Differential response of Candida albicans and Candida glabrata to oxidative and nitrosative stresses.

Invasive candidiasis is associated with high mortality in immunocompromised and hospitalized patients. Candida albicans is the main pathological agent followed by Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. These pathogens colonize different host tissues in humans as they are able to neutralize the reactive species generated from nitrogen and oxygen during the respiratory burst. Among the enzymatic mechanisms that Candida species have developed to protect against free radicals are enzymes with antioxidant and immunodominant functions such as flavohemoglobins, catalases, superoxide dismutases, glutathione reductases, thioredoxins, peroxidases, heat-shock proteins, and enolases. These mechanisms are under transcriptional regulation by factors such as Cta4p, Cwt1p, Yap1p, Skn7p, Msn2p, and Msn4p. However, even though it has been proposed that all Candida species have similar enzymatic systems, it has been observed that they respond differentially to various types of stress. These differential responses may explain the colonization of different organs by each species. Here, we review the enzymatic mechanisms developed by C. albicans and C. glabrata species in response to oxidative and nitrosative stresses. Lack of experimental information for other pathogenic species limits a comparative approach among different organisms.

Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.

We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media.

Concordance and interaction of guanine nucleotide dissociation inhibitor (RhoGDI) with RhoA in oogenesis and early development of the sea urchin.

Rho GTPases are Ras-related GTPases that regulate a variety of cellular processes. In the sea urchin Strongylocentrotus purpuratus, RhoA in the oocyte associates with the membrane of the cortical granules and directs their movement from the cytoplasm to the cell cortex during maturation to an egg. RhoA also plays an important role regulating the Na(+) -H(+) exchanger activity, which determines the internal pH of the cell during the first minutes of embryogenesis. We investigated how this activity may be regulated by a guanine-nucleotide dissociation inhibitor (RhoGDI). The sequence of this RhoA regulatory protein was identified in the genome on the basis of its similarity to other RhoGDI species, especially for key segments in the formation of the isoprenyl-binding pocket and in interactions with the Rho GTPase. We examined the expression and the subcellular localization of RhoGDI during oogenesis and in different developmental stages. We found that RhoGDI mRNA levels were high in eggs and during cleavage divisions until blastula, when it disappeared, only to reappear in gastrula stage. RhoGDI localization overlaps the presence of RhoA during oogenesis and in embryonic development, reinforcing the regulatory premise of the interaction. By use of recombinant protein interactions in vitro, we also find that these two proteins selectively interact. These results support the hypothesis of a functional relationship in vivo and now enable mechanistic insight for the cellular and organelle rearrangements that occur during oogenesis and embryonic development.

Analysis of glycerol dehydrogenase activities present in Mucor circinelloides YR-1.

Two inducible NADP(+)-dependent glycerol dehydrogenase (GlcDH) activities were identified in Mucor circinelloides strain YR-1. One of these, denoted iGlcDH2, was specifically induced by n-decanol when it was used as sole carbon source in the culture medium, and the second, denoted iGlcDH1, was induced by alcohols and aliphatic or aromatic hydrocarbons when glycerol was used as the only substrate. iGlcDH2 was found to have a much broader substrate specificity than iGlcDH1, with a low activity as an ethanol dehydrogenase with NAD(+) or NADP(+) as cofactor. Both isozymes showed an optimum pH for activity of 9.0 in Tris-HCl buffer and are subject to carbon catabolite repression. In contrast, the constitutive NADP(+)-dependent glycerol dehydrogenases (GlcDHI, II, and III) were only present in cell extracts when the fungus was grown in glycolytic carbon sources or glycerol under oxygenation, and their optimum pH was 7.0 in Tris-HCl buffer. In addition to these five NADP(+)-dependent glycerol dehydrogenases, a NAD(+)-dependent alcohol dehydrogenase is also present in glycerol or n-decanol medium; this enzyme was found to have weak activity as a glycerol dehydrogenase.

Intracellular distribution of fatty alcohol oxidase activity in Mucor circinelloides YR-1 isolated from petroleum contaminated soils.

In previous studies, Mucor circinelloides YR-1 isolated from petroleum-contaminated soils grown in decane as sole carbon source, showed fatty alcohol oxidase (FAO) activities in either particulate or soluble fractions from a cell-free extract. One is associated to internal membranes (mFAO) and the other one is soluble (sFAO). Both activities appear to be located in the cells in specific compartments other than peroxisomes. Results suggested that mFAO could be located on the inner face of the membrane of these compartments, and sFAO could be in the lumen of the specific compartments. This study reports on the intracellular distribution of FAO activity and the purification of sFAOs and mFAO after several different procedures for release from the membranous fraction using the mixed membrane fraction (MMF) after cellular homogenization as enzymatic source. Results with the purified mFAO show, by molecular weight criteria, that the enzyme has only one type of subunit with molecular mass of 46 kDa, with two isoelectric point components: 6.0 and 6.3. We found that mFAO is strongly associated to the MMF, possibly in a transitory fashion. Using non-denaturating gels, we suggest that sFAO and mFAO have the same subunits in their native structures, and, due to their native molecular weight of approximately 350 kDa, each could be natively structured as an octameric complex.

Intracellular fate of hydrocarbons: possible existence of specific compartments for their biodegradation.

In previous work, purification procedures and zymogram analysis conducted with supernatants of crude extracts from aerobic mycelium of the YR-1 strain of Mucor circinelloides isolated from petroleum-contaminated soils indicated the existence of only one soluble alcohol oxidase (sAO) activity. In the present work enzymatic activity of alcohol oxidase (AO) was also detected in the mixed membrane fraction (MMF) of a high-speed centrifugation procedure after drastic ballistic cellular homogenization to break the mycelium from strain YR-1. When mycelial cells were gently broken by freezing the mycelium with liquid nitrogen, smashing in a mortar, and submitting the samples to an isopycnic sucrose gradients (10-60% sucrose), AO activity was detected in particular and discrete fractions of the gradient, showing specific density values quite different from the density of peroxisomes. The results suggest that there could be a different intracellular pattern of distribution of the microsomal fraction in aerobically grown mycelium depending on the carbon source used in the culture media, including alcohols and hydrocarbons, but not in glucose. In working with particulate fractions, we found two AO activities: a new membrane alcohol oxidase (mAO) activity and the sAO. Both activities appear to be located in the inner of the cells in specific compartments different from the peroxisomes, so mAO could be in the membrane of these compartments and sAO in the lumen of the vesicles. We also assayed other enzymatic activities involved in hydrocarbon biodegradation to establish its intracellular location and other enzymatic activities such as peroxidase to use them as intracellular markers of different organelles. In the case of monooxygenase, the first enzymatic step in the hydrocarbon biodegradation pathway, its location was in the same fractions where AOs were located, suggesting the existance of a specific organelle that contains the enzymatic activities involved in hydrocarbon biodegradation.

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils.

Different soluble NAD+-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleum- contaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD+-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (approximately 7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD+-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol- dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.

Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 Strain, a filamentous fungus isolated from petroleum-contaminated soils.

Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homo-genization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.

A different method of measuring and detecting monoand dioxygenase activities: key enzymes in hydrocarbon biodegradation.

A spectrophotometric method of measuring oxygenase activity in cell extracts or in zymograms was developed. It is an easy and cheap method that allows spectrophotometric measurement of activity by a colored reaction and reveals activity bands in a polyacrylamide gel electrophoresis (PAGE) gel as brown bands. To prove its usefulness, we report on a study with the oxygenase present in strain YR-1, isolated from petroleum-contaminated soils, that uses hydrocarbons as its sole carbon source. Soluble oxygenase activity was detected (under our conditions of cellular homogenization) in the mycelium of a filamentous fungus strain named YR-1. Oxygenase activity from aerobically grown mycelium was detected in growth medium containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited similar optimum pH for the hydroxylation of different aliphatic or aromatic substrates (decane, hexadecane, benzene, and naphthalene) to the corresponding alcohols. Zymogram analysis conducted with partially purified fractions from cell extracts from the aerobic mycelium of the YR-1 strain indicated the existence of only one oxygenase enzyme. Partially purified samples of enzyme, analyzed by sodium dodecyl sulfate PAGE, indicated the presence of one major protein band with a mol wt of 56 kDa that can be a constituent of the native enzyme. In samples of the enzyme, the 56-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against oxygenase from soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene. Km values obtained for benzene and decane indicated a higher affinity for the latter.

Subcellular localization of the NAD+-dependent alcohol dehydrogenase in Entamoeba histolytica trophozoites.

The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.

Presence and physiologic regulation of alcohol oxidase activity in an indigenous fungus isolated from petroleum-contaminated soils.

A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20,38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. Km values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.