Striking Back against Fungal Infections: The Utilization of Nanosystems for Antifungal Strategies.

Fungal infections have become a major health concern, given that invasive infections by Candida, Cryptococcus, and Aspergillus species have led to millions of mortalities. Conventional antifungal drugs including polyenes, echinocandins, azoles, allylamins, and antimetabolites have been used for decades, but their limitations include off-target toxicity, drug-resistance, poor water solubility, low bioavailability, and weak tissue penetration, which cannot be ignored. These drawbacks have led to the emergence of novel antifungal therapies. In this review, we discuss the nanosystems that are currently utilized for drug delivery and the application of antifungal therapies.

High Efficient Expression and Purification of Human Epidermal Growth Factor in Arachis Hypogaea L.

BACKGROUND: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. METHODS: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. RESULTS: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. CONCLUSION: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.

Transdermal administration of farnesol-ethosomes enhances the treatment of cutaneous candidiasis induced by Candida albicans in mice.

Cutaneous candidiasis, caused by Candida albicans, is a severe and frustrating condition, and finding effective treatments can be challenging. Therefore, the development of farnesol-loaded nanoparticles is an exciting breakthrough. Ethosomes are a novel transdermal drug delivery carrier that incorporates a certain concentration (10-45%) of alcohols into lipid vesicles, resulting in improved permeability and encapsulation rates compared to conventional liposomes. Farnesol is a quorum-sensing molecule involved in morphogenesis regulation in C. albicans, and these ethosomes offer a promising new approach to treating this common fungal infection. This study develops the formulation of farnesol-loaded ethosomes (farnesol-ethosomes) and assesses applications in treating cutaneous candidiasis induced by C. albicans in vitro and in vivo. Farnesol-ethosomes were successfully developed by ethanol injection method. Therapeutic properties of farnesol-ethosomes, such as particle size, zeta potential, and morphology, were well characterized. According to the results, farnesol-ethosomes demonstrated an increased inhibition effect on cells' growth and biofilm formation in C. albicans. In Animal infection models, treating farnesol-ethosomes by transdermal administration effectively relieved symptoms caused by cutaneous candidiasis and reduced fungal burdens in quantity. We also observed that ethosomes significantly enhanced drug delivery efficacy in vitro and in vivo. These results indicate that farnesol-ethosomes can provide future promising roles in curing cutaneous candidiasis. IMPORTANCE: Cutaneous candidiasis attributed to Candida infection is a prevalent condition that impacts individuals of all age groups. As a type of microbial community, biofilms confer benefits to host infections and mitigate the clinical effects of antifungal treatments. In C. albicans, the yeast-to-hypha transition and biofilm formation are effectively suppressed by farnesol through its modulation of multiple signaling pathway. However, the characteristics of farnesol such as hydrophobicity, volatility, degradability, and instability in various conditions can impose limitations on its effectiveness. Nanotechnology holds the potential to enhance the efficiency and utilization of this molecule. Treatment of farnesol-ethosomes by transdermal administration demonstrated a very remarkable therapeutic effect against C. albicans in infection model of cutaneous candidiasis in mice. Many patients suffering fungal skin infection will benefit from this study.

Evolution of mating within the Candida parapsilosis species group.

Candida orthopsilosis and Candida metapsilosis are closely related to Candida parapsilosis, a major cause of infection in premature neonates. Mating has not been observed in these species. We show that ∼190 isolates of C. parapsilosis contain only an MTLa idiomorph at the mating-type-like locus. Here, we describe the isolation and characterization of the MTL loci from C. orthopsilosis and C. metapsilosis. Among 16 C. orthopsilosis isolates, 9 were homozygous for MTLa, 5 were homozygous for MTLα, and 2 were MTLa/α heterozygotes. The C. orthopsilosis isolates belonged to two divergent groups, as characterized by restriction patterns at MTL, which probably represent subspecies. We sequenced both idiomorphs from each group and showed that they are 95% identical and that the regulatory genes are intact. In contrast, 18 isolates of C. metapsilosis contain only MTLα idiomorphs. Our results suggest that the role of MTL in determining cell type is being eroded in the C. parapsilosis species complex. The population structure of C. orthopsilosis indicates that mating may occur. However, expression of genes in the mating signal transduction pathway does not respond to exposure to alpha factor. C. parapsilosis is also nonresponsive, even when the GTPase-activating protein gene SST2 is deleted. In addition, splicing of introns in MTLa1 and MTLa2 is defective in C. orthopsilosis. Mating is not detected. The alpha factor peptide, which is the same sequence in C. parapsilosis, C. orthopsilosis, and C. metapsilosis, can induce a mating response in Candida albicans. It is therefore likely either that mating of C. orthopsilosis takes place under certain unidentified conditions or that the mating pathway has been adapted for other functions, such as cross-species communication.

Maleimide-functionalized phospholipid/Pluronic F127 mixed micelles for efficient ophthalmic delivery of voriconazole against Candida albicans.

Drugs that are topically applied on the eyes have low bioavailability, which has always been an important problem. In this study, maleimide functionalized, voriconazole (VCZ) loaded mixed micelles (Mal-VCZ-MM) were designed. Pluronic F127 and phospholipid were used as materials, and maleimide was used as an adhesive. The prepared Mal-VCZ-MM was nearly spherical with a particle size of 84.45 ± 1.39 nm and a zeta potential of - 20.3 ± 0.29 mV. The encapsulation efficiency of Mal-VCZ-MM was 95.33 ± 0.06%, and it had high stability with a critical micelle concentration value of 1.28 × 10-4 mg/mL. CCK-8 assay showed that its cytotoxicity was lower than that of free VCZ solution (VCZ-Sol). Both quantitative and qualitative analyses of the HCE-T cellular uptake showed that the cellular internalization of Mal-C6-MM was significantly stronger than that of C6-MM. The endocytosis pathway was macropinocytosis-mediated, cavernous-mediated, and energy-dependent. In vitro results against Candida albicans showed that the diameters of the antifungal inhibition zones of VCZ-Sol, VCZ-MM, and Mal-VCZ-MM were 15.5 ± 0.50 mm, 24.0 ± 0.71 mm, and 31.5 ± 1.12 mm, respectively. The antifungal effect of Mal-VCZ-MM was significantly higher than that of VCZ-Sol and VCZ-MM (P < 0.001). This study shows that Mal-VCZ-MM is a highly effective hydrophobic ophthalmic drug-delivery carrier that can improve the therapeutic effect of the drug.

Calcofluor white-cholesteryl hydrogen succinate conjugate mediated liposomes for enhanced targeted delivery of voriconazole into Candida albicans.

Fungal infections gradually lead to a high mortality rate due to difficulties in diagnosis, the limited number of antifungal drugs available, and the appearance of resistant isolates. Here, we developed a calcofluor white-cholesteryl hydrogen succinate conjugate (CFW-CHSc) as a novel nanomaterial that specifically binds to chitin chains in the cell wall. We showed that fluorescent-dye loaded CFW-CHSc-liposomes entered the cytoplasm of Candida albicans cells with increased efficacy. Voriconazole-loaded CFW-CHSc-liposomes displayed an increased antifungal activity against C. albicans yeast cells in an in vitro assay. Animal infection models and animal imaging analysis showed that fluorescent-dye loaded CFW-CHSc-liposomes maintained prolonged residence in rodent tissues. In mouse liver and kidney tissue, voriconazole-loaded CFW-CHSc-liposomes showed significantly enhanced antifungal activity when administered intravenously. Taken together, our studies confirm that CFW-CHSc increases the drug delivery efficacy of nanoparticles in vitro by interacting with chitin chains in the C. albicans cell wall. The fungi-targeting nanoparticles improve the drug delivery efficacy in vivo by enriching the nanoparticles at the site of fungal infection via the blood circulation system. Fungi-targeting nanomaterials have a promising future in the treatment of nosomycosis.

Mucoadhesive phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer for topical ocular delivery of voriconazole: Synthesis, in vitro/vivo evaluation, and mechanism.

Topical eye drops still face challenges of low-drug treatment effects and frequent dosing in ophthalmic applications due to the low preocular retention rate and low transcorneal permeability. Thus, we designed and synthesized a phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer (PBA-CS-VE) for use in mucoadhesive voriconazole (VRC)-loaded nanomicelles for fungal keratitis. In vitro mucin binding and ex vivo eyeball adhesion tests show that the copolymer has strong mucoadhesion. The transportation of coumarin-6 (C6) across a monolayer of HCE-T cells and 3D cell spheroids confirm the strong corneal penetration ability of PBA-CS-VE. The mechanism of promoting corneal penetration was studied in terms of intracellular calcium-ion concentration, cell membrane potential, cell membrane fluidity, and the tight junctions of cells. The pharmacokinetics in the aqueous humor were examined to evaluate the ability of nanomicelles in promoting corneal penetration and prolonging ocular retention. VRC-loaded PBA-CS-VE nanomicelles (PBA-CS-VE-VRC) yielded a very favorable therapeutic effect on a rabbit model of fungal keratitis in vivo as compared to the free drug. Overall, the results indicate that PBA-CS-VE nanomicelles are a mucoadhesive candidate with enhanced transcorneal permeability and prolonged preocular retention for efficient delivery of topical ocular drugs. STATEMENT OF SIGNIFICANCE: Although eye drops are widely used in ocular drug delivery, the disadvantages such as short retention time and weak corneal penetrating ability still seriously affect the therapeutic effect of the drug. Therefore, the mucoadhesive carrier seems to be an interesting strategy for ocular drug delivery. Herein, a novel phenylboronic acid conjugated chitosan oligosaccharide-vitamin E copolymer was designed and constructed as mucoadhesive nanomicelles loaded with voriconazole for fungal keratitis. These nanomicelles were able to improve the in vitro mucin binding and to prolong the residence time of the drug on the surface of the eyeball. Moreover, the nanomicelles exhibited an enhanced drug permeability in cell monolayer models and 3D cell culture models. This work provides a promising ocular drug delivery system.

Cryptococcal Hsf3 controls intramitochondrial ROS homeostasis by regulating the respiratory process.

Mitochondrial quality control prevents accumulation of intramitochondrial-derived reactive oxygen species (mtROS), thereby protecting cells against DNA damage, genome instability, and programmed cell death. However, underlying mechanisms are incompletely understood, particularly in fungal species. Here, we show that Cryptococcus neoformans heat shock factor 3 (CnHsf3) exhibits an atypical function in regulating mtROS independent of the unfolded protein response. CnHsf3 acts in nuclei and mitochondria, and nuclear- and mitochondrial-targeting signals are required for its organelle-specific functions. It represses the expression of genes involved in the tricarboxylic acid cycle while promoting expression of genes involved in electron transfer chain. In addition, CnHsf3 responds to multiple intramitochondrial stresses; this response is mediated by oxidation of the cysteine residue on its DNA binding domain, which enhances DNA binding. Our results reveal a function of HSF proteins in regulating mtROS homeostasis that is independent of the unfolded protein response.

Development of nose-to-brain delivery of ketoconazole by nanostructured lipid carriers against cryptococcal meningoencephalitis in mice.

Cryptococcus neoformans-mediated meningoencephalitis is a critical infectious disorder of the human central nervous system. However, efficient treatment for the disease is limited due to the poor penetration across the blood brain barrier (BBB). Here, we develop a nose-to-brain drug delivery system utilizing nanostructured lipid carriers (NLCs). We demonstrated that fluorescent-dye-loaded NLCs efficiently uptake into the cytoplasm of encapsulated C. neoformans cells. In comparison with current antifungal drugs, the ketoconazole (keto)-NLCs show significantly increased antifungal activity against C. neoformans in vivo under various growth conditions. The NLCs show enhanced tissue colonization properties. Importantly, using animal imaging analyses, NLCs are able to enter brain tissues via the olfactory bulb region by intranasal administration, bypassing the BBB. In addition, NLCs maintain prolonged residence in tissues. In mouse brain tissue, keto-NLCs showed significantly enhanced antifungal activity when administered intranasally, drastically dampening the C. neoformans burden. Taken together, NLCs not only improve the ketoconazole penetration efficiency against capsulated C. neoformans cells, but also boost the efficacy of antifungal drugs. Most importantly, keto-NLCs significantly contribute to the treatment of cryptococcal meningoencephalitis in mice by bypassing the BBB via the olfactory system.

Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence.

Lysine acetylation is critical in regulating important biological processes in many organisms, yet little is known about acetylome evolution and its contribution to phenotypic diversity. Here, we compare the acetylomes of baker's yeast and the three deadliest human fungal pathogens, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using mass spectrometry enriched for acetylated peptides together with public data from Saccharomyces cerevisiae, we show that fungal acetylomes are characterized by dramatic evolutionary dynamics and limited conservation in core biological processes. Notably, the levels of protein acetylation in pathogenic fungi correlate with their pathogenicity. Using gene knockouts and pathogenicity assays in mice, we identify deacetylases with critical roles in virulence and protein translation elongation. Finally, through mutational analysis of deactylation motifs we find evidence of positive selection at specific acetylation motifs in fungal pathogens. These results shed new light on the pathogenicity regulation mechanisms underlying the evolution of fungal acetylomes.

Investigation of Cryptococcus neoformans magnesium transporters reveals important role of vacuolar magnesium transporter in regulating fungal virulence factors.

Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. Recent studies have demonstrated that metals are critical factors for the regulation of fungal virulence in hosts. In this study, we systemically investigated the function of C. neoformans magnesium transporters in controlling the intracellular Mg balance and virulence-associated factors. We identified three Mg transporters in C. neoformans: Mgt1, Mgt2, and Mgt3. While we could not detect a Mg2+ -related growth phenotype in mgt1 and mgt3 knockout strains, a GAL7p-Mgt2 strain showed significant Mg-dependent growth defects in the presence of glucose. Further analysis demonstrated that MGT2 is a homolog of MNR2 in Saccharomyces cerevisiae, which is localized to the vacuolar membrane and participates in intracellular Mg transport. Interestingly, a transcriptome analysis showed that Mgt2 influenced the expression of 19 genes, which were independent of Mg2+ . We showed that melanin synthesis in C. neoformans required Mg2+ and Mgt2, and that capsule production was negatively regulated by Mg2+ and Mgt2. Repressing the expression of MGT2-induced capsule, which resulted in an increased fungal burden in the lungs. Cumulatively, this study sets the stage for further evaluation of the important role of Mg homeostasis in the regulation of melanin and capsule in C. neoformans.

Enhanced antifungal activity of voriconazole-loaded nanostructured lipid carriers against Candida albicans with a dimorphic switching model.

Candida commonly adheres to implanted medical devices and forms biofilms. Due to the minimal activity of current antifungals against biofilms, new drugs or drug-delivery systems to treat these persistent infections are urgently needed. In the present investigation, voriconazole-loaded nanostructured lipid carriers (Vrc-NLCs) were formulated for enhanced drug-delivery efficiency to C. albicans to increase the antifungal activity of Vrc and to improve the treatment of infectious Candida diseases. Vrc-NLCs were prepared by a hot-melt, high-pressure homogenization method, and size distribution, ζ-potential, morphology, drug-encapsulation efficiency, drug loading, and physical stability were characterized. The antifungal activity of Vrc-NLCs in vitro was tested during planktonic and biofilm growth in C. albicans. The mean particle size of the Vrc-NLCs was 45.62±0.53 nm, and they exhibited spheroid-like morphology, smooth surfaces, and ζ-potential of -0.69±0.03 mV. Encapsulation efficiency and drug loading of Vrc-NLCs were 75.37%±2.65% and 3.77%±0.13%, respectively. Physical stability results revealed that despite the low measured ζ-potential, the dispersion of the Vrc-NLCs was stable during their 3-week storage at 4°C. The minimum inhibitory concentration of Vrc-NLCs was identical to that of Vrc. However, the inhibition rate of Vrc-NLCs at lower concentrations was significantly higher than that of Vrc during planktonic growth in C. albicans in yeast-extract peptone dextrose medium. Surprisingly, Vrc-NLCs treatment reduced cell density in biofilm growth in C. albicans and induced more switches form hyphal cells to yeast cells compared with Vrc treatment. In conclusion, Vrc-NLCs maintain antifungal activity of Vrc and increase antifungal drug-delivery efficiency to C. albicans. Therefore, Vrc-NLCs will greatly contribute to the treatment of infectious diseases caused by C. albicans.

[Gene cloning, sequence analysis and tissue expression of estrogen-related receptor alpha (Erralpha) in Japanese medaka and its transcriptional responses after differential EDCs exposure].

Endocrine disrupting chemicals (EDCs) can bind or block nuclear receptors in the body and subsequently affect growth, development and reproduction of fish. Estrogen-related receptors (ERRs), belonging to the nuclear receptor superfamily, have been implicated in diverse physiological processes in estrogen signal pathway in mammals, while little is known about them in fishes. Complete mRNA sequence of ERRalpha from medaka (Oryzias latipes) was cloned, and the sequence is similar to those of other vertebrates, especially that the DNA-binding domain (DBD) of ERRalpha is highly conserved among the vertebrates (97.4%-100% sequence identities) and the ligand-binding domain (LBD) of medaka ERRalpha is 66.4%-67.0% sequence identities with those of mammals. The DBD of medaka ERRalpha is of the same length and has high sequence identity with those of estrogen receptor (ERalpha and ERbeta) and androgen receptor (ARalpha and ARbeta) of medaka, but much difference was found between the LBD of medaka ERRalpha with those of ERalpha, ERbeta, ARalpha and ARbeta. ERRalpha gene is located in chromosome 14 and is consisted of 5 exons. The expressions of ERRalpha in different tissues and the transcriptional responses of ERRalpha in testis of medaka exposed differential EDCs were studied by quantitative real-time RT-PCR. ERRalpha is expressed at apparently high levels in gonad, brain, eye, spleen and intestine, though it was broadly expressed in tissues. Significant transcriptional difference was found between testis and ovary, implying ERRalpha would be involved in sex differentiation and gonad development in fish. After 3 weeks exposure of medaka to 200 ng/L ethynylestradiol (EE2), 200 ng/L estrone (E1), 200 ng/L diethylstilbestrol (DES), 100 microg/L atrazine (AT) and 200 ng/L 17beta-estradiol (E2), transcripts of ERRalpha were significantly decreased to 0.54, 0.56, 0.61, 0.63 and 0.65 of control (p < 0.05) in the testes, respectively. And those in the 1 microg/L tributyltin (TBT) and 1 microg/L triphenyltin (TPT) exposure groups were up-regulated to 1.34 and 1.35 folds of control (p > 0.05), respectively. These results suggested that ERRalpha would take actions in the disruption of sex differentiation and gonad development in fish by EDCs. In addition, no multiple steroid hormone-response element half-sites was found in medaka, which were reported in the upstream of ERRalpha gene in mammals, indicating there would be different regulation patters of ERRalpha between teleost and mammal.