D-glucan Synthase Research Articles

Natural Enfumafungin Analogues from Hormonema carpetanum and Their Antifungal Activities.

Ibrexafungerp, an inhibitor of fungal β-(1,3)-d-glucan synthase, represents the first new class of antifungals to be approved in the last 20 years. Ibrexafungerp is a semisynthetic derivative of the naturally occurring triterpene glycoside enfumafungin. In order to search for new analogues of enfumafungin and to probe its biosynthesis, we undertook a reinvestigation of Hormonema carpetanum, which led to the isolation of two new analogues, enfumafungins B and C, together with enfumafungin. Due to the presence of a hemiacetal moiety in the structure, the enfumafungins appear as a mixture of two interconverting epimers during both the purification process and NMR data acquisition. The structure elucidation, including the differentiation of 25S* and 25R* epimers, was completed by combined analyses of NMR and MS spectroscopic data. The discovery of enfumafungins B and C may have implications for enfumafungin biosynthesis. The antifungal activity of enfumafungins B and C was significantly lower than that of enfumafungin, suggesting that the C-2 substituents and the C-19 carboxy acid are important for activity. Molecular docking simulations revealed significant hydrogen bond interactions between enfumafungins and β-(1,3)-d-glucan synthase, which may be useful for developing new antifungal agents.

Potential Treatment of Dermatophyte Trichophyton rubrum in Rat Model Using Topical Green Biosynthesized Silver Nanoparticles with Achillea santolina Extract

Trichophyton rubrum is the most common dermatophyte, and can cause cutaneous infections in humans and animals (dermatophytosis). In this study, we investigated the anti-dermatophytic potential of green synthesized silver nanoparticles using Achillea santolina extract (AS-AgNPs) in an in vitro and in vivo rat model of dermal T. rubrum dermatophytosis (TRD). The green synthesis of AS-AgNPs was performed using A. santolina extract and characterized by UV-VIS spectroscopy, zeta potential, imaging (transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Energy dispersive X-ray analysis (EDX). The antifungal activity of AS-AgNPs was determined by the broth microdilution method, conidial germination, and hyphal growth inhibition. TEM and SEM were used to study the mode of the antifungal action of AS-AgNPs. AS-AgNPs inhibited the growth of T. rubrum with an MIC of 128 μg/mL, and suppressed the conidial germination and hyphal growth by 55.3% 84.6%, respectively. AS-AgNPs caused modified mycelial structures, increased cell membrane permeability, and cell wall damage. AS-AgNPs significantly increase the permeability of the fungal membrane, as revealed by reducing ergosterol biosynthesis. An increase in the intracellular ROS and the induction of apoptosis were also observed during AS-AgNP treatment. In addition, AS-AgNPs reduced the cell wall integrity, as shown by the reduction in the β-(1,3)-d-glucan synthase and chitin synthase activities. AS-AgNPs showed very low toxicity on primary human dermal fibroblasts (HDF) at the MIC. The topical treatment of the infected skin in the TRD rat model with AS-AgNPs showed a significant reduction in the fugal burden after 7 days and a complete clearance of fungal conidia, with a high recovery of epidermal and dermal structures after 14 days, compared to control rats. Interestingly, AS-AgNPs significantly attenuated the infiltrated inflammatory cells, in association with reducing the tissue proinflammatory cytokines including TNF-α, IL-1, IL-6, MOP and IL-17. In conclusion, our data prove AS-AgNPs to be a novel green topical therapy for dermatophytosis caused by T. rubrum.

Serine/Threonine Phosphatase Calcineurin Orchestrates the Intrinsic Resistance to Micafungin in the Human-Pathogenic Fungus Mucor circinelloides.

Procedures such as solid-organ transplants and cancer treatments can leave many patients in an immunocompromised state. This leads to their increased susceptibility to opportunistic diseases such as fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Recently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available is the echinocandins. Echinocandins seem to be efficacious in the treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have little to no effect on Mucorales fungi. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding the echinocandin target protein β-(1,3)-d-glucan synthase (fksA, fksB, and fksC). Interestingly, we found that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes, resulting in an increased accumulation of β-(1,3)-d-glucan on the cell walls. However, this overexpression of the fks genes is not directly connected to the intrinsic resistance. Subsequent investigation discovered that the serine/threonine phosphatase calcineurin regulates the expression of fksA and fksB, and the deletion of calcineurin results in a decrease in expression of all three fks genes. Deletion of calcineurin also results in a lower minimum effective concentration (MEC) of micafungin. In addition, we found that duplication of the fks gene is also responsible for the intrinsic resistance, in which lack of either fksA or fksB led a lower MEC of micafungin. Together, these findings demonstrate that calcineurin and fks gene duplication contribute to the intrinsic resistance to micafungin we observe in M. circinelloides.

Antibacterial, Anticandidal, and Antibiofilm Potential of Fenchone: In Vitro, Molecular Docking and In Silico/ADMET Study.

The aim of the present study is to investigate the effective antimicrobial and antibiofilm properties of fenchone, a biologically active bicyclic monoterpene, against infections caused by bacteria and Candida spp. The interactions between fenchone and three distinct proteins from Escherichia coli (β-ketoacyl acyl carrier protein synthase), Candida albicans (1, 3-β–D-glucan synthase), and Pseudomonas aeruginosa (Anthranilate-CoA ligase) were predicted using molecular docking and in silico/ADMET methods. Further, to validate the in-silico prediction, the antibacterial and antifungal potential of fenchone was evaluated against E. coli, P. aeruginosa, and C. albicans by determining minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and minimum fungicidal concentration (MFC). The lowest MIC/MBC values of fenchone against E. coli and P. aeruginosa obtained was 8.3 ± 3.6/25 ± 0.0 and 266.6 ± 115.4/533.3 ± 230.9 mg/mL, respectively, whereas the MIC/MFC value for C. albicans was found to be 41.6 ± 14.4/83.3 ± 28.8 mg/mL. It was observed that fenchone has a significant effect on antimicrobial activity (p < 0.05). Our findings demonstrated that fenchone at 1 mg/mL significantly reduced the production of biofilm (p < 0.001) in E. coli, P. aeruginosa, and C. albicans by 70.03, 64.72, and 61.71%, respectively, in a dose-dependent manner when compared to control. Based on these results, it has been suggested that the essential oil from plants can be a great source of pharmaceutical ingredients for developing new antimicrobial drugs.

Review on Current Status of Echinocandins Use.

Fungal infections are rising all over the world every year. There are only five medical compound classes for treatment: triazoles, echinocandins, polyenes, flucytosine and allylamine. Currently, echinocandins are the most important compounds, because of their wide activity spectrum and much lower sides effects that may occur during therapy with other drugs. Echinocandins are secondary metabolites of fungi, which can inhibit the biosynthesis of β-(1,3)-D-glucan. These compounds have fungicidal and fungistatic activity depending on different genera of fungi, against which they are used. Echinocandin resistance is rare—the major cause of resistance is mutations in the gene encoding the β-(1,3)-D-glucan synthase enzyme. In this review of the literature we have summarized the characteristics of echinocandins, the mechanism of their antifungal activity with pharmacokinetics and pharmacodynamics, and the resistance issue.

Virtual screening for potential inhibitors of β(1,3)-D-glucan synthase as drug candidates against fungal cell wall

Background To enhance the outcome in patients with invasive candidiasis, initiation of an efficient antifungal treatment in a suitable dosage is necessary. Echinocandins (e.g. caspofungin) inhibit the enzyme β(1,3)-D-glucan synthase of the fungal cell wall. Compared to azoles and other antifungal agents, echinocandins have lower adverse effects and toxicity in humans. Echinocandins are available in injectable (intravenous) form. Methods In this study, to identify the novel oral drug-like compounds that affect the fungal cell wall, downloaded oral drug-like compounds from the ZINC database were processed with a virtual screening procedure. The docking free energies were calculated and compared with the known inhibitor caspofungin. Four molecules were selected as the most potent ligands and subjected to hydrogen bonds analysis. Results Considering the hydrogen bond analysis, two compounds (ZINC71336662 and ZINC40910772) were predicted to better interact with the active site of β(1,3)-D-glucan synthase compared with caspofungin. Conclusion The introduced compound in this study may be valuable to analyze experimentally as a novel oral drug candidate targeting fungal cell walls.

Effects of microwave exposure on the enzymatic activity of Trichophyton rubrum

Objective To investigate the effects of exposing Trichophyton rubrum fungus to microwaves at different intensities in terms of the activity of succinic dehydrogenase and beta-(1, 3)-D-glucan synthase. Methods Trichophyton rubrum organisms were randomly divided into a control group and experimental groups. The experimental groups were incubated at 27 ℃ after direct radiation with 2450 MHz microwaves at 20, 40, 60 or 80 W for 15 min, repeated 8 times. The control group was incubated without any irradiation. Thirty days later, the beta-(1, 3)-D-glucan synthase and succinate dehydrogenase activities were determined using enzyme-linked immunosorbent assays. Results The enzymatic activity decreased gradually with increasing radiation intensity. When the output power was 80 W, the beta-(1, 3)-glucan-synthase-D activity was 0.730±0.74 U/ml and that of the succinate dehydrogenase was 1.828±1.774 U/L, both significantly lower than in the groups subjected to less powerful irradiation. Conclusions Microwave radiation can decrease the enzymatic activity of Trichophyton rubrum in a dose-dependent manner. Higher intensity is more effective. Microwave irradiation can decrease the activity of succinate dehydrogenase and beta-(1, 3)-glucan synthase from Trichophyton rubrum in vitro, resulting in the destruction of fungal cell walls and interfering with the tricarboxylic acid cycle, furthering cell death. Moreover, the temperature change possibly also helps promote the biological effects of microwave radiation. Key words: Microwaves; Trichophyton rubrum

Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain fks1 mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins.IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

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

Micafungin belongs to the antifungal family of echinocandins, which act as noncompetitive inhibitors of the fungal cell wall β-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 β-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 β-glucans on the cell wall surface.

Emodin Reduces the Activity of (1,3)-β-D-glucan Synthase from Candida albicans and Does Not Interact with Caspofungin.

Candidiasis is the most common opportunistic yeast infection, with Candida albicans as a paramount causative species. (1,3)-β-D-glucan is one of the three main targets of clinically available antifungal agents used to treat Candida infections. It is one of the most abundant fungal cell wall components. Echinocandins represent the newest class of antifungals affecting cell wall biosynthesis through non-competitive inhibition of (1,3)-β-D-glucan synthase. Therefore, treatment with echinocandins causes defects in fungal cell integrity. In the present study, similar activity of emodin (6-methyl-1,3,8-trihydroxyanthraquinone) has been revealed. Many reports have already shown the antifungal potential of this pleiotropic molecule, including its activity against C. albicans. The aim of this report was to evaluate the activity of emodin towards a new molecular target, i.e. (1,3)-β-D-glucan synthase isolated from Candida cells. Moreover, given the identical mechanism of the activity of both molecules, interaction of emodin with caspofungin was determined. The study revealed that emodin reduced (1,3)-β-D-glucan synthase activity and increased cell wall damage, which was evidenced by both a sorbitol protection assay and an aniline blue staining assay. Furthermore, the synergy testing method showed mainly independence of the action of both tested antifungal agents, i.e. emodin and caspofungin used in combination.

De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That both Overlap and Are Distinct from Those Conferring Echinocandin Resistance.

SCY-078 is an orally active antifungal whose target is the β-(1,3)-d-glucan synthase (GS). We evaluated the spontaneous emergence of SCY-078-resistant Candida glabrata isolates following drug exposure in vitro Resistant isolates were analyzed using broth microdilution methodology and FKS sequencing. The kinetic inhibition parameter IC50 (50% inhibitory concentration) was also determined from GS complexes. The spectrum of resistance mutations found suggested a partially overlapping but independent binding site for SCY-078 relative to echinocandins on GS.

Antifungal Activity of Salvia miltiorrhiza Against Candida albicans Is Associated with the Alteration of Membrane Permeability and (1,3)-β-D-Glucan Synthase Activity.

Candidiasis has posed a serious health risk to immunocompromised patients owing to the increase in resistant yeasts, and Candida albicans is the prominent pathogen of fungal infections. Therefore, there is a critical need for the discovery and characterization of novel antifungals to treat infections caused by C. albicans. In the present study, we report on the antifungal activity of the ethanol extract from Salvia miltiorrhiza against C. albicans and the possible mode of action against C. albicans. The increase in the membrane permeability was evidenced by changes in diphenylhexatriene binding and release of both 260-nm-absorbing intracellular materials and protein. In addition, inhibition of cell wall synthesis was demonstrated by the enhanced minimal inhibitory concentration in the presence of sorbitol and reduced (1,3)-β-D-glucan synthase activity. The above evidence supports the notion that S. miltiorrhiza has antifungal activity against C. albicans by the synergistic activity of targeting the cell membrane and cell wall. These findings indicate that S. miltiorrhiza displays effective activity against C. albicans in vitro and merits further investigation to treat C. albicans-associated infections.

Antifungal activities of SCY-078 (MK-3118) and standard antifungal agents against clinical non-Aspergillus mold isolates.

The limited armamentarium of active and oral antifungal drugs against emerging non-Aspergillus molds is of particular concern. Current antifungal agents and the new orally available beta-1,3-d-glucan synthase inhibitor SCY-078 were tested in vitro against 135 clinical non-Aspergillus mold isolates. Akin to echinocandins, SCY-078 showed no or poor activity against Mucoromycotina and Fusarium spp. However, SCY-078 was highly active against Paecilomyces variotii and was the only compound displaying some activity against notoriously panresistant Scedosporium prolificans.

Antifungal Breakpoints of Non-albicans Candida Clinical Isolates in Vitek-2 Compact.

Sir, The incidence and diversity of infections caused by non-albicans Candida species (NAC) have shown a substantial increase in recent years. Given the growing number of susceptible patients with compromised immunity as a consequence of HIV infection, cancer chemotherapy and transplantation associated immunosuppression, NAC has emerged as an eminent fungal pathogen worldwide. Pertaining to the diverse mechanisms of resistance to various antifungals agents among NAC, it has become imperative to identify the effective antifungals. A prospective study was conducted in Department of Microbiology in our tertiary care hospital from May to August, 2014. A total of 36 candida isolates (16 C. albicans, 11 C. tropicalis, 5 C. krusei, 2 C. glabrata and 2 C. parapsilosis) recovered from blood and urine were identified as per standard procedures. The NAC isolates (n=20) were selected for antifungal susceptibility testing (AFST) in Vitek-2 Compact using AST-YS07 card. MIC for fluconazole, voriconazole, caspofungin, micafungin and flucytosine were interpreted [Table/Fig-1] as per CLSI guideline [1]. Among the various AFST methods currently in use for yeasts, macrobroth dilution, microbroth dilution and disc diffusion tests are recommended by CLSI [2]. However, the utility of these tests are limited by the technically challenging procedure, difficulty of inoculums standardization and subjective variation in interpretation. Unlike bacterial pathogens, the slow growth rate of candida also precludes a short turnaround time. Early initiation of appropriate therapy is a critical factor determining the clinical outcome. The introduction of automated AFST has ensured prompt results in a standardized platform. However, there is a lack of published data of fungal resistance pattern determined by automated system in India. In this study, the mean analysis time for determining antifungal susceptibility by Vitek-2 Compact was 14.15 ± 2.49 hour. Vitek-2 utilizes a self-calibrating optic system comprising of light emitting diodes and photo detectors to continually monitor the growth of organisms inside the wells of the test cards, reducing the analysis time [3]. All five C. krusei isolates had fluconazole and flucytosine resistance with MIC > 8μg/dl. Two C. tropicalis strains showed fluconazole resistance and one had intermediate susceptibility to flucytosine. The median MICs for fluconazole, voriconazole, caspofungin, micafungin and flucytosine among C. tropicalis were 1, 0.12, 0.25, 0.06 and 18μg/dl respectively. In C. krusei the median MICs for these drugs were 16, 0.12, 0.25, 0.12 and 8μg/dl respectively. Our findings are in accordance with other studies. Tellapragada et al., found all NAC isolates except C. krusei were uniformly sensitive to fluconazole, while one C. parapsilosis and one C. tropicalis strain displayed voriconazole and caspofungin resistance respectively [4]. Echinocandins are novel synthetic lipopeptide antifungals which non-competitively inhibit the fungal β [1,3]-D-glucan synthase enzyme [5]. Echinocandin resistance among NAC species have been mainly reported in C. parapsilosis with FKS1 mutation [5]. In our study, caspofungin and micafungin were sensitive in all isolates, indicating their critical role in treatment of infections by azole resistant strains. However, further study with a larger sample size comparing Vitek-2 Compact results with other methods of MIC testing is required to confirm our findings. In conclusion, Vitek-2 Compact is essential in rapid determination of antifungal susceptibility of candida. However, the cost constraints may deter its incorporation in routine diagnostic protocol in resource poor settings. [Table/Fig-1]: Resistance pattern of non-albicans candida isolates All isolates were uniformly sensitive to voriconazole, caspofungin and micafungin

Deletion of AIF1 but not of YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death.

Caspofungin was the first member of a new class of antifungals called echinocandins to be approved by a drug regulatory authority. Like the other echinocandins, caspofungin blocks the synthesis of β(1,3)-D-glucan of the fungal cell wall by inhibiting the enzyme, β(1,3)-D-glucan synthase. Loss of β(1,3)-D-glucan leads to osmotic instability and cell death. However, the precise mechanism of cell death associated with the cytotoxicity of caspofungin was unclear. We now provide evidence that Saccharomyces cerevisiae cells cultured in media containing caspofungin manifest the classical hallmarks of programmed cell death (PCD) in yeast, including the generation of reactive oxygen species (ROS), the fragmentation of mitochondria, and the production of DNA strand breaks. Our data also suggests that deleting AIF1 but not YCA1/MCA1 protects S. cerevisiae and Candida albicans from caspofungin-induced cell death. This is not only the first time that AIF1 has been specifically tied to cell death in Candida but also the first time that caspofungin resistance has been linked to the cell death machinery in yeast.

Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-D-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Computational analysis of phytocompounds with 1,3-\�-D-Glucan synthase for antidermatophytic activity

Anti-dermatophytic activity of some traditionally used medicinal plants of North Karnataka RegionKavitha Sagar and Vidyasagar G.M.

Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence.

In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme β-1,3-d-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, β-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.

Frequency of fks Mutations among Candida glabrata Isolates from a 10-Year Global Collection of Bloodstream Infection Isolates

Among 119 echinocandin non-wild-type (non-WT) Candida glabrata strains from two global surveys, mutations in fks hot spots (HSs) were detected in 28 (from 7 countries and 8 U.S. states): 24 strains (85.7%) had non-WT MICs for micafungin, 22 (78.6%) for anidulafungin, and 25 (89.3%) for caspofungin. The most common FKS substitutions among non-WT strains were at positions F659 (n = 7) and S663 (n = 7). Three isolates displaying WT MIC results had F625Y, L630I, and D632Y substitutions or non-HS mutations. Mutations that have been reported to decrease the echinocandin binding to the 1,3-β-d-glucan synthase were categorized as resistant by applying the new CLSI breakpoint criteria for all three echinocandins.

Characterization of Putative a (1,3)-β-D-glucan (curdlan) Synthase for a Low Molecular Weight Curdlan Biosynthesis from Agrobacterium sp. M503

A β-(1,3)-D-glucan (curdlan) synthase gene for a low molecular weight curdlan biosynthesis, crdSAg, from Agrobacterium sp. M503 was cloned and its encoding protein was characterized by several online protein analysis softwares. The crdSAg consists of 1965-base-pairs Open Reading Frame (ORF) encoding a protein with molecular weight approximate 73.5 kDa, which contains the conserved domain of CESA-CelA_like belonging to glycosyltransferase family 2 (GT2). Moreover, CrdSAg was a membrane protein with seven hydrophobic transmembrance domains. The second structure analysis indicated it was composed of 43.12% α-helix, 17.89% β-sheet, and 38.99% random coil structure. These data will lay a foundation to clarify the biosynthesis mechanism of the low molecular weight curdlan.