Natural Substances as Valuable Alternative for Improving Conventional Antifungal Chemotherapy: Lights and Shadows.

Fungi are eukaryotic organisms with relatively few pathogenic members dangerous for humans, usually acting as opportunistic infections. In the last decades, several life-threatening fungal infections have risen mostly associated with the worldwide extension of chronic diseases and immunosuppression. The available antifungal therapies cannot combat this challenge because the arsenal of compounds is scarce and displays low selective action, significant adverse effects, and increasing resistance. A growing isolation of outbreaks triggered by fungal species formerly considered innocuous is being recorded. From ancient times, natural substances harvested from plants have been applied to folk medicine and some of them recently emerged as promising antifungals. The most used are briefly revised herein. Combinations of chemotherapeutic drugs with natural products to obtain more efficient and gentle treatments are also revised. Nevertheless, considerable research work is still necessary before their clinical use can be generally accepted. Many natural products have a highly complex chemical composition, with the active principles still partially unknown. Here, we survey the field underlying lights and shadows of both groups. More studies involving clinical strains are necessary, but we illustrate this matter by discussing the potential clinical applications of combined carnosic acid plus propolis formulations.

Insight into the Antifungal Effects of Propolis and Carnosic Acid-Extension to the Pathogenic Yeast Candida glabrata: New Propolis Fractionation and Potential Synergistic Applications.

Fungi have traditionally been considered opportunistic pathogens in primary infections caused by virulent bacteria, protozoan, or viruses. Consequently, antimycotic chemotherapy is clearly less developed in comparison to its bacterial counterpart. Currently, the three main families of antifungals (polyenes, echinocandins, and azoles) are not sufficient to control the enormous increase in life-threatening fungal infections recorded in recent decades. Natural substances harvested from plants have traditionally been utilized as a successful alternative. After a wide screening of natural agents, we have recently obtained promising results with distinct formulations of carnosic acid and propolis on the prevalent fungal pathogens Candida albicans and Cryptococcus neoformans. Here, we extended their use to the treatment against the emerging pathogenic yeast Candida glabrata, which displayed lower susceptibility in comparison to the fungi mentioned above. Taking into account the moderate antifungal activity of both natural agents, the antifungal value of these combinations has been improved through the obtention of the hydroethanolic fractions of propolis. In addition, we have demonstrated the potential clinical application of new therapeutical designs based on sequential pre-treatments with carnosic/propolis mixtures, followed by exposure to amphotericin B. This approach increased the toxic effect induced by this polyene.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Lack of Functional Trehalase Activity in Candida parapsilosis Increases Susceptibility to Itraconazole.

Central metabolic pathways may play a major role in the virulence of pathogenic fungi. Here, we have investigated the susceptibility of a Candida parapsilosis mutant deficient in trehalase activity (atc1Δ/ntc1Δ strain) to the azolic compounds fluconazole and itraconazole. A time-course exposure to itraconazole but not fluconazole induced a significant degree of cell killing in mutant cells compared to the parental strain. Flow cytometry determinations indicated that itraconazole was able to induce a marked production of endogenous ROS together with a simultaneous increase in membrane potential, these effects being irrelevant after fluconazole addition. Furthermore, only itraconazole induced a significant synthesis of endogenous trehalose. The recorded impaired capacity of mutant cells to produce structured biofilms was further increased in the presence of both azoles, with itraconazole being more effective than fluconazole. Our results in the opportunistic pathogen yeast C. parapsilosis reinforce the study of trehalose metabolism as an attractive therapeutic target and allow extending the hypothesis that the generation of internal oxidative stress may be a component of the antifungal action exerted by the compounds currently available in medical practice.

A Specific Mixture of Propolis and Carnosic Acid Triggers a Strong Fungicidal Action against Cryptococcus neoformans.

Current antifungal chemotherapy against the prevalent basidiomycete Cryptococcus neoformans displays some drawbacks. This pathogenic fungus is refractory to echinocandins, whereas conventional treatment with amphotericin B plus 5-fluorocytosine has a limited efficacy. In this study, we explored the potential cryptococcal activity of some natural agents. After conducting a screening test with a set of propolis from different geographical areas, we selected an extract from China, which displayed a certain cytotoxic activity against C. neoformans, due to this extract being cheap and easily available in large amounts. The combination of this kind of propolis with carnosic acid in a 1:4 ratio induced a stronger fungicidal effect, which occurred following a synergistic pattern, without visible alterations in external cell morphology. Furthermore, several carnosic acid-propolis formulations applied onto preformed biofilms decreased the metabolic activity of the sessile cells forming biofilms. These data support the potential application of mixtures containing these two natural extracts in the design of new antifungal strategies in order to combat opportunistic infections caused by prevalent pathogenic fungi.

Trehalase inhibition by validamycin A may be a promising target to design new fungicides and insecticides.

The introduction of insecticides and fungicides in agriculture has improved crop yields and, consequently, the quality of life for many people, especially in what is widely considered as the 'first world'. However, the indiscriminate use of dangerous chemical insecticides has led to pest resistance, human and animal poisoning and environmental pollution. Biochemical and genetic evidence concludes that the non-reducing disaccharide trehalose plays an essential role in the pathobiology of many insects and fungi. Both organisms share identical pathway for trehalose biosynthesis (the TPS/TPP pathway), while a high degree of homology in their trehalose hydrolysis capacity (trehalase activities) has also been demonstrated. In the search for new, effective and environmentally sustainable compounds, a set of trehalase inhibitors has emerged as a potentially interesting antifungal and insecticidal target. In particular, the trehalose analogue, Validamycin A, which has a strong inhibitory effect on several trehalases, has been successfully introduced for the treatment of various diseases caused by insects and fungi. Herein, we review the main features of the specific interaction between Validamycin A and trehalase as well as the expected advantages of the applications based on trehalase inhibition as insecticides and fungicides. © 2021 Society of Chemical Industry.

The Fungicidal Action of Micafungin is Independent on Both Oxidative Stress Generation and HOG Pathway Signaling in Candida albicans.

In fungi, the Mitogen-Activated Protein kinase (MAPK) pathways sense a wide variety of environmental stimuli, leading to cell adaptation and survival. The HOG pathway plays an essential role in the pathobiology of Candida albicans, including the colonization of the gastrointestinal tract in a mouse model, virulence, and response to stress. Here, we examined the role of Hog1 in the C. albicans response to the clinically relevant antifungal Micafungin (MF), whose minimum inhibitory concentration (MIC) was identical in the parental strain (RM100) and in the isogenic homozygous mutant hog1 (0.016 mg/L). The cell viability was impaired without significant differences between the parental strain, the isogenic hog1 mutant, and the Hog1+ reintegrant. This phenotype was quite similar in a collection of hog1 mutants constructed in a different C. albicans background. MF-treated cells failed to induce a relevant increase of both reactive oxygen species (ROS) formation and activation of the mitochondrial membrane potential in parental and hog1 cells. MF was also unable to trigger any significant activation of the genes coding for the antioxidant activities catalase (CAT1) and superoxide dismutase (SOD2), as well as on the corresponding enzymatic activities, whereas a clear induction was observed in the presence of Amphotericin B (AMB), introduced as a positive control of Hog1 signaling. Furthermore, Hog1 was not phosphorylated by the addition of MF, but, notably, this echinocandin caused Mkc1 phosphorylation. Our results strongly suggest that the toxic effect of MF on C. albicans cells is not mediated by the Hog1 MAPK and is independent of the generation of an internal oxidative stress in C. albicans.

Trends in Microbiology publications: are classic scientific journals condemned to extinction?

Scientific journals have played an essential role in the diffusion of research breakthroughs. For many years there was no competition between journals, but, in recent decades they have become categorized by a careful assessment of their published contents based on several metric parameters. Of greater note, the 'prestige index' has become an essential tool used by public and private institutions to develop their scientific policy. Thus, the evaluation of research staffs, the concession of grants or fellowships and even the scholarly reputation and academic positions are mainly founded on a given journal's 'quality'. As a consequence, the prestige of some journals has gone up, based on the assumption that they publish cutting-edge science, while the reputation of others has gone down. Within the field of Microbiology, we have carried out a direct analysis by monitoring several representative classic journals according to customary metric parameters over 20 years. This analysis also covers another set of journals of recent appearance (novel journals). Although a direct comparison between both groups is not possible, this approach serves to perceive the trends of publication among microbiologists. Our preliminary conclusion is that the continued existence of many so-termed classic journals devoted to Microbiology is seriously threatened.

Novel Bi-Factorial Strategy against Candida albicans Viability Using Carnosic Acid and Propolis: Synergistic Antifungal Action.

The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees' panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C. albicans strain caused a moderate degree of cell death at relatively high concentrations. However, the combination of both extracts, especially in a 1:4 ratio, induced a potent synergistic pattern, leading to a drastic reduction in cell survival even at much lower concentrations. The result of a mathematical analysis by isobologram was consistent with synergistic action of the combined extracts rather than a merely additive effect. In turn, the capacity of SC5314 cells to form in vitro biofilms was also impaired by the simultaneous presence of both agents, supporting the potential application of carnosic acid and propolis mixtures in the prevention and treatment of clinical infections as an alternative to antibiotics and other antifungal agents endowed with reduced toxic side effects.

The MAPK Hog1 mediates the response to amphotericin B in Candida albicans.

The HOG MAP kinase pathway plays a crucial role in the response to different stresses in the opportunistic pathogen Candida albicans. The polyene amphotericin B (AMB) has been reported to trigger oxidative stress in several pathogenic fungi, including C. albicans. In the present work, we have analyzed the role of the MAPK Hog1 in sensing and survival to AMB treatment. Mutants lacking Hog1 are more susceptible to AMB than their parental strains and Hog1 became phosphorylated in the presence of this polyene. A set of mutated versions of Hog1 revealed that both the kinase activity and phosphorylation of Hog1 are required to cope with AMB treatment. Flow cytometry analysis showed that AMB induced intracellular ROS accumulation in both parental and hog1 null mutant strains. In addition, AMB triggered a Hog1-independent synthesis of trehalose. The addition of rotenone to AMB-treated cells improved cell viability, decreased intracellular ROS and prevented intracellular trehalose accumulation, suggesting that AMB-induced ROS is associated to a functional electron transport chain but the presence of rotenone did not impair Hog1 phosphorylation in AMB-treated cells. Our results indicate that Hog1 is necessary during AMB treatment to increase its survival.

Sensitivity of the Candida albicans trehalose-deficient mutants tps1Δ and tps2Δ to amphotericin B and micafungin.

Purpose. Fungal infections have increased in recent decades, with Candida albicans being the fourth most common aetiological agent of nosocomial infections. Disaccharide trehalose has been proposed as a target for the development of new antifungals. In C. albicans we have examined the susceptibility shown by two mutants deficient in trehalose biosynthesis, namely tps1Δ and tps2Δ, to amphotericin B (AmB) and micafungin (MF).Methodology. Minimum inhibitory concentrations (MICs) were calculated according to the Clinical and Laboratory Standards Institute (CLSI) criteria. Cell viability was assessed by cell counting. Intracellular reactive oxygen species (ROS) and the mitochondrial membrane potential were measured by flow cytometry, while the trehalose content and biofilm formation were determined by enzymatic assays.Results. While the tps1Δ mutant was highly sensitive to AmB exposure, its resistance to MF was similar to that of the wild-type. Notably, the opposite phenotype was recorded in the tps2Δ mutant. In turn, MF induced a significant level of endogenous ROS production in the parental SC5314 and tps2Δ cells, whereas the ROS formation in tps1Δ cells was virtually undetectable. The level of endogenous ROS correlated positively with the rise in mitochondrial activity. Only AmB was able to promote intracellular synthesis of trehalose in the parental strain; it was absent from tps1Δ cells and showed low levels in tps2Δ, confirming the unspecific dephosphorylation of trehalose-6P in C. albicans. Furthermore, the capacity of both tps1Δ and tps2Δ mutants to form biofilms was drastically reduced after AmB exposure, whereas it increased in tps1Δ cells treated with MF.Conclusion. Our data lend weight to the idea of using trehalose biosynthesis as a potential target for antifungal therapy.

The impact factor: implications for research policy, editorial rules and scholarly reputation.

The assessment of scientific research is essentially based on several metric parameters, the so-termed Impact Factor perhaps being the predominant one. Despite well-founded criticisms and the wide opposition of reputed scientists, this procedure has become a tool of scientific policy, and is applied in editorial procedures for scientific publication, the evaluation of research groups, the concession of grants, fellowships or even academic positions. Indeed, cutting-edge research is today a competitive and exigent task, where the legitimacy and restrictions of such metric factors remain a preoccupation. However, whatever the policy of evaluation implemented, most breakthroughs are revolutionary, and involve a change in a given paradigm, usually being made by unorthodox scientists, whose scholarly reputation may be questioned by the establishment, and who may often be excluded as a result of the current system of highly productive research.

Micafungin Enhances the Human Macrophage Response to Candida albicans through ß-Glucan Exposure.

Micafungin belongs to the antifungal family of echinocandins, which act as noncompetitive inhibitors of the fungal cell wall ß-1,3-d-glucan synthase. Since Candida albicans is the most prevalent pathogenic fungus in humans, we study the involvement of micafungin in the modulation of the inflammatory response developed by human tissue macrophages against C. albicans The MIC for micafungin was 0.016 µg/ml on the C. albicans SC5314 standard strain. Micafungin induced a drastic reduction in the number of exponential SC5314 viable cells, with the fungicidal effect being dependent on the cellular metabolic activity. Notably, micafungin also caused a structural remodelling of the cell wall, leading to exposure of the ß-glucan and chitin content on the external surface. At the higher doses used (0.05 µg/ml), the antifungal also induced the blowing up of budding yeasts. In addition, preincubation with micafungin before exposure to human tissue macrophages enhanced the secretion of tumor necrosis factor alpha (TNF-α), interleukin-17A (IL-17A), and IL-10 cytokines. Our results strongly suggest that in C. albicans treatment with micafungin, in addition to having the expected toxic antifungal effect, it potentiates the immune response, improving the interaction and activation of human macrophages, probably through the unmasking of ß-glucans on the cell wall surface.

Arsenic inorganic compounds cause oxidative stress mediated by the transcription factor PHO4 in Candida albicans.

Arsenic is a toxic metalloid widespread in nature. Recently, it has been demonstrated a main role of the transcription factor Pho4 in the acquisition of tolerance to arsenic-derived compounds, arsenite and arsenate in Candida albicans. Here, the effect of these compounds on this pathogenic yeast has been analyzed. In wild type cells, both arsenite and arsenate induced a marked increase in the endogenous production of Reactive Oxygen Species (ROS), together with the accumulation of intracellular trehalose and the activation of catalase, suggesting their role as generators of oxidative stress in this yeast. However, a pho4 null mutant showed a minor increase of intracellular ROS and a different kinetics of catalase activation upon exposure to arsenite and arsenate. Interestingly, the enzymatic activity of glutathione reductase and superoxide dismutase were exclusively triggered by arsenite but not by arsenate. pho4 mutant cells were also found to be sensitive to azide but significantly resistant to arsenate through a process dependent on an active electron transport chain and the alternative oxidase system. Therefore, arsenic-derived compounds induce a strong antioxidant response in C.albicans via different mechanisms.

ROS formation is a differential contributory factor to the fungicidal action of Amphotericin B and Micafungin in Candida albicans.

The hypothetical role played by the intracellular formation of reactive oxygen species (ROS) in the fungicidal action carried out by Amphotericin B (AmB) and Micafungin (MF) was examined in Candida albicans, which remains the most prevalent fungal pathogen. The clinical MICs for MF and AmB were 0.016 and 0.12µg/ml, respectively. Whereas AmB (0.5-1.0×MIC) induced a marked production of intracellular ROS accompanied by a high degree of cell killing in the C. albicans SC5314 strain, the fungicidal effect of MF was still operative, but ROS generation was slight. Preincubation with thiourea suppressed the formation of ROS and caused a marked increase in cell viability, regardless of the antifungal used. Simultaneous measurement of several well established antioxidant enzymes (catalase, glutathione reductase and superoxide dismutase) revealed strong AmB-induced activation of the three enzymatic activities, whereas MF only had a weak stimulating effect. Likewise, AmB but not MF promoted a conspicuous rise in the mitochondrial membrane potential together with the intracellular synthesis of trehalose, the non-reducing disaccharide which acts as a specific protector against oxidative stress in C. albicans. Optical and electronic microscopy analysis revealed a significant damage to cell integrity and structural alterations caused by both antifungals. Taken together, our results strongly suggest that the induction of an internal oxidative stress in C. albicans through the accumulation of ROS is a preferential contributory factor to the antifungal action of a widely used polyene (AmB) but not of MF (echinocandin).

Homozygous deletion of ATC1 and NTC1 genes in Candida parapsilosis abolishes trehalase activity and affects cell growth, sugar metabolism, stress resistance, infectivity and biofilm formation.

A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further CpNTC1/NTC1 disruption. Furthermore, Cpatc1Δ/ntc1Δ KO cells had difficulty in resuming active growth in fresh YPD. This homozygous mutant also lacked any in vitro measurable trehalase activity, whether acid or neutral, suggesting that a single gene codes for each enzyme. By contrast, in Cpatc1Δ/ntc1Δ KO strain the resistance to oxidative and heat stress displayed by atc1Δ mutant was suppressed. Cpatc1Δ/ntc1Δ KO cells showed a significant decrease in virulence as well as in the capacity to form biofilms. These results point to a major role for acid trehalase (Atc1p) in the pathobiology of C. parapsilosis, whereas the activity of neutral trehalase can only partially counteract Atc1p deficiency. They also support the use of ATC1 and NTC1 genes as interesting antifungal targets.