Comparative Biophysical and Ultrastructural Analysis of Melanins Produced by Clinical Strains of Different Species From the Trichosporonaceae Family.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii, which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii, Trichosporon japonicum, Apiotrichum montevideense, Trichosporon inkin, Trichosporon faecale, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.

Surface, adhesiveness and virulence aspects of Candida haemulonii species complex.

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

Phosphorus-rich structures and capsular architecture in Cryptococcus neoformans.

AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.

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

UNLABELLED: 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.

Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans.

Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 µM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.