Application of 6-nitrocoumarin as a substrate for the fluorescent detection of nitroreductase activity in Sporothrix schenckii.

INTRODUCTION: Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii.
METHODS: Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light.
RESULTS: All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure.
CONCLUSION: The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

INTRODUCTION

Sporothrix schenckii is considered as a complex of species composed of S. brasiliensis, S. mexicana, S. globosa, S. schenckii sensu stricto and S. schenckii var. luriei 17, thermal dimorphic pathogenic fungi that may usually be implanted through the skin causing a subcutaneous mycosis14 22. Classically, the infection is caused by traumatic inoculation with soil, and plants and organic matter contaminated with the fungus16. Some leisure and occupational activities, such as agriculture and floriculture, have been associated with the transmission of the disease4 11.

Chromogenic and fluorogenic substrates are useful tools for the research of enzymatic action. They facilitate purification and identification, as well as kinetic studies of the enzymes27. Although most chromogenic and fluorogenic substrates rely on known chromophores or fluorophores, there is still progress to be made in developing new aromatic systems as revealing groups13.

Nitroreductase is a member of a group of enzymes that reduces the wide range of nitroaromatic compounds and has potential industrial applications10 12 26. Nitroreductase activity has been detected in a diverse range of bacteria8 15 25 and in yeast9 28 by using chromogenic or fluorogenic substrates. For this purpose, nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several aminocoumarin derivatives, already used in several other enzyme assays.

The purpose of this work was the use of 6-NC as a novel fluorescent substrate to study its susceptibility to the intact cell system of S. schenckii, aiming to evaluate the nitroreductase activity in that fungus by the analysis of the reaction products formed in the culture media.

MATERIALS AND METHODS

6-Nitrocoumarin and 6-aminocoumarin synthesis: 6-Nitrocoumarin was prepared using the methodology described by MORGAN & MICKLETHWAIT (1904)18, then purified by column chromatography using silicagel (Merck), and then eluted with chloroform (Synth).

6-Aminocoumarin was obtained by the reduction of 6-nitrocoumarin with iron, under a standard procedure; it was purified by column chromatography with silicagel (Merck) and a gradient of ethyl acetate-hexane (Synth).

The purity of the compounds was checked by Thin Layer Cromatography (TLC), and the structures were confirmed by the melting points and the usual spectroscopic techniques (IR, UV-Vis, 1H- and 13C-NMR).

Microorganisms: In this work, 33 clinical isolates of the S. schenckii complex, obtained in the Brazilian states of São Paulo, Rio Grande do Sul and Minas Gerais, and two strains reference (American Type Culture Collection - ATCC 201681 and 201679) from Rockville, MD, USA were utilized.

The strains were cultured in potato dextrose agar (Difco) containing polysorbate 80 1% (v/v) (Vetec). Each strain was inoculated in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (8×10–2 mol/L) dissolved in dimethyl sulfoxide (Vetec), or only dimethyl sulfoxide as a negative control. The strains were incubated for seven, 14 and 21 days at 35 °C and examined under an Epifluorescence Microscope - EFM (Nikon) with a UV-2A (330-380 nm) filter block.

Chromatographic analysis: The strains were cultured in Sabouraud dextrose agar (Difco) for five days in Roux flasks and then were treated with 100 mL of a solution of 6-nitrocoumarin (1 mmol) in a sodium phosphate buffer (10 mmol) and glucose (4% w/v) (Synth), and incubated for seven, 14 and 21 days. Afterwards, the samples were treated with 100 mL of an aqueous saturated saline solution and extracted three times with chloroform (15 mL portion). The extract was concentrated in a rotary evaporator and analyzed by TLC (Silicagel), using hexane-ethyl acetate (1:4 v/v) as the eluent, and visualized by UV light (365 nm). Controls were 6-NC and 6-AC.

The crude product was dried with anhydrous sodium sulfate, purified by column chromatography using hexane-ethyl acetate (1:4 v/v) as the eluent, and the reaction products were characterized by the usual spectroscopic techniques.

RESULTS

: All strains were able to reduce the 6-nitrocoumarin compound to generate significant levels of fluorescence when compared with the organism-free control (Fig. 1). There was no difference between the intensity of fluorescence produced in the different periods analyzed.

Fig. 1

Fungal structures visualized under epifluorescence microscope: (A) 6-aminocoumarin substrate (positive control); (B) and (C) 6-nitrocoumarin substrate; (D) without substrate (negative control).

Chromatographic analysis: All the extracts obtained showed two bands: one corresponding to 6-aminocoumarin, and the other to 6-hydroxyaminocoumarin (Fig. 2).

Fig. 2

Thin Layer Cromatography carried out after incubation in different periods: (1) negative control; (2) 6-nitrocoumarin control; (3) 6-aminocoumarin control; extraction after incubation for (4) 7 days; (5) 14 days; (6) 21 days. (7): control of the absence of fungus growth.

DISCUSSION

In microbiology, the use of fluorescent compounds has found practical applications not only in the evaluation of cellular feasibility of yeast and spores, but also in the detection of bacterial growth and in the chemiotaxonomic differentiation of microorganisms9 23. In clinical specimens (biological liquids, skin biopsy and other tissues), these compounds have been used for the diagnosis of cutaneous, subcutaneous and systemic mycosis2 21.

Coumarin composes one class of heterocycles used as fluorofor and pigment for LASER. There has been a proposal for the use of coumarin as fluorogenic substrates for detecting the nitroreductase activity in bacteria and in yeast9 28.

The nitroreductase acts on aromatic nitro compounds not only in the presence, but also in the absence of oxygen1. The type I (oxygen-insensitive) catalyses the reduction of nitro groups, through sequential two-electron reductions, to nitroso, hydroxylamine intermediates and finally primary amines7 24; and the type II (oxygen-sensitive) catalyses one electron reduction of the nitro group, producing a nitro anion radical that subsequently reacts with oxygen, forming a superoxide radical and regenerating the original nitroaromatic compound20.

Metronidazole and related N-1 substitutes 5-nitroimidazoles like ornidazole, secnidazole and tinidazole are widely used in the treatment of diseases caused by protozoa and anaerobic bacteria19. It has been speculated that a reactive intermediate, formed in the microbial reduction of the 5-nitro group of nitroimidazoles, covalently binds to the DNA of the microorganism, triggering the lethal effect. Potential reactive intermediates include the nitroxide, nitroso, hydroxylamine and amine6. However, assays that were carried out have shown that S. schenckii is not responsive to metronidazole (data not shown). This fact emphasizes the necessity of testing different fluorogenic substrates for selecting a specific enzymatic activity, particularly the nitroreductase.

Through EFM it was possible to differentiate cell structures as hyphae, conidiophores and conidia. Compared to positive controls showing yellow fluorescence, the dye tested showed an intermediate blue fluorescence that was further investigated. Reduced 6-nitrocoumarin obtained better yield in fluorescence in reproductive cells, indicating that it is useful for a better visualization and identification of the species. GAZENKO et al. (1998)5 observed that the comparison of the actinomycetes enzymatic activities of dormant spores with vegetative cells showed similarity of the enzymatic profiles, but higher activity for vegetative cells.

TLC eluted a sub-product with blue fluorescence, with a retention factor (Rf) value higher than aminocoumarin (yellow band), possibly related to the partial biological reduction of 6-nitrocoumarin. The investigation of the arylbenzothiazole, the benzoxazole and the benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase in Gram-negative and Gram-positive bacteria has shown differences of colonies fluorescence3. Similar to our study, they concluded that the origin of this effect has not yet been established, but may be a consequence of either the localization of the fluorophore in different regions of the organism or the result of further metabolic transformation of the initially formed fluorophore.

In this study, nitroreduction was active in all times of incubation tested, indicating a concurrent production of both reduction products. The results indicate that nitroreductase activity is widespread in this species. 6-NC has shown to be interesting as a fluorogenic substrate for the detection of nitroreductase activity and for a better visualization of Sporothrix schenckii reproductive cells. From the data obtained during this study, we intend to design a standard method for quantifying the nitroreductase activity, as well as for isolating the enzymes related to this activity.