Controlling the Interaction between Fluorescent Gold Nanoclusters and Biointerfaces for Rapid Discrimination of Fungal Pathogens.

Nondestructive detection and discrimination of fungal pathogens is essential for rapid and precise treatment, which further effectively prevents antifungal resistance from overused drugs. In this work, fluorescent gold nanoclusters served as the basis for discriminating Candida species. Varied on surface ligands, these gold nanoclusters demonstrated different optical properties as a result of the perturbation effects of ligands. The biointerface interaction between the surface ligands of gold nanoclusters and the cell walls of Candida species can be constructed, and their restriction on ligands perturbation effect produced enhanced fluorescence signals. Owing to the variation of the cell wall composition, cells of different Candida species demonstrated different degrees of association with the gold nanoclusters, leading to discriminable amounts of fluorescence enhancements. The reverse signal response from these gold nanoclusters gives rise to a synergistic and effective assay that allows identification of Candida species.

Predicting microbial growth dynamics in response to nutrient availability.

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker's yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed.

Candida kefyr in Kuwait: Prevalence, antifungal drug susceptibility and genotypic heterogeneity.

OBJECTIVE: Candida kefyr causes invasive candidiasis in immunocompromised patients, particularly among those with oncohematological diseases. This study determined the prevalence of C. kefyr among yeast isolates collected during 2011-2018 in Kuwait. Antifungal susceptibility testing (AST) and genotypic heterogeneity among C. kefyr was also studied. METHODS: Clinical C. kefyr isolates recovered from bloodstream and other specimens during 2011 to 2018 were retrospectively analyzed. All C. kefyr isolates were identified by CHROMagar Candida, Vitek2 and PCR amplification of rDNA. AST was performed by Etest. Molecular basis of resistance to fluconazole and echinocandins was studied by PCR-sequencing of ERG11 and FKS1, respectively. Genotypic heterogeneity was determined with microsatellite-/minisatellite-based primers and for 27 selected isolates by PCR-sequencing of IGS1 region of rDNA. RESULTS: Among 8257 yeast strains, 69 C. kefyr (including four bloodstream) isolates were detected by phenotypic and molecular methods. Isolation from urine and respiratory samples from female and male patients was significantly different (P = 0.001). Four isolates showed reduced susceptibility to amphotericin B and one isolate to all (amphotericin B, fluconazole, voriconazole and caspofungin/micafungin) antifungals tested. Fluconazole-resistant isolate contained only synonymous mutations in ERG11. Echinocandin-resistant isolate contained wild-type hotspot-1 and hotspot-2 of FKS1. Fingerprinting with microsatellite-/minisatellite-based primers identified only three types. IGS1 sequencing identified seven haplotypes among 27 selected isolates. CONCLUSIONS: The overall prevalence of C. kefyr among clinical yeast isolates and among candidemia cases was recorded as 0.83% and 0.32%, respectively. The frequency of isolation of C. kefyr from bloodstream and other invasive samples was stable during the study period. The C. kefyr isolates grown from invasive (bloodstream, bronchoalveolar lavage, abdominal drain fluid, peritonial fluid and gastric fluid) samples and amphotericin B-resistant isolates were genotypically heterogeneous strains.

Transforming Candida hispaniensis, a promising oleaginous and flavogenic yeast.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity.

Translation Inhibition by Rocaglates Activates a Species-Specific Cell Death Program in the Emerging Fungal Pathogen Candida auris.

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen.IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.

Candida auris and Nosocomial Infection.

The existence of the multi-drug resistant (MDR) pathogenic fungus, Candida auris came to light in 2009. This particular organism is capable of causing nosocomial infections in immunecompromised persons. This pathogen is associated with consistent candidemia with high mortality rate and presents a serious global health threat. Whole genome sequence (WGS) investigation detected powerful phylogeographic Candida auris genotypes which are specialized to particular geological areas indicating dissemination of particular genotype among provinces. Furthermore, this organism frequently exhibits multidrug-resistance and displays an unusual sensitivity profile. Identification techniques that are commercialized to test Candida auris often show inconsistent results and this misidentification leads to treatment failure which complicates the management of candidiasis. Till date, Candida auris has been progressively recorded from several countries and therefore its preventive control measures are paramount to interrupt its transmission. In this review, we discussed prevalence, biology, drug-resistance phenomena, virulence factors and management of Candida auris infections.

Arginolipid: A membrane-active antifungal agent and its synergistic potential to combat drug resistance in clinical Candida isolates.

Antifungal drug resistance exhibits a major clinical challenge for treating nosocomial fungal infections. To find a possible solution, we synthesized and studied the antifungal activities of three different arginolipids (Nα -acyl-arginine ethyl ester) against clinical drug-resistant isolates of Candida. The most active arginolipid, oleoyl arginine ethyl ester (OAEE) consisting of a long unsaturated hydrophobic chain, was tested for its mode of action, which revealed that it altered ergosterol biosynthesis and compromised the fungal cell membrane. Also, OAEE was found to exhibit synergistic interactions with fluconazole (FLU) or amphotericin B (AmB) against planktonic Candida cells, wherein it reduced the inhibitory concentrations of these drugs to their in vitro susceptible range. Studies conducted against the C. tropicalis biofilm revealed that the OAEE+AmB combination synergistically reduced the metabolic activity and hyphal density in biofilms, whereas OAEE+FLU was found to be additive against most cases. Finally, the evaluated selective toxicity of OAEE toward fungal cells over mammalian cells could establish it as an alternative treatment for combating drug-resistant Candida infections.

Excision of endosymbiotic bacteria from yeast under aging and starvation stresses.

Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners.

Validation of methylene blue viability staining with the emerging pathogen Candida auris.

Methylene blue viability staining has been traditionally used to assess viability of Saccharomyces cerevisiae in brewing and wine making. Here, this method was tested and validated with the emerging fungal pathogen Candida auris to determine if this species would also deferentially stain, which could provide utility in assessing microbial control and disinfectant efficacy.

The Pitfall of Utilizing a Commercial Biochemical Yeast Identification Kit to Detect Candida auris.

Candida auris is an emerging pathogenic yeast responsible for nosocomial infections with high mortality, on a global scale. A 65-year-old woman with hypovolemic shock and severe metabolic acidosis was intubated and admitted to the intensive care unit (ICU). Shortly after admission, she developed ventilator-associated pneumonia caused by multidrug-resistant Acinetobacter baumannii, which necessitated treatment with high-dose ampicillin-sulbactam. Two weeks later, a yeast was cultured from her blood. It formed pale pink colonies on CHROMagar Candida medium and produced predominantly oval budding yeast cells with the occasional rudimentary pseudohyphae on cornmeal agar. ID 32 C identified the yeast as Candida sake However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and sequencing of the D1/D2 region of the 28S rRNA gene identified the yeast as C. auris.

Anticandidal activity of biosynthesized silver nanoparticles: effect on growth, cell morphology, and key virulence attributes of Candida species.

Purpose: The pathogenicity in Candida spp was attributed by several virulence factors such as production of tissue damaging extracellular enzymes, germ tube formation, hyphal morphogenesis and establishment of drug resistant biofilm. The objective of present study was to investigate the effects of silver nanoparticles (AgNPs) on growth, cell morphology and key virulence attributes of Candida species. Methods: AgNPs were synthesized by the using seed extract of Syzygium cumini (Sc), and were characterized by UV-Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM). ScAgNPs were used to evaluate their antifungal and antibacterial activity as well as their potent inhibitory effects on germ tube and biofilm formation and extracellular enzymes viz. phospholipases, proteinases, lipases and hemolysin secreted by Candida spp. Results: The MICs values of ScAgNPs were ranged from 0.125-0.250 mg/ml, whereas the MBCs and MFCs were 0.250 and 0.500 mg/ml, respectively. ScAgNPs significantly inhibit the production of phospholipases by 82.2, 75.7, 78.7, 62.5, and 65.8%; proteinases by 82.0, 72.0, 77.5, 67.0, and 83.7%; lipase by 69.4, 58.8, 60.0, 42.9, and 65.0%; and hemolysin by 62.8, 69.7, 67.2, 73.1, and 70.2% in C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis and C. krusei, respectively, at 500 µg/ml. ScAgNPs inhibit germ tube formation in C. albicans up to 97.1% at 0.25 mg/ml. LIVE/DEAD staining results showed that ScAgNPs almost completely inhibit biofilm formation in C. albicans. TEM analysis shows that ScAgNPs not only anchored onto the cell surface but also penetrated and accumulated in the cytoplasm that causes severe damage to the cell wall and cytoplasmic membrane. Conclusion: To summarize, the biosynthesized ScAgNPs strongly suppressed the multiplication, germ tube and biofilm formation and most importantly secretion of hydrolytic enzymes (viz. phospholipases, proteinases, lipases and hemolysin) by Candia spp. The present research work open several avenues of further study, such as to explore the molecular mechanism of inhibition of germ tubes and biofilm formation and suppression of production of various hydrolytic enzymes by Candida spp.

Antifungal activity, mode of action variability, and subcellular distribution of coumarin-based antifungal azoles.

Azole antifungals inhibit the biosynthesis of ergosterol, the fungal equivalent of cholesterol in mammalian cells. Here we report an investigation of the activity of coumarin-substituted azole antifungals. Screening against a panel of Candida pathogens, including a mutant lacking CYP51, the target of antifungal azoles, revealed that this enzyme is inhibited by triazole-based antifungals, whereas imidazole-based derivatives have more than one mode of action. The imidazole-bearing antifungals more effectively reduced trailing growth associated with persistence and/or recurrence of fungal infections than triazole-based derivatives. The imidazole derivatives were more toxic to mammalian cells and more potently inhibited the activity of CYP3A4, which is one of the main causes of azole toxicity. Using live cell imaging, we showed that regardless of the type of azole ring fluorescent 7-diethylaminocoumarin-based azoles localized to the endoplasmic reticulum, the organelle that harbors CYP51. This study suggests that the coumarin is a promising scaffold for development of novel azole-based antifungals that effectively localize to the fungal cell endoplasmic reticulum.

Automated "pick and transfer" of targeted cells using dielectrophoresis.

Selective manipulation of single cells is an important step in sample preparation for biological analysis. A highly specific and automated device is desired for such an operation. An ideal device would be able to selectively pick several single cells in parallel from a heterogeneous population and transfer those to designated sites for further analysis without human intervention. The robotic manipulator developed here provides the basis for development of such a device. The device in this work is designed to selectively pick cells based on their inherent properties using dielectrophoresis (DEP) and automatically transfer and release those at a transfer site. Here we provide proof of concept of such a device and study the effect of different parameters on its operation. Successful experiments were conducted to separate Candida cells from a mixture with 10 µm latex particles and a viability assay was performed for separation of viable rat adipose stem cells (RASCs) from non-viable ones. The robotic DEP device was further used to pick and transfer single RASCs. This work also discusses the advantages and disadvantages of our current setup and illustrates the future steps required to improve the performance of this robotic DEP technology.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

Synthesis and immunological studies of ß-1,2-mannan-peptide conjugates as antifungal vaccines.

Fungal cell surface carbohydrates and proteins are useful antigens for the development of antifungal vaccines. In this study, glycopeptides consisting of the ß-1,2-mannan and N-terminal peptide epitopes of Candida albicans (C. albicans) cell wall phosphomannan complex and Als1p (rAls1p-N) protein, respectively, were synthesized and covalently conjugated with keyhole limpet hemocyanin (KLH) and human serum albumin (HSA) through homobifunctional disuccinimidyl glutarate. The resultant KLH-conjugates were immunologically evaluated using Balb/c mice to reveal that they induced high levels of IgG antibodies. Furthermore, these conjugates showed self-adjuvanting property, as they could promote robust antibody responses without the presence of an external adjuvant. More significantly, the obtained antisera could effectively recognize both the carbohydrate and the Als1 peptide epitopes and immunofluorescence and flow cytometry assays also demonstrated that the elicited antibodies could react with the cell surface of a number of fungi, including C. albicans, C. tropicalis, C. lustaniae and C. glabrata. These results suggested the great potential of these conjugates as antifungal vaccines.

Copolymeric micelles as efficient inert nanocarrier for hypericin in the photodynamic inactivation of Candida species.

Aim: To evaluate the efficacy of photodynamic inactivation (PDI) mediated by hypericin encapsulated in P-123 copolymeric micelles (P123-Hyp) alone and in combination with fluconazole (FLU) against planktonic cells and biofilm formation of Candida species Materials & methods: PDI was performed using P123-Hyp and an LED device with irradiance of 3.0 mW/cm2 . Results: Most of isolates (70%) were completely inhibited with concentrations up to 2.0 µmol/l of HYP and light fluence of 16.2 J/cm2. FLU-resistant strains had synergic effect with P123-HYP-PDI and FLU. The biofilm formation was inhibited in all species, in additional the changes in Candida morphology observed by scanning electron microscopy. Conclusion: P123-Hyp-PDI is a promising option to treat fungal infections and medical devices to prevent biofilm formation and fungal spread.

Lipidomics Approaches: Applied to the Study of Pathogenesis in Candida Species.

High rate of reported cases of infections in humans caused by fungal pathogens pose serious concern. Potentially these commensal fungi remain harmless to the healthy individuals but can cause severe systemic infection in patients with compromised immune system. Effective drug remedies against these infections are rather limited. Moreover, frequently encountered multidrug resistance poses an additional challenge to search for alternate and novel targets. Notably, imbalances in lipid homeostasis which impact drug susceptibility of Candida albicans cells do provide clues of novel therapeutic strategies. Sphingolipids (SPHs) are unique components of Candida cells, hence are actively exploited as potential drug targets. In addition, recent research has uncovered that several SPH intermediates and of other lipids as well, govern cell signaling and virulence of C. albicans. In this chapter, we highlight the role of lipids in the physiology of Candida, particularly focusing on their roles in the development of drug resistance. Considering the importance of lipids, the article also highlights recent high-throughput analytical tools and methodologies, which are being employed in our understanding of structures, biosynthesis, and roles of lipids in fungal pathogens.

Improved smallest peptides based on positive charge increase of the γ-core motif from PνD1 and their mechanism of action against Candida species.

BACKGROUND: Plant defensins have a hallmark γ-core motif (GXCX3-9C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the PvD1 defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity. METHODS: We made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected PvD1 γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ33-41 PvD1 ++ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties. RESULTS: The γ33-41 PvD1 ++ peptide caused the most toxic effects in the yeast Candida buinensis, leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis. CONCLUSION: We observed that the antifungal activity of PvD1 is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity.

Cell wall mannan of Candida krusei mediates dendritic cell apoptosis and orchestrates Th17 polarization via TLR-2/MyD88-dependent pathway.

Dendritic cells (DCs) abundantly express diverse receptors to recognize mannans in the outer surface of Candida cell wall, and these interactions dictate the host immune responses that determine disease outcomes. C. krusei prevalence in candidiasis worldwide has increased since this pathogen has developed multidrug resistance. However, little is known how the immune system responds to C. krusei. Particularly, the molecular mechanisms of the interplay between C. krusei mannan and DCs remain to be elucidated. We investigated how C. krusei mannan affected DC responses in comparison to C. albicans, C. tropicalis and C. glabrata mannan. Our results showed that only C. krusei mannan induced massive cytokine responses in DCs, and led to apoptosis. Although C. krusei mannan-activated DCs underwent apoptosis, they were still capable of initiating Th17 response. C. krusei mannan-mediated DC apoptosis was obligated to the TLR2 and MyD88 pathway. These pathways also controlled Th1/Th17 switching possibly by virtue of the production of the polarizing cytokines IL-12 and IL-6 by the C. krusei mannan activated-DCs. Our study suggests that TLR2 and MyD88 pathway in DCs are dominant for C. krusei mannan recognition, which differs from the previous reports showing a crucial role of C-type lectin receptors in Candida mannan sensing.

The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.