A protocol for ultrastructural study of Candida albicans biofilm using transmission electron microscopy.

This protocol describes how to analyze C. albicans biofilm using transmission electron microscopy. We present two approaches to observe the ultrastructure of fungal cells within unperturbed biofilms, as well as an immunogold labeling procedure. This approach maintains the architecture of the fungal biofilm close to its native state by growing C. albicans biofilm on a plastic surface. After the freeze substitution procedure, classical transmission electron microscopy or electron tomography will allow the ultrastructural analysis of the microbial community.

Metabolic, structural, and proteomic changes in Candida albicans cells induced by the protein-carbohydrate fraction of Dendrobaena veneta coelomic fluid.

The isolated protein-polysaccharide fraction (AAF) from the coelomic fluid of Dendrobaena veneta earthworm shows effective activity against Candida albicans yeast. Fungal cells of the clinical strain after incubation with the active fraction were characterized by disturbed cell division and different morphological forms due to the inability to separate the cells from each other. Staining of the cells with acridine orange revealed a change in the pH of the AAF-treated cells. It was observed that, after the AAF treatment, the mitochondrial DNA migrated towards the nuclear DNA, whereupon both merged into a single nuclear structure, which preceded the apoptotic process. Cells with a large nucleus were imaged with the scanning electron cryomicroscopy (Cryo-SEM) technique, while enlarged mitochondria and the degeneration of cell structures were shown by transmission electron microscopy (TEM). The loss of the correct cell shape and cell wall integrity was visualized by both the TEM and SEM techniques. Mass spectrometry and relative quantitative SWATH MS analysis were used to determine the reaction of the C. albicans proteome to the components of the AAF fraction. AAF was observed to influence the expression of mitochondrial and oxidative stress proteins. The oxidative stress in C. albicans cells caused by the action of AAF was demonstrated by fluorescence microscopy, proteomic methods, and XPS spectroscopy. The secondary structure of AAF proteins was characterized by Raman spectroscopy. Analysis of the elemental composition of AAF confirmed the homogeneity of the preparation. The observed action of AAF, which targets not only the cell wall but also the mitochondria, makes the preparation a potential antifungal drug killing the cells of the C. albicans pathogen through apoptosis.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

First report of antifungal activity of CecropinA-Magenin2 (CE-MA) hybrid peptide and its truncated derivatives.

The use of natural antimicrobial peptides (AMPs) is limited. Modifications of peptides by in silico predictions and computational methods can lead to more accurate designs and reducing their high synthesis costs, instability, and cytotoxicity. In this study, the antifungal properties of CecropinA-Magenin2 (CE-MA) hybrid peptide and its truncated derivatives were evaluated. Eleven C-terminal-truncated derivatives were designed and three of them with 10, 8 and 6 residues namely CMt1, CMt2 and CMt3 were selected through an initial screening based on the prediction of antimicrobial and antifungal activities, toxicity and physicochemical properties. These derivatives and the parental CE-MA peptide were synthesized. Then, based on molecular docking studies, antimicrobial tests and cytotoxicity assays, CMt1 peptide was selected for further studies such as time of killing, combinatorial effects with other drugs and the mechanism of action. The results showed that CE-MA is a weak antifungal peptide but its truncated derivative, CMt1 showed a strong antifungal activity with less toxicity. The results of the ergosterol assay, confocal microscopy and FE-SEM studies indicated that invasion to cell wall and membrane components were the main antifungal mechanisms of CMt1 peptide. Altogether, here we introduce a new truncated peptide with a strong antifungal activity with less toxicity which can be a good candidate for further in vivo and clinical studies to be used as an antifungal drug.

Structural analyses of PCNA from the fungal pathogen Candida albicans identify three regions with species-specific conformations.

An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. DATABASES: The PDB and SASBDB accession codes for CaPCNA are 7BUP and SASDHQ9, respectively.

In Situ Imaging of Candida albicans Hyphal Growth via Atomic Force Microscopy.

Candida albicans is an opportunistic fungal pathogen of humans known for its ability to cause a wide range of infections. One major virulence factor of C. albicans is its ability to form hyphae that can invade host tissues and cause disseminated infections. Here, we introduce a method based on atomic force microscopy to investigate C. albicans hyphae in situ on silicone elastomer substrates, focusing on the effects of temperature and antifungal drugs. Hyphal growth rates differ significantly for measurements performed at different physiologically relevant temperatures. Furthermore, it is found that fluconazole is more effective than caspofungin in suppressing hyphal growth. We also investigate the effects of antifungal drugs on the mechanical properties of hyphal cells. An increase in Young's modulus and a decrease in adhesion force are observed in hyphal cells subjected to caspofungin treatment. Young's moduli are not significantly affected following treatment with fluconazole; the adhesion force, however, increases. Overall, our results provide a direct means of observing the effects of environmental factors and antifungal drugs on C. albicans hyphal growth and mechanics with high spatial resolution.IMPORTANCECandida albicans is one of the most common pathogens of humans. One important virulence factor of C. albicans is its ability to form elongated hyphae that can invade host tissues and cause disseminated infections. Here, we show the effect of different physiologically relevant temperatures and common antifungal drugs on the growth and mechanical properties of C. albicans hyphae using atomic force microscopy. We demonstrate that minor temperature fluctuations within the normal range can have profound effects on hyphal cell growth and that different antifungal drugs impact hyphal cell stiffness and adhesion in different ways.

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

A designed antifungal peptide with therapeutic potential for clinical drug-resistant Candida albicans.

Due to the increasing drug-resistant of Candida albicans (C. albicans), there is an urgent need to develop a novel therapeutic agent for C. albicans induced inflammatory disease treatment. Antimicrobial peptides (AMPs) are regarded as one of the most promising antifungal drugs. However, most of the designed AMPs showed side-effects. In the present study, 10 novel peptides were designed based on the sequence of frog skin secretions peptide (Ranacyclin AJ). Among them, AKK8 (RWRFKWWKK) exhibited the strongest antifungal effect against both standard and clinically isolated drug-resistant C. albicans. AKK8 killed C. albicans (within 30 min), and the antifungal effect lasted for 24 h, showed an efficient and long lasted antifungal effect against C. albicans. Notably, AKK8 showed low toxicity to human red blood cells and high stability in human serum. Moreover, AKK8 administration showed therapeutic effects on systemic infections mice induced by the clinical drug-resistant C. albicans, in a dose-depended manner. These findings suggested that AKK8 may be a potential candidate for the anti-inflammation treatments for diseases caused by clinical drug-resistant C. albicans.

The effect of benzyl isothiocyanate on Candida albicans growth, cell size, morphogenesis, and ultrastructure.

Candida albicans is a commensal yeast that may become pathogenic and even lethal to the host. Over the last few decades, antifungal resistance has increased, promoting screening of the antifungal potential of old and new substances. This study investigates the antifungal potential of isothiocyanates (ITCs) against C. albicans oral isolates. A preliminary susceptibility disk diffusion test (DD) was performed using allyl isothiocyanate (AITC), benzyl isothiocynanate (BITC) and phenyl ethyl isothiocyanate (PEITC) at a fixed concentration range (0.001-0.1 M). Because C. albicans isolates were more susceptible to BITC and PEITC, their effect on cell size and on germ tube formation (GTF) were tested. The most promising molecule, BITC, was further tested for effects on cell viability, oxidative stress and for ultrastructure. ITCs, especially the aromatic ones, had a significant type-, dose- and isolate-dependent anti-Candida activity. Although BITC and PEITC had similar activity against the yeast cells, BITC had a more pronounced effect on cell size and GTF. Furthermore, BITC appears to induce oxidative stress and promote changes in the cell ultrastructure, interfering with cell wall structure. Our work showed that aromatic ITCs have the potential to effect C. albicans cells in multiple ways, including size, shape and GTF (BITC and PEITC), oxidative stress, and ultrastructure (BITC). Overall, our results suggest that BITC may be effectively used against C. albicans to modulate its growth, and control or suppress its invasive potential.

Comparative Evaluations of the Pathogenesis of Candida auris Phenotypes and Candida albicans Using Clinically Relevant Murine Models of Infections.

The newly emerged Candida species Candida auris is associated with an exponential rise in life-threatening invasive disease in health care facilities worldwide. Unlike other species, C. auris exhibits a high level of transmissibility, multidrug resistance, and persistence in the environment, yet little is known about its pathogenesis largely due to limited data from animal models. Based on in vitro biofilm evaluations and confocal laser scanning microscopy, C. auris phenotypes with different biofilm-forming abilities were identified, indicating potential clinical implications. Using clinically relevant murine models of implanted catheter, oral, and intraperitoneal infections, we comparatively evaluated the host site-specific pathogenic potential of C. auris phenotypes and Candida albicans Based on the results of microbial recovery and scanning electron microscopy analysis of explanted catheters, compared to C. albicans, C. auris more avidly adhered and formed biofilms on catheters. However, although C. auris adhered to oral tissue ex vivo, unlike C. albicans, it failed to colonize the oral cavity in vivo, as demonstrated by microbial recovery and tissue histopathology analysis. In contrast, recovery from peritoneal lavage fluid and kidneys during time course experiments demonstrated that C. auris persisted longer in the peritoneal cavity and kidneys. Although there were clear niche-specific differences in pathogenic features between C. auris and C. albicans, no significant differences were noted between the C. auris phenotypes in vivo The combined findings highlight unique niche-specific pathogenic traits for C. auris warranting further investigations. Understanding the factors contributing to the rise of C. auris as a nosocomial pathogen is critical for controlling the spread of this species.IMPORTANCE The newly emerged Candida species C. auris has been associated with an exponential rise in invasive disease in health care facilities worldwide with a mortality rate approaching 60%. C. auris exhibits a high level of transmissibility, multidrug resistance, and persistence in hospital environments, yet little is known about its pathogenesis largely due to limited data from animal studies. We used clinically relevant murine models of infection to comparatively evaluate the host niche-specific pathogenic potential of C. auris and C. albicans Findings demonstrated that C. auris adheres more avidly, forming robust biofilms on catheters implanted in mice. However, although C. auris adhered to oral tissue ex vivo, it failed to colonize the oral cavity in vivo In contrast, in the intraperitoneal infection model, C. auris persisted longer in the peritoneal cavity and kidneys. Understanding the host-pathogen factors contributing to the rise of C. auris as a nosocomial pathogen is critical for controlling the spread of this species.

Punicalagin triggers ergosterol biosynthesis disruption and cell cycle arrest in Cryptococcus gattii and Candida albicans : Action mechanisms of punicalagin against yeasts.

Punicalagin is a phenolic compound extracted from Lafoensia pacari A. St.-Hil (Lythraceae) leaves. It has demonstrated interesting activity against pathogenic fungi, e.g., Cryptococcus gattii and Candida albicans, by inhibiting fungi growth in a minimum inhibitory concentration (MIC) at 4 µg/mL. However, the mechanisms behind its antifungal action are not well understood. In this study, certain parameters were investigated, by transmission electron microscopy, ergosterol synthesis inhibition, and flow cytometry analyses, to gain insight into the possible biological targets of punicalagin (4 or 16 µg/mL) against yeast cells. Data showed that, in contrast to untreated cells, punicalagin triggered severe ultrastructural changes in C. gattii and C. albicans, such as disorganization of cytoplasmic content and/or thickened cell walls. In addition, it caused a decrease in yeast plasma membrane ergosterol content in a concentration-dependent manner. However, it was unable to bring about significant fungal cell membrane rupture. On the other hand, punicalagin (16 µg/mL) significantly arrested C. albicans and C. gattii cells at the G0/G1 phase, with a consequent reduction in cells at the G2/M phase in both fungi isolates, and thereby prevented progression of the normal yeast cell cycle. However, these alterations showed no involvement of reactive oxygen species overproduction in C. albicans and C. gattii cells, although punicalagin triggered a significant loss of mitochondrial membrane potential in C. albicans. These findings suggest that punicalagin is a promising plant-derived compound for use in developing new antifungal therapies.

Immune modulation by complement receptor 3-dependent human monocyte TGF-ß1-transporting vesicles.

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-ß1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ß-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-ß1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-ß1 to the TGF-ß receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-ß1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-ß1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.

Dodonaea viscosa var angustifolia derived 5,6,8-trihydroxy-7,4' dimethoxy flavone inhibits ergosterol synthesis and the production of hyphae and biofilm in Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: Candida albicans is developing resistance to existing drugs increasing morbidity and mortality, which elevates an immediate need to explore new antifungal agents. Phytochemicals are an excellent source of therapeutic agents. We previously reported the antifungal activity of the crude extract of Dodonaea viscosa var. angustifolia Jacq. (DVA) from which a beneficial compound flavone: 5,6,8-trihydroxy-7,4' dimethoxy flavone (5,6,8-trihydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) abbreviated as TMMC, was extracted. AIM OF THE STUDY: The present study evaluated the effect of a TMMC subfraction on biofilms, membrane stability, ergosterol biosynthesis and germ tube (GT) formation in Candida albicans. MATERIALS AND METHODS: Extracts were prepared and fractionated to obtain purified TMMC. Minimum inhibitory concentrations of TMMC were obtained and subinhibitory concentrations were selected for further studies. Confocal laser scanning microscopy (CLSM) was performed to assess the effect of TMMC on membrane permeability and sterol deposition using propidium iodide (PI) and filipin stains, respectively. RESULTS: Minimum inhibitory concentrations (MIC) and Minimum Fungicidal concentrations (MFC) of TMMC were 0.39 mg/mL and 1.56 mg/mL, respectively. TMMC inhibited biofilm formation and damaged mature biofilms at 0.39 mg/mL and 1.56 mg/mL, respectively. CLSM further confirmed the disruption and architectural changes in biofilms following treatment with TMMC. TMMC also inhibited GT formation and ergosterol biosynthesis in a concentration dependent manner, which was further confirmed by varying sterol distribution and membrane disruption in treated and untreated cells. CONCLUSIONS: With the multiple targets at different concentrations, TMMC warrants its potential use as antifungal drug against C. albicans. However further studies using animal models and more mechanistic approaches will be required.

The Interactions between the Antimicrobial Peptide P-113 and Living Candida albicans Cells Shed Light on Mechanisms of Antifungal Activity and Resistance.

In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi's resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.

A semisynthetic borrelidin analogue BN-3b exerts potent antifungal activity against Candida albicans through ROS-mediated oxidative damage.

In the process of investigating the antifungal structure-activity relationships (SAR) of borrelidin and discovering antifungal leads, a semisynthetic borrelidin analogue, BN-3b with antifungal activity against Candida albicans, was achieved. In this study, we found that oxidative damage induced by endogenous reactive oxygen species (ROS) plays an important role in the antifungal activity of BN-3b. Further investigation indicated that BN-3b stimulated ROS accumulation, increased malondialdehyde (MDA) levels, and decreased reduced/oxidized glutathione (GSH/GSSG) ratio. Moreover, BN-3b decreased mitochondrial membrane potential (MMP) and ATP generation. Ultrastructure analysis revealed that BN-3b severely damaged the cell membrane of C. albicans. Quantitative PCR (RT-qPCR) analysis revealed that virulence factors of C. albicans SAPs, PLB1, PLB2, HWP1, ALSs, and LIPs were all down-regulated after BN-3b exposure. We also found that BN-3b markedly inhibited the hyphal formation of C. albicans. In addition, in vivo studies revealed that BN-3b significantly prolonged survival and decreased fungal burden in mouse model of disseminated candidiasis.

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 µg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens.

INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Gemini quaternary ammonium compound PMT12-BF4 inhibits Candida albicans via regulating iron homeostasis.

Quaternary ammonium compounds (QACs) are classified as cationic surfactants, and are known for their biocidal activity. However, their modes of action are thus far not completely understood. In this study, we synthesized a gemini QAC, PMT12-BF4 and found that it exerted unsurpassed broad-spectrum antifungal activity against drug susceptible and resistant Candida albicans, and other pathogenic fungi, with a minimal inhibitory concentration (MIC) at 1 or 2 µg/mL. These results indicated that PMT12-BF4 used a mode of action distinct from current antifungal drugs. In addition, fungal pathogens treated with PMT12-BF4 were not able to grow on fresh YPD agar plates, indicating that the effect of PMT12-BF4 was fungicidal, and the minimal fungicidal concentration (MFC) against C. albicans isolates was 1 or 2 µg/mL. The ability of yeast-to-hyphal transition and biofilm formation of C. albicans was disrupted by PMT12-BF4. To investigate the modes of action of PMT12-BF4 in C. albicans, we used an RNA sequencing approach and screened a C. albicans deletion mutant library to identify potential pathways affected by PMT12-BF4. Combining these two approaches with a spotting assay, we showed that the ability of PMT12-BF4 to inhibit C. albicans is potentially linked to iron ion homeostasis.