Evaluation of the in vitro and in vivo dimorphism ofSporothrix schenckii,Blastomyces dermatitidis, andParacoccidioides brasiliensisisolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 °C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 °C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 °C and their micromorphology showed thin filaments with single hyaline conidia. At 36 °C the colonies did not differ from those grown at 25 °C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 °C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.Key words: pathogenic fungi, in vitro storage, in vivo passage, morphogenesis.

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Synthesis of Melanin-Like Pigments by Sporothrix schenckii In Vitro and during Mammalian Infection

Infection and Immunity ◽

10.1128/iai.71.7.4026-4033.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 4026-4033 ◽

Cited By ~ 61

Author(s):

Rachael Morris-Jones ◽

Sirida Youngchim ◽

Beatriz L. Gomez ◽

Phil Aisen ◽

Roderick J. Hay ◽

...

Keyword(s):

Fungal Pathogens ◽

Proteolytic Enzymes ◽

Sporothrix Schenckii ◽

Brain Heart Infusion ◽

Pathogenic Fungi ◽

Yeast Cells ◽

Important Virulence Factor ◽

Mammalian Infection

ABSTRACT Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

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Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

mBio ◽

10.1128/mbio.00647-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 64

Author(s):

Visesato Mor ◽

Antonella Rella ◽

Amir M. Farnoud ◽

Ashutosh Singh ◽

Mansa Munshi ◽

...

Keyword(s):

Cell Cycle Progression ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Sequencing Analysis ◽

Fungal Cell ◽

New Class ◽

Narrow Spectrum

ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

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DC-SIGN targets amphotericin B-loaded liposomes to diverse pathogenic fungi

Fungal Biology and Biotechnology ◽

10.1186/s40694-021-00126-3 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Suresh Ambati ◽

Tuyetnhu Pham ◽

Zachary A. Lewis ◽

Xiaorong Lin ◽

Richard B. Meagher

Keyword(s):

Amphotericin B ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Protein Isoforms ◽

Viral Pathogens ◽

Life Threatening ◽

Better Than

Abstract Background Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans. Results We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs. Conclusions DC-SIGN’s CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.

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Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00165-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 7

Author(s):

Mariane C. Bagatin ◽

Arethusa L. Pimentel ◽

Débora C. Biavatti ◽

Ernani A. Basso ◽

Erika S. Kioshima ◽

...

Keyword(s):

Tertiary Structure ◽

Fungal Infections ◽

Paracoccidioides Brasiliensis ◽

Pathogenic Fungi ◽

Dynamics Simulation ◽

Homoserine Dehydrogenase ◽

Content Type ◽

Zinc Database ◽

Etiological Agents

ABSTRACT This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 μg · ml−1, respectively, for the Pb18 isolate of P. brasilensis, 64 μg · ml−1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

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Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.565571 ◽

2021 ◽

Vol 10 ◽

Author(s):

Diego de Souza Gonçalves ◽

Claudia Rodriguez de La Noval ◽

Marina da Silva Ferreira ◽

Leandro Honorato ◽

Glauber Ribeiro de Sousa Araújo ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Histoplasma Capsulatum ◽

Galleria Mellonella ◽

Paracoccidioides Brasiliensis ◽

Pathogenic Fungi ◽

Database Evaluation ◽

Serological Reactivity ◽

Macrophage Killing

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

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In vivo and In vitro Antifungal Activity of 2,3-Dimethylquinoxline

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i31a31684 ◽

2021 ◽

pp. 198-207

Author(s):

Abdelbagi Alfadil ◽

Hamoud A. Alsamhan ◽

Ahmed S. Ali ◽

Huda M. Alkreathy ◽

Mohammad W. Alrabia ◽

...

Keyword(s):

Antifungal Activity ◽

Fungal Infections ◽

Oral Candidiasis ◽

Pathogenic Fungi ◽

Clinical Problem ◽

Preclinical Study ◽

Mice Model ◽

Wide Range

Aims: To explore the antifungal activity of 2,3-dimethylquinoxaline. Study Design: A preclinical study of a compound against 10 fungal species. Backgrounds: Severe fungal infections cause significant clinical problem and need more effort to search for new antifungals. Methodology: We evaluated the susceptibility of 2,3-dimethylquinoxaline in vitro against a wide range of pathogenic fungi, including six Candida species, two Aspergillus species, one Cryptococcus species, and one Trichophyton species. Also, we evaluated the susceptibility of 2,3-dimethylquinoxaline in vivo against oral candidiasis using a mice model. Results: The highest score of the minimum inhibitory concentration was 9 µg/ml against Cryptococcus neoformans. While, the lowest score was 1125 µg/ml against Candida tropicalis. The oral candidiasis in a mouse model was resolved using 2,3-dimethylquinoxaline 1% gel. Conclusion: The 2,3-Dimethyquinoxaline has interesting antifungal activity. Quinoxalines in general need to be further developed as a promising antifungal candidate.

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Recognition of Fungal Components by the Host Immune System

Current Protein and Peptide Science ◽

10.2174/1389203721666191231105546 ◽

2020 ◽

Vol 21 (3) ◽

pp. 245-264 ◽

Cited By ~ 3

Author(s):

Laura C. García-Carnero ◽

José A. Martínez-Álvarez ◽

Luis M. Salazar-García ◽

Nancy E. Lozoya-Pérez ◽

Sandra E. González-Hernández ◽

...

Keyword(s):

Immune System ◽

Histoplasma Capsulatum ◽

Paracoccidioides Brasiliensis ◽

Sporothrix Schenckii ◽

Clinical Importance ◽

Pathogenic Fungi ◽

Diagnostic Tools ◽

Future Research ◽

Host Immune System ◽

Fungal Antigens

: By being the first point of contact of the fungus with the host, the cell wall plays an important role in the pathogenesis, having many molecules that participate as antigens that are recognized by immune cells, and also that help the fungus to establish infection. The main molecules reported to trigger an immune response are chitin, glucans, oligosaccharides, proteins, melanin, phospholipids, and others, being present in the principal pathogenic fungi with clinical importance worldwide, such as Histoplasma capsulatum, Paracoccidioides brasiliensis, Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Sporothrix schenckii. Knowledge and understanding of how the immune system recognizes and responds to fungal antigens are relevant for the future research and development of new diagnostic tools and treatments for the control of mycosis caused by these fungi.

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Conifers Phytochemicals: A Valuable Forest with Therapeutic Potential

Molecules ◽

10.3390/molecules26103005 ◽

2021 ◽

Vol 26 (10) ◽

pp. 3005

Author(s):

Kanchan Bhardwaj ◽

Ana Sanches Silva ◽

Maria Atanassova ◽

Rohit Sharma ◽

Eugenie Nepovimova ◽

...

Keyword(s):

Experimental Data ◽

Potential Role ◽

Therapeutic Potential ◽

Fungal Infections ◽

Cell Damage ◽

Cancer Growth ◽

Naturally Occurring ◽

Antioxidant Effects

Conifers have long been recognized for their therapeutic potential in different disorders. Alkaloids, terpenes and polyphenols are the most abundant naturally occurring phytochemicals in these plants. Here, we provide an overview of the phytochemistry and related commercial products obtained from conifers. The pharmacological actions of different phytochemicals present in conifers against bacterial and fungal infections, cancer, diabetes and cardiovascular diseases are also reviewed. Data obtained from experimental and clinical studies performed to date clearly underline that such compounds exert promising antioxidant effects, being able to inhibit cell damage, cancer growth, inflammation and the onset of neurodegenerative diseases. Therefore, an attempt has been made with the intent to highlight the importance of conifer-derived extracts for pharmacological purposes, with the support of relevant in vitro and in vivo experimental data. In short, this review comprehends the information published to date related to conifers’ phytochemicals and illustrates their potential role as drugs.

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Techniques for the Assessment of In Vitro and In Vivo Antifungal Combinations

Journal of Fungi ◽

10.3390/jof7020113 ◽

2021 ◽

Vol 7 (2) ◽

pp. 113

Author(s):

Anne-Laure Bidaud ◽

Patrick Schwarz ◽

Guillaume Herbreteau ◽

Eric Dannaoui

Keyword(s):

Animal Models ◽

Fungal Infections ◽

Acquired Resistance ◽

Adequate Treatment ◽

Combination Treatments ◽

Target Organs ◽

Fungal Load ◽

Time Kill

Systemic fungal infections are associated with high mortality rates despite adequate treatment. Moreover, acquired resistance to antifungals is increasing, which further complicates the therapeutic management. One strategy to overcome antifungal resistance is to use antifungal combinations. In vitro, several techniques are used to assess drug interactions, such as the broth microdilution checkerboard, agar-diffusion methods, and time-kill curves. Currently, the most widely used technique is the checkerboard method. The aim of all these techniques is to determine if the interaction between antifungal agents is synergistic, indifferent, or antagonistic. However, the interpretation of the results remains difficult. Several methods of analysis can be used, based on different theories. The most commonly used method is the calculation of the fractional inhibitory concentration index. Determination of the usefulness of combination treatments in patients needs well-conducted clinical trials, which are difficult. It is therefore important to study antifungal combinations in vivo, in experimental animal models of fungal infections. Although mammalian models have mostly been used, new alternative animal models in invertebrates look promising. To evaluate the antifungal efficacy, the most commonly used criteria are the mortality rate and the fungal load in the target organs.

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Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

Journal of Fungi ◽

10.3390/jof7020130 ◽

2021 ◽

Vol 7 (2) ◽

pp. 130

Author(s):

Nathan P. Wiederhold

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Candida Species ◽

Mammalian Cells ◽

Mitochondrial Membrane ◽

Fungal Infections ◽

Published Data ◽

Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

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