Biofilm Formation as a Pathogenicity Factor of Medically Important Fungi

To cause disease, the infectious agent makes use of both invasiveness factors—the pathogen virulence factors—and the ability to resist and evade the host immune system. The success of the infection process is the result of a complex equation involving pathogen interaction with the host, wherein the expression of several virulence factors (and not just one or the other) will favor the establishment of the pathogen in the host. Fungal pathogens are frequently associated with biofilm formation

Beneficial Effects of HIV Peptidase Inhibitors on Fonsecaea pedrosoi: Promising Compounds to Arrest Key Fungal Biological Processes and Virulence

BACKGROUND: Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Current therapy for chromoblastomycosis is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the fact that endemic countries and regions are economically poor. PURPOSE AND PRINCIPAL FINDINGS: In the present work, we have investigated the effect of human immunodeficiency virus (HIV) peptidase inhibitors (PIs) on the F. pedrosoi conidial secreted peptidase, growth, ultrastructure and interaction with different mammalian cells. All the PIs impaired the acidic conidial-derived peptidase activity in a dose-dependent fashion, in which nelfinavir produced the best inhibitory effect. F. pedrosoi growth was also significantly reduced upon exposure to PIs, especially nelfinavir and saquinavir. PIs treatment caused profound changes in the conidial ultrastructure as shown by transmission electron microscopy, including invaginations in the cytoplasmic membrane, disorder and detachment of the cell wall, enlargement of fungi cytoplasmic vacuoles, and abnormal cell division. The synergistic action on growth ability between nelfinavir and amphotericin B, when both were used at sub-inhibitory concentrations, was also observed. PIs reduced the adhesion and endocytic indexes during the interaction between conidia and epithelial cells (CHO), fibroblasts or macrophages, in a cell type-dependent manner. Moreover, PIs interfered with the conidia into mycelia transformation when in contact with CHO and with the susceptibility killing by macrophage cells. CONCLUSIONS/SIGNIFICANCE: Overall, by providing the first evidence that HIV PIs directly affects F. pedrosoi development and virulence, these data add new insights on the wide-spectrum efficacy of HIV PIs, further arguing for the potential chemotherapeutic targets for aspartyl-type peptidase produced by this human pathogen

Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals

<p>Abstract</p> <p>Background</p> <p>The pathogenic fungus <it>Fonsecaea pedrosoi </it>constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.</p> <p>We evaluated the production of nitric oxide (NO) in macrophages, the oxidative burst and the inducible nitric oxide synthase (i-NOS) activity in interactions between activated murine macrophages and <it>F. pedrosoi</it>. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in <it>F. pedrosoi</it>. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H₂O₂and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated.</p> <p>Results</p> <p>Melanised <it>F. pedrosoi </it>cells were more resistant to both H₂O₂and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control <it>F. pedrosoi </it>or TC-treated <it>F. pedrosoi</it>. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages.</p> <p>Conclusion</p> <p>The NO-trapping ability of <it>F. pedrosoi </it>melanin is an important mechanism to escape the oxidative burst of macrophages.</p

Growth inhibition and ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors in Candida spp. isolates, including non-albicans organisms

<p>Abstract</p> <p>Background</p> <p>Although <it>Candida </it>species are commensal microorganisms, they can cause many invasive fungal infections. In addition, antifungal resistance can contribute to failure of treatment.</p> <p>The purpose of this study was to evaluate the antifungal activity of inhibitors of Δ²⁴⁽²⁵⁾-sterol methyltransferase (24-SMTI), 20-piperidin-2-yl-5α-pregnan-3β-20(R)-diol (AZA), and 24(R,S),25-epiminolanosterol (EIL), against clinical isolates of <it>Candida </it>spp., analysing the ultrastructural changes.</p> <p>Results</p> <p>AZA and EIL were found to be potent growth inhibitors of <it>Candida </it>spp. isolates. The median MIC₅₀was 0.5 μg.ml^-1for AZA and 2 μg.ml^-1for EIL, and the MIC₉₀was 2 μg.ml^-1for both compounds. All strains used in this study were susceptible to amphotericin B; however, some isolates were fluconazole- and itraconazole-resistant. Most of the azole-resistant isolates were <it>Candida </it>non-<it>albicans </it>(CNA) species, but several of them, such as <it>C. guilliermondii, C. zeylanoides</it>, and <it>C. lipolytica</it>, were susceptible to 24-SMTI, indicating a lack of cross-resistance. Reference strain <it>C. krusei </it>(ATCC 6258, FLC-resistant) was consistently susceptible to AZA, although not to EIL. The fungicidal activity of 24-SMTI was particularly high against CNA isolates. Treatment with sub-inhibitory concentrations of AZA and EIL induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. Fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts. The selectivity of 24-SMTI for fungal cells versus mammalian cells was assessed by the sulforhodamine B viability assay.</p> <p>Conclusion</p> <p>Taken together, these results suggest that inhibition of 24-SMT may be a novel approach to control <it>Candida </it>spp. infections, including those caused by azole-resistant strains.</p

The role of hydrophobicity and surface receptors at hyphae of <i>Lyophyllum</i> sp. strain Karsten in the interaction with <i>Burkholderia terrae</i> BS001:Implications for interactions in soil

The soil bacterium Burkholderia terrae strain BS001 can interact with varying soil fungi, using mechanisms that range from the utilization of carbon/energy sources such as glycerol to the ability to reach novel territories in soil via co-migration with growing fungal mycelia. Here, we investigate the intrinsic properties of the B. terrae BS001 interaction with the basidiomycetous soil fungus Lyophyllum sp. strain Karsten. In some experiments, the ascomycetous Trichoderma asperellum 302 was also used. The hyphae of Lyophyllum sp. strain Karsten were largely hydrophilic on water-containing media versus hydrophobic when aerial, as evidenced by contact angle analyses (CA). Co-migration of B. terrae strain BS001 cells with the hyphae of the two fungi occurred preferentially along the - presumably hydrophilic - soil-dwelling hyphae, whereas aerial hyphae did not allow efficient migration, due to reduced thickness of their surrounding mucous films. Moreover, the cell numbers over the length of the hyphae in soil showed an uneven distribution, i.e. the CFU numbers increased from minima at the inoculation point to maximal numbers in the middle of the extended hyphae, then decreasing towards the terminal side. Microscopic analyses of the strain BS001 associations with the Lyophyllum sp. strain Karsten hyphae in the microcosms confirmed the presence of B. terrae BS001 cells on the mucous matter that was present at the hyphal surfaces of the fungi used. Cell agglomerates were found to accumulate at defined sites on the hyphal surfaces, which were coined ‘fungal-interactive’ hot spots. Evidence was further obtained for the contention that receptors for a physical bacterium-fungus interaction occur at the Lyophyllum sp. strain Karsten hyphal surface, in which the specific glycosphingolipid ceramide mono hexoside (CMH) plays an important role. Thus, bacterial adherence may be mediated by heterogeneously-distributed fungal-specific receptors, implying the CMH moieties. This study sheds light on the physical aspects of the B. terrae BS001 – L. sp strain Karsten interaction, highlighting heterogeneity along the hyphae with respect to hydrophobicity and the presence of potential anchoring sites

Proanthocyanidins polymeric tannin from Stryphnodendron adstringens are active against Candida albicans biofilms

Abstract\ud \ud Background\ud Biofilm formation is important in Candida albicans pathogenesis and constitutes a mechanism of antifungal resistance. Thus, we evaluated the effect of proanthocyanidins polymer-rich fractions from Stryphnodendron adstringens (fraction F2 and subfraction F2.4) against C. albicans biofilms.\ud \ud \ud Methods\ud Firstly, the antifungal activity of F2 and F2.4 against planktonic cells of Candida albicans (ATCC 10231) was determined using broth microdilution method. Anti-biofilm effect of F2 and F2.4 was evaluated during biofilm formation or on mature biofilm of C. albicans and compared with standard antifungals amphotericin B and fluconazole. Metabolic activity of sessile and dispersion cells from biofilms after antifungal treatments were measured using a tetrazolium reduction assay and the biofilm total biomass was quantified by crystal violet-based assay. Morphological alterations after treatments were observed using scanning electron microscopy.\ud \ud \ud Results\ud The anti-biofilm effect of F2 and F2.4 were comparable to standard antifungals (amphotericin B and fluconazole). F2 and F2.4 treatments reduced biofilm metabolic activity (in sessile and in dispersion cells) during biofilm formation, and in mature biofilms, unlike fluconazole, which only prevents the biofilm formation. Treatments with F2, F2.4 or fluconazole reduced biofilm biomass during biofilm formation, but not in mature biofilm. Amphotericin B presented higher inhibitory effect on biofilm formation and on mature biofilm of C. albicans. F2 and F2.4 treatments led to the appearance of dumbbell-shaped blastoconidia and of blastoconidia clusters in biofilms.\ud \ud \ud Conclusion\ud Proanthocyanidins polymer-rich fractions from S. adstringens successfully inhibited C. albicans planktonic growth and biofilm development, and they represent a potential new agent for the treatment of biofilm-associated candidiasis.This work was supported by Fundação de Amparo à Pesquisa\ud do Estado de São Paulo (FAPESP - Processo no. 2013/11232-0), Fundação\ud Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro\ud (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior\ud (CAPES), and Conselho Nacional de Desenvolvimento e Pesquisa (CNPq).\ud RLFL and TVMV are fellows of the CNPq

Synergism effect of the essential oil from Ocimum basilicum var. Maria Bonita and its major components with fluconazole and its influence on ergosterol biosynthesis

The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 mg/mL against C. neoformans and 152 mg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 mg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics