁺ and oxidative stress-mediated apoptosis mechanisms. This study deciphered the mode of action of PAE, which will be useful in the design of improved antifungal therapies.

     

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C₆ to C₁₃ were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C₁₁) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C₁₀) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C₁₂) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H⁺-ATPase. Octanol (C₈) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C₉–C₁₂) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.     

Broad-spectrum antifungal-producing lactic acid bacteria and their application in fruit models

A large-scale screen of some 7,000 presumptive lactic acid bacteria (LAB), isolated from animal, human, or plant origin, identified 1,149 isolates with inhibitory activity against the food-spoilage mould Penicillium expansum. In excess of 500 LAB isolates were subsequently identified to produce a broad spectrum of activity against P. expansum, Penicillium digitatum, Penicillium notatum, Penicillium roqueforti, Rhizopus stolonifer, Fusarium culmorum, Aspergillus fumigatus and Rhodotorula mucilaginosa. Partial 16S rRNA sequencing of 94 broad spectrum isolates revealed that the majority of antifungal producers were strains of Lactobacillus plantarum. The remaining population was composed of Weissella confusa and Pediococcus pentosaceous isolates. Characterization of six selected broad-spectrum antifungal LAB isolates revealed that antifungal activity is maximal at a temperature of 30 °C, a pH of 4.0 and is stable across a variety of salt concentrations. The antifungal compound(s) was shown to be neither proteinaceous nor volatile in nature. P. pentosaceous 54 was shown to have protective properties against P. expansum spoilage when applied in pear, plum and grape models, therefore representing an excellent candidate for food-related applications.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.     

Actividad antifúngica in vitro de la micafungina

BackgroundMicafungin is a new and very useful pharmacological tool for the treatment of invasive mycoses with a wide antifungal spectrum for the most common pathogenic fungi. Micafungin is especially active against the genera Candida and Aspergillus. Its antifungal mechanism is based on the inhibition of the β-1,3- D-glucan synthesis, an essential molecule for the cell wall architecture, with different con sequences for Candida and Aspergillus, being micafungin fungicide for the former and fungistatic for the latter.AimTo describe the in vitro antifungal spectrum of micafungin based in the scientific and medical lite rature of recent years.MethodsWe have done a bibliographic retrieval using the scientific terms, “micafungin”, “activity”, “Candida”, “Aspergillus”, “fungi”, “mycos*”, “susceptibility”, in PubMed/Medline from the National Library of Medicine de EE.UU. from 2005 to 2009.ResultsWe can underline that most than 99% of Candida isolates are susceptible to ≤ 2 μg/ml of micafungin. MIC are very low (≤ 0.125 μg/ml) for most clinical isolates of the species Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei while Candida parapsilosis and Candida guilliermondii isolates are susceptible to anidulafungin concentrations ≤ 2 μg/ml. The activity of micafungin is excellent against those medical important species of Aspergillus. However, its activity is very low against Cryptococcus and the Zygomycetes.ConclusionsThe excellent activity of micafungin has made this antifungal a first line therapeutic indication for candidemia and invasive candidiasis in non-neutropenic patients.     

Reactive oxygen species modulate itraconazole-induced apoptosis via mitochondrial disruption in Candida albicans

Itraconazole (ITC), a well-known fungistatic agent, has potent fungicidal activity against Candida albicans. However, its mechanism of fungicidal activity has not been elucidated yet, and we aimed to identify the mechanism of ITC against C. albicans. ITC caused cell shrinkage via potassium leakage through the ion channel. Since shrunken cells could indicate apoptosis, we investigated apoptotic features. Annexin V-FITC and TUNEL assays indicated that fungicidal activity of ITC was involved in apoptosis. Subsequently, we confirmed an intracellular factor that could cause apoptosis. ITC treatment caused reactive oxygen species (ROS) accumulation. To confirm whether ROS is related with ITC-triggered cell death, cell viability was examined using the ROS scavenger N-acetylcysteine (NAC). NAC pretreatment recovered ITC-induced cell death, indicating that antifungal activity of ITC is associated with ROS, which is also confirmed by impaired glutathione-related antioxidant system and oxidized intracellular lipids. Moreover, ITC-induced mitochondrial dysfunction, in turn, triggered cytochrome c release and metacaspase activation, leading to apoptosis. Unlike the only ITC-treatment group, cells with NAC pretreatment did not show significant damage to mitochondria, and attenuated apoptotic features. Therefore, our results suggest that ITC induces apoptosis as fungicidal mechanism, and intracellular ROS is major factor to trigger the apoptosis by ITC in C. albicans.     

MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical,Veterinary and Environmental Strains,and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome.     

Virulence Factors and Anti Fungal Sensitivity Pattern of<Emphasis Type="Italic"> Candida</Emphasis> Sp. Isolated from HIV and TB Patients

The study comprised of 60 Candida spp., 50 isolates from HIV and TB positive individuals (immunocompromised) and 10 isolates from non-HIV and -TB patients (immunocompetent). Among the 60 Candidal isolates, 83.3% were identified as C. albicans, 11.6% as C. glabrata and rest 5% as C. krusei. There is no study in production pattern of extracellular enzymes of Candida spp. isolated from HIV and TB patients in comparison with non-HIV and -TB patients in India. The comparison of phospholipase activities showed that there was a significant difference between the groups at (P = 0.001). The non-HIV and -TB groups of C. glabrata and C. krusei did not show detectable phospholipase activity when compared to the HIV and TB groups. The mean difference in the phospholipase activities of these two groups was significant (P = <0.001). Candida spp. of both the groups do not possess the ability to hydrolyze gelatin. All the strains possessed the ability to show alpha haemolysis. Even though it had shown alpha haemolysis, the significant difference in haemolytic activity was observed only in C. albicans (P = <0.001). None of the isolates from the two groups possessed the ability to hydrolyze gelatin. In the resistance profile of Candida spp., C. albicans of HIV and TB groups had shown resistance to fluconazole, Itraconazole, ketaconazole, nystatin but showed 100% sensitivity towards amphotericin-B. The isolates of C. krusei and C. glabrata showed no resistance to any of the drugs tested. In the case of, non-HIV and -TB patients the resistance pattern was low.     

Quercetin-induced yeast apoptosis through mitochondrial dysfunction under the accumulation of magnesium in Candida albicans

Latterly, the upsurge in use of antifungal drugs has brought about the emergence of several drug-resistance strains, making it skeptical to continue relying on current therapeutic regime. In the necessity of resistance-free antifungal agent, flavonoids presented possibilities of replacing existing drugs, displaying antifungal activity against pathogenic fungi. Among them, quercetin, one of the most representative flavonoids, exhibited antifungal activity against Candida albicans. To inspect the further understanding regarding quercetin, the antifungal mode of action of quercetin was investigated. In the initial step, the apoptosis was monitored after quercetin treatment. Moreover, intracellular levels of Mg²⁺ was assessed and was determined that Mg²⁺ increase occurred under the influence of quercetin. In addition, several features of mitochondrial dysfunction were monitored. Mitochondrial dysfunction triggers decrease in mitochondrial redox levels and leads to disruption in mitochondrial antioxidant system. Increased intracellular ROS and decreased intracellular redox levels were also displayed, indicating the occurrence of overall disruption in antioxidant systems. Sequentially, DNA fragmentation was observed and this DNA damage in turn induces apoptosis. In analyses, hexaamminecobalt(III) chloride (Cohex) was applied to inhibit Mg²⁺ transport between cytosol and mitochondria. Cohex attenuated the effects induced by quercetin, which demonstrates that the presence of Mg²⁺ is essential in quercetin-induced apoptosis.     

Terpenoids with antifungal activity trigger mitochondrial dysfunction in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A number of pathogenic fungi like Candida, cannot survive upon damage to mitochondrial DNA (mtDNA) while the budding yeast can tolerate the damage therefore we chose Saccharomyces cerevisiae as a model system for this study. Since a number of potent antifungals have originated from various natural sources, we decided to use a triterpenoid and tetraterpenoid in this study as an antifungal agent. Our data clearly indicates that terpenoids play a role in diminishing the mitochondrial content which results in altered level of reactive oxygen species (ROS) and ATP generation. Here, we report that triterpenoid and tetraterpenoid display MIC at 100 and 120 μg /mL respectively against S. cerevisiae. At MIC dose triterpenoid (Lupeol) treated cells showed relatively higher mitochondrial dysfunction as compared to tetraterpenoid, resulting high level of ROS generation in triterpenoid in comparison to tetraterpenoid treated cells. Whereas the ATP level decreases in triterpenoid treated cells while it remains same in tetraterpenoid treated cells. Hence triterpenoid showed more potent antifungal activity as compared to the tetraterpenoid at their MIC by targeting mitochondrial integrity. The outcome of the study is to decipher the mode of action of terpenoids which will be useful in designing of improved antifungal therapies and also accelerate the development of translational applications.     

Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures

Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.