Process Biochemistry 39 (2004) 633–636

Study of different nitrogen sources on glucose uptake and productionof melanin precursors and fungal mass ofFonsecaea pedrosoi

cultured in tricyclazole

J.M. Costaa, V.A. Corbellinia,∗, M.L. Scrofernekerb

a Department of Physics and Chemistyr, Universidade de Santa Cruz do Sul, State of Rio Grande do Sul, Av. Independˆencia,No. 2293, sala 1212, Santa Cruz do Sul 96815-900, RS, Brazil

b Department of Microbiology, Institute of Basic Sciences, Universidade Federal do Rio Grande do Sul, RS, Brazil

Received 12 March 2003; accepted 7 May 2003

Abstract

The influence of tricyclazole on the production of phenolic precursors, glucose uptake and production of fungal mass ofFonsecaea pedrosoiwas assessed in the presence of phenylalanine, sodium nitrate and tryptophan. Photocolorimetry was used for the quantitation of glucoseand total phenol, and the dry weight method for biomass. The glucose uptake with phenylalanine was 99.85×103 mg/ml; with tryptophan,99.88×103 mg/ml and with sodium nitrate, 99.90×103 mg/ml. Production of biomass: with tryptophan, 2.7×10−1 mg; with sodium nitrate,3.0×10−1 mg and with phenylalanine, 3.4×10−1 mg. Tricyclazole induced higher phenol accumulation, lower glucose uptake, inhibition ofmelanin deposition on the cell and higher production and higher biomass production in relation to glucose uptake for all nitrogen sources testedunder these conditions. A negative correlation between biomass production and glucose uptake was observed in the presence of secondarymetabolism.© 2003 Elsevier Ltd. All rights reserved.

Keywords: Fonsecaea pedrosoi; Melanin; Nitrogen source; Tricyclazole; Fungal mass; Chromoblastomycosis

1. Introduction

Chromoblastomycosis is a ubiquitous, chronic fungal in-fection, restricted to the skin and subcutaneous tissues, es-pecially found in tropical areas, and often caused byFonse-caea pedrosoi[1,2]. The main characteristic of this fungus isthe production of DHN melanin[3]. These pigments are notessential to the growth and development of the fungus but,on the other hand, they increase its life span and capacity forfood competition in certain environments, thus causing in-juries to the human host[4]. This consequence seems to berelated to the structure of melanins, which depends on themetabolic pathway used by the fungus for their synthesis.A priori, these pathways can compete with other metabolicpathways involved in the formation of components that arenecessary for cell viability. Therefore, a pathway can be re-

∗ Corresponding author.E-mail addresses:valer@dquimfis.unisc.br (V.A. Corbellini),

scrofern@vortex.ufrgs.br (M.L. Scroferneker).

placed or disabled by another one depending on the nutrientsto which the fungus has access[5].

The present study aimed at investigating the effect of threedifferent nitrogen sources (sodium nitrate, phenylalanine andtryptophan) on mycelial production and glucose uptake inculture media in the presence or absence of tricyclazole, aninhibitor of DHN melanin synthesis[4].

2. Materials and methods

2.1. Preparation of culture media

The sample ofF. pedrosoiATCC46428 was kept on Cza-peck agar (Oxoid) at 25◦C. The culture medium used forthe tests with different nitrogen sources basically consistedof glucose monohydrate (11.1 g), magnesium sulphate hep-tahydrate (0.75 g), copper sulphate (0.1 mg), manganese sul-phate (0.1 mg), zinc sulphate (0.1 mg), ferrous sulphate (0.1mg), monobasic potassium phosphate (1.5 g) and distilled

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0032-9592(03)00191-2

634 J.M. Costa et al. / Process Biochemistry 39 (2004) 633–636

water q.s.p. (1 l). 3 l of basic culture medium was pre-pared and the nitrogen source (phenylalanine, tryptophanand sodium nitrate) was added to each litre in an amountthat was sufficient to provide 0.5 g/l of nitrogen. From eachmedium, with its respective nitrogen source, 500 ml wasseparated and 8 mg of tricyclazole was added.

2.2. Preparation of the inoculum

200 ml of Czapeck’s medium (Oxoid) was prepared, 3 gof agar was added and one sample ofF. pedrosoiwas seeded.The culture was kept for 20 days at 37◦C. After 20 daysof growth, the inoculum was standardized at 550 nm, andan absorbance of 1.12 was obtained. Each flask of culturemedium, with different nitrogen sources, received 0.25 mlof this suspension. The flasks were kept in a hothouse, inthe dark, at 37◦C for 8 days, with no shaking. Each flaskreceived 0.5 ml of sodium azide at 6%, and was left to restfor 24 h in order to inhibit growth.

2.3. Determination of dry weight

After 8 days of growth, the cultures were filtered withWhatman #1 filter with the aid of a Büchner funnel. Afterfiltration, the papers containing the residue were placed ina hothouse at 50◦C for 48 h. After that, the cultures wereplaced in a desiccator for 12 h. The dry weight was then de-termined. The filtrate was used to assess melanin precursors.

2.4. Extraction of melanin precursors

The filtrate of each sample was placed in a 250-ml beaker,saturated with 20 g of sodium chloride and acidified up topH 1 with 1 ml of concentrated hydrochloric acid. Two vol-umes of 20 ml of ethyl acetate were used for the extractionand a funnel was used for decantation. The extracted ma-terial was placed in a rotary evaporator. This material wascharacterized as organic extract. The residue obtained afterthe extraction with ethyl acetate was denominated modifiedfiltrate.

2.5. Qualitative assessment of the organic extract

In order to test for phenols, the organic extracts wereplaced in a hothouse at 50◦C until the ethyl acetate totallyevaporated. Each sample was then suspended in 10 ml dis-tilled water. Solutions with phenylalanine and tryptophanwere prepared with the same concentration used in the cul-ture media. 2 ml of each sample were placed in differenttest tubes and 1 ml of modified Barton’s reagent[6] wasadded with some shaking. The test for phenols was consid-ered positive when a blue to green colour appeared.

2.6. Biochemical assessment of modified filtrates

For the quantitation of glucose, the modified filtrateswere grouped into triplicate samples and were diluted up to

250 ml. The concentration of glucose in each sample wasquantitated by the method proposed by Morris[7], whichmeasures the intensity of the green colour produced by thecondensation of anthrone in a sulphuric acid solution withsugar derivatives, under heating conditions. 5 ml of anthronewas added to 1 ml of sample in an ice bath. After cool-ing, absorbance was read at 625 nm against a control withdeionized water. The samples were quantitated in triplicateand the concentration of carbohydrates was determined bycomparison with the analytical curve for glucose.

3. Results

After 8 days of culture in media without tricyclazole thefungus presented a darker colour, whereas in media with theinhibitor, the fungus showed a hyaline aspect.

In media without tricyclazole, there was less accumulationof fungal mass, while in media containing the inhibitor therewas a higher accumulation of fungal mass. In the presenceof phenylalanine, the accumulation of fungal mass was moreevident, followed by the culture in sodium nitrate and by theculture in tryptophan.Fig. 1 shows the difference in fungalmass accumulation in the different culture media tested.

The qualitative assay for phenolic compounds showed apositive reaction in all tested media. In the medium contain-ing sodium nitrate as nitrogen source, a more intense colourwas observed in the presence of the inhibitor, which couldindicate a greater amount of phenols.

The present study also assessed glucose uptake by thefungus in the presence or absence of tricyclazole.Fig. 2shows the difference in glucose uptake in different culturemedia.

The results obtained for the colorimetric quantitation ofglucose firstly show that, in culture media with tricyclazole,F. pedrosoigrew with a lower concentration of glucose thanin those without the inhibitor in the following order: pheny-lalanine, tryptophan and sodium nitrate. This order was thesame in the absence of tricyclazole.Fig. 3shows the resultsobtained for absorbed glucose in different culture media.

To better understand the behaviour of the fungus in theassessed conditions, a relationship was established between

Fig. 1. Dry weights obtained in six different culture media.

J.M. Costa et al. / Process Biochemistry 39 (2004) 633–636 635

Fig. 2. Residual glucose obtained in six different culture media.

Fig. 3. Absorbed glucose in six different culture media.

the amount of absorbed glucose and the value for dryweight. Fig. 4 shows the values obtained between the dryweight/absorbed glucose ratio in different culture media.

A comparison of dry weight and glucose in different cul-ture media indicated that among the three nitrogen sourcestested, sodium nitrate produces more fungal mass in the ab-sence of the inhibitor, whereas the phenylalanine-containingmedium was more effective in the presence of tricyclazole.

4. Discussion

The present experiment shows the influence of three dif-ferent nitrogen sources and the use of an inhibitor on glu-cose uptake, accumulation of fungal mass and production ofmelanin precursors inF. pedrosoi.

Fig. 4. Dry weight/absorbed glucose ratio regarding six different culturemedia.

The colour observed in the cultures after 8 days showsthat, in culture media containing the inhibitor, pigmentedsubstances, such as scytalone, vermelone, flaviolin, 2-hydro-xyjuglone and 3-hydroxyjuglone, were released. Thesesubstances possibly mediate DHN synthesis, as already ob-served under other experimental conditions[8]. They aredirectly involved in melanin synthesis, causing hyphae topresent a dark colour depending on culture conditions, asassessed by Alviano et al.[3]. The fact that fungi culturedin tricyclazole-free medium have a dark aspect shows thatthey possibly accumulate melanin in their cell wall.

The dry weight analysis revealed different weight for thedistinct culture media used; the accumulation of fungal masswas higher in the presence of the inhibitor than in its ab-sence. This difference was clearer for phenylalanine, trypto-phan and sodium nitrate. These findings can be explained bythe following hypotheses: (a) the fungus when cultured in aliquid medium and in the presence of the inhibitor showed anextended log phase, allowing for increased mycelial branch-ing, whereas the fungus that grows free of the inhibitorreaches the stationary phase earlier and (b) the possibilitythat the fungus was in the log phase on the 8th day of culturein both media may be ruled out. In this case, the differencein dry weight occurs because of the accumulation of differ-ent macromolecules. The fungus that grew in the presenceof the inhibitor did not accumulate melanin, but it accumu-lated other substances, such as polysaccharides and lipids,which contribute significantly to dry weight accumulation.

In addition, two other situations should not be ruled out:the fungus was in the stationary phase under both cultureconditions or the fungus was in the stationary phase in thepresence of the inhibitor and in the log phase in its ab-sence. In the former situation, the fungus that grew in thepresence of the inhibitor had a lower production period ora greater specific growth rate. In the latter situation, therewas increased accumulation of macromolecules, which con-tributed towards dry weight accumulation when comparedto melanin accumulation, even after exponential growth hasceased.

By considering the amount of glucose uptake, the mostprobable hypothesis was that the fungus was in the station-ary phase under both conditions, with reduction of primarymetabolism in the presence of tricyclazole. This is due tothe fact that, given the physiology ofF. pedrosoi, culturesof 6–8 days are normally used to isolate metabolic anti-gens released into the medium when the fungus has alreadyreached the stationary phase[3]. Also, the fungus that grewin the presence of the inhibitor adapted more easily to themedium, since it did not depend on the synthesis of a groupof melanins involved in the pathway of phenolic precursors.However, this hypothesis can only be confirmed by obtain-ing a growth curve for each nitrogen source in the presenceor absence of the inhibitor, in addition to quantitation of car-bohydrates, proteins and lipids in the fungal mass and othermarkers of microbial metabolism, such as ATP productionand respiration rate (CO2 production)[9].

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The results obtained for the qualitative determination ofphenolic compounds showed increased production of phe-nols in the presence of the inhibitor in the medium con-taining sodium nitrate as nitrogen source. This finding isin agreement with others obtained for fungal culture in thepresence of tricyclazole in liquid medium[10] and for theculture ofF. pedrosoiin solid medium[2]. In culture mediawith phenylalanine and tryptophan, the characteristic colourof phenolic compounds was also observed; however, thisresult might have been masked by the composition of theculture medium.

The fact that the tricyclazole-containing medium showeda better glucose uptake implies a lower production of fun-gal mass, since glucose is essential for the supply of car-bon sources for the production of other molecules such aspolysaccharides, proteins and lipids. Nevertheless, accord-ing to the results obtained, the lower glucose uptake didnot imply lower production of fungal mass in the presenceof tricyclazole. These data suggest that, in such situation,the distribution of glucose degradation pathways favors theproduction of macromolecules, which contribute to the ac-cumulation of dry weight. As melanin synthesis is blocked,the accumulation of phenolic precursors should have lessinfluence on fungal mass production, because part of thesecompounds is released into the medium.

Considering the efficiency of the production of fungalmass as the ratio between dry weight and absorbed glucose,it is possible to say that there is greater influence of theproduction of fungal mass in the presence of an inhibitorregardless of the nitrogen source analyzed (phenylalanine,tryptophan and sodium nitrate). The media in which glucoseuptake for the production of fungal mass proved better werethose with the inhibitor, showing that the catabolism of thenitrogen source was more associated with the anabolism oforganic matter. Among the different nitrogen sources, wecan observe that in the absence of the inhibitor the mediumcontaining sodium nitrate allowed for a higher production offungal mass, whereas the phenylalanine-containing mediumis more effective in the presence of tricyclazole. Probablythere was higher uptake of nitrogen sources in the pres-ence of the inhibitor, since they also contributed as carbonsources.

The results presented above illustrate the importance ofnutritional factors in the production of dematiaceous fungi in

adverse situations, such as cultivation in the presence of aninhibitor of secondary metabolism. Under these conditions,the fungus mobilizes new mechanisms in order to accumu-late intracellular components (other than melanin), whichcontribute towards the increase of their biomass, avoidingthe toxic effect of tricyclazole. Therefore, the efficiency ofprimary metabolism increases in an “aggressive” situation,leading to a higher accumulation of biomass. The secondarymetabolism (melanin accumulation) seems to make the fun-gus use a larger amount of energy with low efficiency inbiomass production. In the presence of the inhibitor, this sit-uation is changed to low energy consumption (ATP, oxida-tive phosphorylation), especially for those nitrogen sourcesthat also contribute as a carbon source.

Acknowledgements

We would like to express our thanks to Mr. Gilson Mattosfor revising the English translation of the manuscript.

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