Ludmila de Matos Baltazar

The role of oxidative and nitrosative bursts caused by azoles and amphotericin B against the fungal pathogen Cryptococcus gattii

OBJECTIVES Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined. METHODS We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger. RESULTS The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger. CONCLUSIONS Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death

OBJECTIVES Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. METHODS We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. RESULTS The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. CONCLUSIONS Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.

Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Cryptococcus gattii: In Vitro Susceptibility to Photodynamic Inactivation

Cryptococus gattii is an emergent primary human pathogen that causes meningismus, papilledema, high intracranial pressure and focal involvement of the central nervous system in immunocompetent hosts. Prolonged antifungal therapy is the conventional treatment, but it is highly toxic, selects for resistant strains, contributes to therapy failure and has a poor prognosis. Photodynamic inactivation (PDI) offers a promising possibility for the alternative treatment of cryptococcosis. The aim of this study was to test the effectiveness of toluidine blue O (TBO) and light‐emitting diode (LED) against C. gattii strains with distinct susceptibility profile to antifungal drugs (amphotericin B: 0.015–1.0 μg mL−1; itraconazole: 0.015–2 μg mL−1; fluconazole: 4–64 μg mL−1). Using 25 μm (6.76 μg mL−1) TBO and LED energy density of 54 J cm−2 these fungal isolates presented variable susceptibility to PDI. The production of reactive oxygen species (ROS)/peroxynitrite was determined, and the catalase and peroxidase activities were measured. After PDI, high amounts of ROS/peroxynitrite are produced and higher catalase and peroxidase activities could be correlated with a lower susceptibility of C. gattii isolates to PDI. These results indicate that PDI could be an alternative to C. gattii growth inhibition, even of isolates less susceptible to classical antifungal drugs, also pointing to mechanisms related to their variable susceptibility behavior.

Photodynamic therapy efficiently controls dermatophytosis caused by Trichophyton rubrum in a murine model

DEAR EDITOR, Dermatophytes are filamentous fungi that use keratin to colonize the host, causing superficial infections called dermatophytoses. Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum spp. and Epidermophyton spp. are important aetiological agents worldwide. A few drugs, including azoles (itraconazole), allylamines (terbinafine) and hydroxypiridones [ciclopirox olamine (CPX)], are available for the treatment of dermatophytoses, but the widespread use of antifungals has been linked to the emergence of resistant strains of T. rubrum. This problem may jeopardize treatment and thereby justifies the search for new therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer and a nonthermal light source to induce a reaction that results in cell death. In the presence of molecular oxygen, aPDT triggers the production of reactive oxygen (ROS) and reactive nitrogen species, which have short lifetimes and limited diffusion through the tissue, explaining the localized damage induced by aPDT. Previously, we have shown that T. rubrum is susceptible to photodynamic inhibition using toluidine blue (TBO) and light-emitting diode (LED) at 630 nm; however, there is a lack of studies regarding its efficacy in vivo. Therefore, the aim of this study was to evaluate the effects of aPDT, using TBO and LED, in controlling T. rubrum infection in a murine model of dermatophytosis. In this study, C57BL/6 mice were infected with 1 9 10 conidia per animal of T. rubrum American Type Culture Collection 28189 and treated with aPDT daily [using TBO 0 2% in a gel formulation (Sigma-Aldrich, St Louis, MO, U.S.A.) and 630-nm LED (Fisioled; MMoptics, S~ao Paulo, Brazil) at a dose of 42 J cm] or CPX [10 mg g 1 (dose of 0 65 mg 1 per mouse); Medley, S~ao Paulo, Brazil] for 48 h, over a period of 7 days (Comiss~ao de Etica no Uso de Animals/Universidade Federal de Minas Gerais ethics protocol approval no. 040/ 2011). Histopathology of the skin, fungal burden in potato agar, and myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity were evaluated. The influence of aPDT on the viability of intraperitoneal macrophages, intracellular formation of hyphae and oxidative burst (ROS) was determined. For this purpose, TBO (10 lg mL ) and LED (48 J cm) were used. The groups of mice and macrophages used in this work are described in Table 1. Histopathological analyses showed that aPDT reduced the signs of dermatitis and recovered tissue architecture (Fig. 1c, g,i) compared with the infected and nontreated group (Fig. 1b,f,i). Interestingly, fungal cells were found mainly in hair follicles, corroborating the keratinophilic aspect of T. rubrum (Fig. 1f–h). Indeed, aPDT significantly reduced the fungal burden by 87% compared with the untreated group (P < 0 01) and by 64% compared with the CPX group (P < 0 05) (Fig. 1d,f–h,j). LED or TBO alone did not reduce the fungal burden (Fig. 1j). In addition, aPDT significantly

Melanin Protects Paracoccidioides brasiliensis from the Effects of Antimicrobial Photodynamic Inhibition and Antifungal Drugs

ABSTRACT Paracoccidioidomycosis (PCM) is a public health concern in Latin America and South America that when not correctly treated can lead to patient death. In this study, the influence of melanin produced by Paracoccidioides spp. on the effects of treatment with antimicrobial photodynamic inhibition (aPI) and antifungal drugs was evaluated. aPI was performed using toluidine blue (TBO) as a photosensitizer and a 630-nm light-emitting diode (LED) light. The antifungals tested were itraconazole and amphotericin B. We evaluated the effects of each approach, aPI or antifungals, against nonmelanized and melanized yeast cells by performing susceptibility tests and by quantifying oxidative and nitrosative bursts during the experiments. aPI reduced nonmelanized cells by 3.0 log units and melanized cells by 1.3 log units. The results showed that melanization protects the fungal cell, probably by acting as a scavenger of nitric oxide and reactive oxygen species, but not of peroxynitrite. Melanin also increased the MICs of itraconazole and amphotericin B, and the drugs were fungicidal for nonmelanized and fungistatic for melanized yeast cells. Our study shows that melanin production by Paracoccidioides yeast cells serves a protective function during aPI and treatment with itraconazole and amphotericin B. The results suggest that melanin binds to the drugs, changing their antifungal activities, and also acts as a scavenger of reactive oxygen species and nitric oxide, but not of peroxynitrite, indicating that peroxynitrite is the main radical that is responsible for fungal death after aPI.

Heterocycle Thiazole Compounds Exhibit Antifungal Activity through Increase in the Production of Reactive Oxygen Species in the Cryptococcus neoformans-Cryptococcus gattii Species Complex

ABSTRACT Human cryptococcosis can occur as a primary or opportunistic infection and develops as an acute, subacute, or chronic systemic infection involving different organs of the host. Given the limited therapeutic options and the occasional resistance to fluconazole, there is a need to develop novel drugs for the treatment of cryptococcosis. In this report, we describe promising thiazole compounds 1, 2, 3, and 4 and explore their possible modes of action against Cryptococcus. To this end, we show evidence of interference in the Cryptococcus antioxidant system. The tested compounds exhibited MICs ranging from 0.25 to 2 μg/ml against Cryptococcus neoformans strains H99 and KN99α. Interestingly, the knockout strains for Cu oxidase and sarcosine oxidase were resistant to thiazoles. MIC values of thiazole compounds 1, 2, and 4 against these mutants were higher than for the parental strain. After the treatment of C. neoformans ATCC 24067 (or C. deneoformans) and C. gattii strain L27/01 (or C. deuterogattii) with thiazoles, we verified an increase in intracellular reactive oxygen species (ROS). Also, we verified the synergistic interactions among thiazoles and menadione, which generates superoxides, with fractional inhibitory concentrations (FICs) equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazole compounds 1, 2, 3, and 4, respectively. In addition, thiazoles exhibited antagonistic interactions with parasulphonatephenyl porphyrinato ferrate III (FeTPPS). Thus, in this work, we showed that the action of these thiazoles is related to an interference with the antioxidant system. These findings suggest that oxidative stress may be primarily related to the accumulation of superoxide radicals.

Butenafine and analogues: An expeditious synthesis and cytotoxicity and antifungal activities

Graphical abstract