Antifungal Tetrahydrocarbazole Compound CAR-8 Induces Endoplasmic Reticulum Stress in Candida albicans.

The development of new effective antifungal agents is essential to combat fungal infections. Tetrahydrocarbazole has been exploited as a promising skeleton against various pathogenic microorganisms and is used to search for novel active antifungal compounds. In this study, a library composed of small tetrahydrocarbazole compounds was screened, and a potent antifungal agent, CAR-8, was identified with a minimum inhibitory concentration of 2-4 µg/mL against Candida albicans. CAR-8 showed strong fungicidal activities and killed almost all C. albicans within 3 h at a concentration of 16 µg/mL. At concentrations of 2 and 8 µg/mL, CAR-8 significantly inhibited the formation of hyphae and biofilms. Moreover, CAR-8 at 10 and 20 mg/kg reduced the fungal load and improved the survival in the C. albicans infection model in the invertebrate Galleria mellonella. Transcriptome analysis revealed significant changes in the expression of genes associated with protein processing in the endoplasmic reticulum (ER), ER-associated degradation, and unfolded protein response (UPR), which suggested that CAR-8 treatment induced ER stress. Moreover, CAR-8 treatment resulted in various phenotypes similar to tunicamycin, a classical ER stress inducer. These included nonconventional splicing of HAC1 mRNA, the fragmented morphology of ER, the distribution changes of GFP-Snc1 in Saccharomyces cerevisiae, and cell apoptosis probably caused by ER stress. More importantly, the disruption of IRE1 or HAC1 increased the sensitivity of C. albicans to CAR-8, confirming that the UPR signaling pathway was critical for CAR-8 resistance. Overall, our study identifies a potent ER stress-induced antifungal compound that will help the discovery of new antifungal drugs.

Sodium Houttuyfonate Ameliorates DSS-induced Colitis Aggravated by Candida albicans through Dectin-1/NF-κB/miR-32-5p/NFKBIZ Axis Based on Intestinal microRNA Profiling.

Our previous research indicated that Sodium houttuyfonate (SH) can effectively ameliorate dextran sulfate sodium (DSS)-induced colitis exacerbated by Candida albicans. However, the underlying protective mechanism of SH remains unclear. Therefore, in this study, a mice colitis model was infected with C. albicans, and the total colonic miRNAs were assessed. Furthermore, the differentially expressed miRNAs were enriched, clustered, and analyzed. Moreover, based on the dual luciferase analysis of NFKBIZ modulation by miR-32-5p, the in vitro and in vivo therapeutic effects of SH on inflammatory response, fungal burden, oxidative stress, and apoptosis were assessed at transcriptional and translational levels in the presence of agonist and antagonist. A total of 1157 miRNAs were identified, 84 of which were differentially expressed. Furthermore, qRT-PCR validated that SH treatment improved 17 differentially expressed miRNAs with > fourfold upregulation or > sixfold downregulation. Similar to most differentially altered miRNA, C. albicans significantly increased Dectin-1, NF-κB, TNF-α, IL-1ß, IL-17A, and decreased miR-32-5p which negatively targeted NFKBIZ. In addition, SH treatment reduced inflammatory response and fungal burden in a colitis model with C. albicans infection. Further analyses indicated that in C. albicans infected Caco2 cells, SH inhibited fungal growth, oxidative stress, and apoptosis by increasing Dectin-1, NF-κB, NFKBIZ, TNF-α, IL-1ß, IL-17A, and decreasing miR-32-5p. Therefore, SH can ameliorate the severity of colitis aggravated by C. albicans via the Dectin-1/NF-κB/miR-32-5p/NFKBIZ axis.

The Candida albicans quorum-sensing molecule farnesol alters sphingolipid metabolism in human monocyte-derived dendritic cells.

Candida albicans, an opportunistic fungal pathogen, produces the quorum-sensing molecule farnesol, which we have shown alters the transcriptional response and phenotype of human monocyte-derived dendritic cells (DCs), including their cytokine secretion and ability to prime T cells. This is partially dependent on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which has numerous ligands, including the sphingolipid metabolite sphingosine 1-phosphate. Sphingolipids are a vital component of membranes that affect membrane protein arrangement and phagocytosis of C. albicans by DCs. Thus, we quantified sphingolipid metabolites in monocytes differentiating into DCs by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Farnesol increased the activity of serine palmitoyltransferase, leading to increased levels of 3-keto-dihydrosphingosine, dihydrosphingosine, and dihydrosphingosine 1-phosphate and inhibited dihydroceramide desaturase by inducing oxidative stress, leading to increased levels of dihydroceramide and dihydrosphingomyelin species and reduced ceramide levels. Accumulation of dihydroceramides can inhibit mitochondrial function; accordingly, farnesol reduced mitochondrial respiration. Dihydroceramide desaturase inhibition increases lipid droplet formation, which we observed in farnesol-treated cells, coupled with an increase in intracellular triacylglycerol species. Furthermore, inhibition of dihydroceramide desaturase with either farnesol or specific inhibitors impaired the ability of DCs to prime interferon-γ-producing T cells. The effect of farnesol on sphingolipid metabolism, triacylglycerol synthesis, and mitochondrial respiration was not dependent on PPAR-γ. In summary, our data reveal novel effects of farnesol on sphingolipid metabolism, neutral lipid synthesis, and mitochondrial function in DCs that affect their instruction of T cell cytokine secretion, indicating that C. albicans can manipulate host cell metabolism via farnesol secretion.IMPORTANCECandida albicans is a common commensal yeast, but it is also an opportunistic pathogen which is one of the leading causes of potentially lethal hospital-acquired infections. There is growing evidence that its overgrowth in the gut can influence diseases as diverse as alcohol-associated liver disease and COVID-19. Previously, we found that its quorum-sensing molecule, farnesol, alters the phenotype of dendritic cells differentiating from monocytes, impairing their ability to drive protective T cell responses. Here, we demonstrate that farnesol alters the metabolism of sphingolipids, important structural components of the membrane that also act as signaling molecules. In monocytes differentiating to dendritic cells, farnesol inhibited dihydroceramide desaturase, resulting in the accumulation of dihydroceramides and a reduction in ceramide levels. Farnesol impaired mitochondrial respiration, known to occur with an accumulation of dihydroceramides, and induced the accumulation of triacylglycerol and oil bodies. Inhibition of dihydroceramide desaturase resulted in the impaired ability of DCs to induce interferon-γ production by T cells. Thus, farnesol production by C. albicans could manipulate the function of dendritic cells by altering the sphingolipidome.

Mechanism of Vaginal Epithelial Cell Pyroptosis Induced by the NLRP3 Inflammasome in Vulvovaginal Candidiasis.

PROBLEM: Vulvovaginal candidiasis (VVC) is a common mucosal fungal infection, and Candida albicans is the main causative agent. The NLRP3 inflammasome plays an important role in VVC, but the underlying mechanism is unknown. METHOD OF STUDY: Vaginal epithelial cells were divided into three groups: control, C. albicans strain SC5314 (wild-type, WT), and WT+ Matt Cooper Compound 950 (MCC950, a specific NLRP3 inhibitor). After human vaginal epithelial cells were pretreated with 1 µmol/L MCC950 for 2 h, C. albicans (MOI = 1) was cocultured with the human vaginal epithelial cells for 12 h. The cell supernatants were collected, LDH was detected, and the IL-1ß and IL-18 levels were determined by ELISA. The expression of the pyroptosis-related proteins NLRP3, Caspase-1 p20 and GSDMD was measured by Western blotting analysis. The protein expression of the pyroptosis-related N-terminus of GSDMD (GSDMD-N) was detected by immunofluorescence. RESULTS: In this study, we showed that the WT C. albicans strain induced pyroptosis in vaginal epithelial cells, as indicated by the LDH and proinflammatory cytokine levels and the upregulated levels of the pyroptosis-related proteins NLRP3, Caspase-1 p20, and GSDMD-N. MCC950 reversed the changes in the expression of these proteins and proinflammatory cytokines in vaginal epithelial cells. CONCLUSION: C. albicans activated the NLRP3 inflammasome to induce vaginal epithelial cell pyroptosis. MCC950 inhibited the NLRP3 inflammasome, reduced vaginal epithelial cell pyroptosis, and decreased the release of inflammatory cytokines.

Fleagrass (Adenosma buchneroides Bonati) Acts as a Fungicide Against Candida albicans by Damaging Its Cell Wall.

Fleagrass, a herb known for its pleasant aroma, is widely used as a mosquito repellent, antibacterial agent, and for treating colds, reducing swelling, and alleviating pain. The antifungal effects of the essential oils of fleagrass and carvacrol against Candida albicans were investigated by evaluating the growth and the mycelial and biofilm development of C. albicans. Transmission electron microscopy was used to evaluate the integrity of the cell membrane and cell wall of C. albicans. Fleagrass exhibited high fungicidal activity against C. albicans at concentrations of 0.5% v/v (via the Ras1/cAMP/PKA pathway). Furthermore, transmission electron microscopy revealed damage to the cell wall and membrane after treatment with the essential oil, which was further confirmed by the increased levels of ß-1,3-glucan and chitin in the cell wall. This study showed that fleagrass exerts good fungicidal and hyphal growth inhibition activity against C. albicans by disrupting its cell wall, and thus, fleagrass may be a potential antifungal drug.

Antimicrobial gel with silver vanadate and silver nanoparticles: antifungal and physicochemical evaluation.

Aim: To develop a ß-AgVO3 gel and evaluate its physicochemical stability and antifungal activity against Candida albicans. Materials & methods: The gel was prepared from the minimum inhibitory concentration (MIC) of ß-AgVO3. The physicochemical stability was evaluated by centrifugation, accelerated stability (AS), storage (St), pH, syringability, viscosity and spreadability tests and antifungal activity by the agar diffusion. Results: The MIC was 62.5 µg/ml. After centrifugation, AS and St gels showed physicochemical stability. Lower viscosity and higher spreadability were observed for the higher ß-AgVO3 concentration and the minimum force for extrusion was similar for all groups. Antifungal effect was observed only for the ß-AgVO3 gel with 20xMIC. Conclusion: The ß-AgVO3 gel showed physicochemical stability and antifungal activity.

We used silver and vanadium to make a gel that can kill fungi in the mouth. We looked at the color of the gel, it's smell and also checked how well it lasted. The gel turned yellow and had no smell and did not spoil for at least 2 months. When we tested the gel against a type of fungus, it worked as well as another medicine called chlorhexidine, which is sold in pharmacies. But when we compared it with another medicine called nystatin, our gel was not as effective in killing the fungus.

The spliceosome impacts morphogenesis in the human fungal pathogen Candida albicans.

At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

Strain variation in the Candida albicans iron limitation response.

Iron acquisition is critical for pathogens to proliferate during invasive infection, and the human fungal pathogen Candida albicans is no exception. The iron regulatory network, established in reference strain SC5314 and derivatives, includes the central player Sef1, a transcription factor that activates iron acquisition genes in response to iron limitation. Here, we explored potential variation in this network among five diverse C. albicans strains through mutant analysis, Nanostring gene expression profiling, and, for two strains, RNA-Seq. Our findings highlight four features that may inform future studies of natural variation and iron acquisition in this species. (i) Conformity: In all strains, major iron acquisition genes are upregulated during iron limitation, and a sef1Δ/Δ mutation impairs that response and growth during iron limitation. (ii) Response variation: Some aspects of the iron limitation response vary among strains, notably the activation of hypha-associated genes. As this gene set is tied to tissue damage and virulence, variation may impact the progression of infection. (iii) Genotype-phenotype variation: The impact of a sef1Δ/Δ mutation on cell wall integrity varies, and for the two strains examined the phenotype correlated with sef1Δ/Δ impact on several cell wall integrity genes. (iv) Phenotype discovery: DNA repair genes were induced modestly by iron limitation in sef1Δ/Δ mutants, with fold changes we would usually ignore. However, the response occurred in both strains tested and was reminiscent of a much stronger response described in Cryptococcus neoformans, a suggestion that it may have biological meaning. In fact, we observed that the iron limitation of a sef1Δ/Δ mutant caused recessive phenotypes to emerge at two heterozygous loci. Overall, our results show that a network that is critical for pathogen proliferation presents variation outside of its core functions.IMPORTANCEA key virulence factor of Candida albicans is the ability to maintain iron homeostasis in the host where iron is scarce. We focused on a central iron regulator, SEF1. We found that iron regulator Sef1 is required for growth, cell wall integrity, and genome integrity during iron limitation. The novel aspect of this work is the characterization of strain variation in a circuit that is required for survival in the host and the connection of iron acquisition to genome integrity in C. albicans.

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis.

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

Candesartan Attenuates Vasculitis in a Mouse Model of Kawasaki Disease Induced by Candida albicans Water-Soluble Fraction.

BACKGROUND: The standard treatment for Kawasaki disease is immunoglobulin therapy, but the high frequency of coronary sequelae in immunoglobulin-refractory cases indicates a need for further improvement in treatment. METHODS: Kawasaki disease-like vasculitis was induced in 5-week-old DBA/2 mice by intraperitoneal administration of 0.5 mg Candida albicans water-soluble fraction (CAWS) daily for 5 days followed by daily administration of candesartan, an angiotensin receptor blocker. The vasculitis suppression effect was confirmed histologically and serologically in mice sacrificed at 28 days after the start of candesartan. RESULTS: The area of inflammatory cell infiltration at the aortic root was 2.4±1.4% in the Control group, 18.1±1.9% in the CAWS group, and 7.1±2.3%, 5.8±1.4%, 7.6±2.4%, and 7.9±5.0% in the CAWS+candesartan 0.125-mg/kg, 0.25-mg/kg, 0.5-mg/kg, and 1.0-mg/kg groups, respectively (p=0.0200, p=0.0122, p=0.0122, and p=0.0200 vs. CAWS, respectively). The low-dose candesartan group also showed significantly reduced inflammatory cell infiltration. A similar trend was confirmed by immunostaining of macrophages and TGFß receptors. Measurement of the inflammatory cytokines IL-1ß, IL-6, and TNF-α confirmed the anti-vasculitis effect of candesartan. CONCLUSIONS: Candesartan inhibited vasculitis even at clinical doses used in children, making it a strong future candidate as an additional treatment for immunoglobulin-refractory Kawasaki disease.

Improvement of a low-cost protocol for a simultaneous comparative evaluation of hydrolytic activity between sessile and planktonic cells: Candida albicans as a study model.

Candida albicans is often implicated in nosocomial infections with fatal consequences. Its virulence is contributed to hydrolytic enzymes and biofilm formation. Previous research focused on studying these virulence factors individually. Therefore, this study aimed to investigate the impact of biofilm formation on the hydrolytic activity using an adapted low-cost method. Eleven strains of C. albicans were used. The biofilms were formed on pre-treated silicone discs using 24-well plates and then deposited on the appropriate agar to test each enzyme, while the planktonic cells were conventionally seeded. Biofilms were analysed using Raman spectroscopy, fluorescent and scanning electron microscopy. The adapted method provided an evaluation of hydrolytic enzymes activity in C. albicans biofilm and showed that sessile cells had a higher phospholipase and proteinase activities compared with planktonic cells. These findings were supported by spectroscopic and microscopic analyses, which provided valuable insights into the virulence mechanisms of C. albicans during biofilm formation.

Formulation and Antimycotic Evaluation of Colloidal Itraconazole-Loaded Metered Dose Sprays for Treating Superficial Mycoses.

Commercial topical formulations containing itraconazole (poorly water soluble), for mycotic infections, have poor penetration to infection sites beneath the nails and skin thereby necessitating oral administration. To improve penetration, colloidal solutions of itraconazole (G1-G4) containing Poloxamer 188, tween 80, ethanol, and propylene glycol were prepared and incorporated into HFA-134-containing sprays. Formulations were characterized using particle size, drug content, and Fourier-transform infrared spectroscopy (FTIR). In vitro permeation studies were performed using Franz diffusion cells for 8 h. Antimycotic activity on Candida albicans and Trichophyton rubrum was performed using broth micro-dilution and flow cytometry, while cytotoxicity was tested on HaCaT cell lines. Particle size ranged from 39.35-116.80 nm. FTIR and drug content revealed that G1 was the most stable formulation (optimized formulation). In vitro release over 2 h was 45% for G1 and 34% for the cream. There was a twofold increase in skin permeation, fivefold intradermal retention, and a sevenfold increase in nail penetration of G1 over the cream. Minimum fungicidal concentrations (MFC) against C. albicans were 0.156 and 0.313 µg/mL for G1 and cream, respectively. The formulations showed optimum killing kinetics after 48 h. MFC values against T. rubrum were 0.312 and 0.625 µg/mL for the G1 and cream, respectively. Transmission electron microscopy revealed organelle destruction and cell leakage for G1 in both organisms and penetration of keratin layers to destroy T. rubrum. Cytotoxicity evaluation of G1 showed relative safety for skin cells. The G1 formulation showed superior skin permeation, nail penetration, and fungicidal activity compared with the cream formulation.

Computational Evaluation on the Interactions of an Opaque-Phase ABC Transporter Associated with Fluconazole Resistance in Candida albicans, by the Psidium guajava Bio-Active Compounds.

Objectives: Candida albicans is an opportunistic pathogen that occurs as harmless commensals in the intestine, urogenital tract, and skin. It has been influenced by a variety of host conditions and has now evolved as a resistant strain. The aim of this study was thus detect the fluconazole resistant C. albicans from the root caries specimens and to computationally evaluate the interactions of an opaque-phase ABC transporter protein with the Psidium guajava bio-active compounds. Methods: 20 carious scrapings were collected from patients with root caries and processed for the isolation of C. albicans and was screened for fluconazole resistance. Genomic DNA was extracted and molecular characterization of Cdrp1 and Cdrp2 was done by PCR amplification. P. guajava methanolic extract was checked for the antifungal efficacy against the resistant strain of C. albicans. Further in-silico docking involves retrieval of ABC transporter protein and ligand optimization, molinspiration assessment on drug likeness, docking simulations and visualizations. Results: 65% of the samples showed the presence of C.albicans and 2 strains were fluconazole resistant. Crude methanolic extract of P. guajava was found to be promising against the fluconazole resistant strains of C. albicans. In-silico docking analysis showed that Myricetin was a promising candidate with a high docking score and other drug ligand interaction scores. Conclusion: The current study emphasizes that bioactive compounds from Psidium guajava to be a promising candidate for treating candidiasis in fluconazole resistant strains of C. albicans However, further in-vivo studies have to be implemented for the experimental validation of the same in improving the oral health and hygiene.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Candida albicans in the oral cavities of pets: biofilm formation, putative virulence, antifungal resistance profiles and classification of the isolates.

The study aimed to investigate Candida albicans presence, antifungal resistance, biofilm formation, putative virulence genes, and molecular characterization in oral samples of dogs and cats. A total of 239 oral samples were collected from cats and dogs of various breeds and ages at Erciyes University, Faculty of Veterinary Medicine Clinics, between May 2017 and April 2018. Among 216 isolates obtained, 15 (6.95%) were identified as C. albicans, while 8 (3.7%) were non-albicans Candida species. Antifungal susceptibility testing revealed sensitivities to caspofungin, fluconazole, and flucytosine in varying proportions. Molecular analysis indicated the presence of fluconazole and caspofungin resistance genes in all C. albicans isolates. Additionally, virulence genes ALS1, HWP1, and HSP90 showed variable presence. Biofilm formation varied among isolates, with 46.7% strong, 33.3% moderate, and 20% weak producers. PCA analysis categorized isolates into two main clusters, with some dog isolates grouped separately. The findings underscore the significance of oral care and protective measures in pets due to C. albicans prevalence, biofilm formation, virulence factors, and antifungal resistance in their oral cavity, thereby aiding clinical diagnosis and treatment in veterinary medicine.

Anticandidal applications of selenium nanoparticles biosynthesized with Limosilactobacillus fermentum (OR553490).

Candida albicans is one of the most dangerous pathogenic fungi in the world, according to the classification of the World Health Organization, due to the continued development of its resistance to currently available anticandidal agents. To overcome this problem, the current work provided a simple, one-step, cost-effective, and safe technique for the biosynthesis of new functionalized anticandidal selenium nanoparticles (Se NPs) against C. albicans ATCC10231 using the cell-free supernatant of Limosilactobacillus fermentum (OR553490) strain. The bacterial strain was isolated from yogurt samples available in supermarkets, in Damietta, Egypt. The mixing ratio of 1:9 v/v% between cell-free bacterial metabolites and sodium selenite (5 mM) for 72 h at 37 °C were the optimum conditions for Se NPs biosynthesis. Ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta analyses, and elemental analysis system (EDS) were used to evaluate the optimized Se NPs. The Se NPs absorption peak appeared at 254 nm. Physicochemical analysis of Se NPs revealed the crystalline-shaped and well-dispersed formation of NPs with an average particle size of 17-30 nm. Se NPs have - 11.8 mV, as seen by the zeta potential graph. FT-IR spectrum displayed bands of symmetric and asymmetric amines at 3279.36 cm-1 and 2928.38 cm-1, aromatic and aliphatic (C-N) at 1393.32 cm-1 and 1237.11.37 cm-1 confirming the presence of proteins as stabilizing and capping agents. Se NPs acted as a superior inhibitor of C. albicans with an inhibition zone of 26 ± 0.03 mm and MIC value of 15 µg/mL compared to one of the traditional anticandidal agent, miconazole, which revealed 18 ± 0.14 mm and 75 µg/mL. The cytotoxicity test shows that Se NPs have a low toxic effect on the normal keratinocyte (IC50 ≈ 41.5 µg/mL). The results indicate that this green synthesis of Se NPs may have a promising potential to provide a new strategy for drug therapy.

Miconazole induces aneuploidy-mediated tolerance in Candida albicans that is dependent on Hsp90 and calcineurin.

Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.

Comparative study of antifungal susceptibility testing methods for clinical Candida albicans isolates.

PURPOSE: Candida albicans is the second most common cause of candidemia in Malaysia. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the gold standard for determining its minimum inhibitory concentration (MIC); however, it is laborious and time-consuming. This study was conducted to evaluate the usefulness of alternative methods, namely Sensititre YeastOne (SYO), VITEK 2 system, and E-test for determining the MIC of clinical C. albicans isolates. MATERIALS AND METHODS: The susceptibilities of 95 C. albicans isolates were compared between SYO, VITEK 2 system, and E-test with CLSI broth microdilution method. The categorical agreement (CA), essential agreement (EA), very major errors (VME), major errors (ME) and minor errors (MiE) were calculated. RESULTS: Our finding showed the CA varied for SYO from 96.8% to 100%, while the EA ranged from 91.6% to 100%. The SYO method showed 1.1% of VME and ME, and up to 3.2% of MiE. Next, the CA and EA ranges for the VITEK 2 system were 97.8%-100% and 23.2%-100%, respectively. In the VITEK 2 technique, 1.1% of VME were found. For the E-test, the CA varied from 83.2% to 100% while the EA ranged from 64.2% to 98.9%. The E-test method showed 1.1% of VME and up to 16.8% of MiE. CONCLUSIONS: In conclusion, SYO and VITEK 2 (except flucytosine) could be potential alternatives to the CLSI broth microdilution method in determining the MIC of C. albicans.

Hemiprotonic ph-ph+ with two targets inhibits metastatic breast cancer and concurrent candidiasis.

Concurrent infection in breast cancer patients is the direct cause of the high mortality rate of the disease. However, there is no available method to increase the survival rate until now. To address the problem, we propose one drug with two target strategy to treat the refractory disease. A small chemical, ph-ph+, was attempted to be used in the study to explore the feasibility of the approach in anticancer and antifungus at the same time. The results showed that ph-ph+ could prevent the proliferation and metastasis of breast cancer cells, and kill C. albicans simultaneously. The molecular mechanism was associated with the activation of an evolutionarily conserved protease CLpP in the cancer and C. albicans cells. Also, the signaling pathway mediated by PLAGL2 that highly expressed in cancer cells participated in preventing cell metastasis and inducing apoptosis of ph-ph+. The one drug with dual targets inhibited the growth and metastasis of the cancer cells, and meanwhile eliminated C. albicans in tissues in the experimental animals. The results suggested that ph-ph+ with dual targets of CLpP and PLAGL2 would be a feasible approach to prolong the survival rate in patients with metastatic breast cancer and pathogenic infection.

Tyrosol-gold nanoparticle functionalized acacia gum-PVA nanofibers for mitigation of Candida biofilm.

Increasing incidences of fungal infections and prevailing antifungal resistance in healthcare settings has given rise to an antifungal crisis on a global scale. The members of the genus Candida, owing to their ability to acquire sessile growth, are primarily associated with superficial to invasive fungal infections, including the implant-associated infections. The present study introduces a novel approach to combat the sessile/biofilm growth of Candida by fabricating nanofibers using a nanoencapsulation approach. This technique involves the synthesis of tyrosol (TYS) functionalized chitosan gold nanocomposite, which is then encapsulated into PVA/AG polymeric matrix using electrospinning. The FESEM, FTIR analysis of prepared TYS-AuNP@PVA/AG NF suggested the successful encapsulation of TYS into the nanofibers. Further, the sustained and long-term stability of TYS in the medium was confirmed by drug release and storage stability studies. The prepared nanomats can absorb the fluid, as evidenced by the swelling index of the nanofibers. The growth and biofilm inhibition, as well as the disintegration studies against Candida, showed 60-70 % biofilm disintegration when 10 mg of TYS-AuNP@PVA/AG NF was used, hence confirming its biological effectiveness. Subsequently, the nanofibers considerably reduced the hydrophobicity index and ergosterol content of the treated cells. Considering the challenges associated with the inhibition/disruption of fungal biofilm, the fabricated nanofibers prove their effectiveness against Candida biofilm. Therefore, nanocomposite-loaded nanofibers have emerged as potential materials that can control fungal colonization and could also promote healing.