Microbial metabolism

THE ROLE OF TREHALOSE DURING DIMÓRFICO CHANGE AND THE RESPONSE TO OXIDATIVE STRESS IN CANDIDA ALBICANS.

Author: ALVAREZ PERAL FRANCISCO JOSÉ.
Year: 2002.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of preparation: ESCUELA SUPERIOR DE INGENIEROS AGRÓNOMOS.

Summary: The genus Candida includes yeast ascomicetáceas no real sexual cycle. Candida albicans is a yeast commensal man and animals homeostermos. Its definition includes taxonomic cells vegetative levaduriformes with gemación multilateral without mating. Presents requirements nutritious relatively simple and can grow in a medium containing a basic source of organic carbon (sugars), a source of nitrogen (ammonium salts), phosphate and specific requirements of certain vitamins and amino acids (biotin, thiamin). It grows well in the temperature range inlcuidos between 20Â ° C and 40Â ° C, likewise made between 2 and 8 units of pH (Odd, 1988, 1994). While in yeast of gemación, is well documented intracellular accumulation of disacárdido not reducer trehalose (D-glucopiranosil-D-glucopiranosido), the information available at C.albicans is more scarce (Thevelein, 1996, François et al., 1997; ArgÃ ¼ elles , 2000). The trehalose synthesis occurs primarily under conditions of lack of growth (stationary phase or sudden removal of nutrients), acting primarily as a resistance factor, increasing the protection and stability in a variety of cellular conditions nutritional and environmental stress (fasting nutritional clash thermal osmotic or oxidative processes and freezing or drying). In fungi and yeasts, hydrolysis of physiological trehalose is almost exclusively entrusted to the enzyme trehalasa, desdoble the trehalose into its component parts: 2 molecules of glucose. It is known the existence of two trehalasas with different subcellular localization (cytosolic and vacuolar or outside depending on the species), and kinetic parameters (neutrona and acid) (Thevelein; 1996; Nwaka and Holzer, 1998; ArgÃ ¼ elles, 2000). C.albicans is achieving at present a tremendous clinical importance, as the number of candiddiasis has been increasing dramatically due on the one hand to the growing number of pacietnes inmunocompreometidos case of AIDS patients, treated with anti-cancer therapies, transplant recipients , infants and the elderly. Moreover, the proliferation of resistant strains is a result of the use (abuse) of antibiotics and chemotherapeutic agents. So appear very often chronic oral candidiasis, which is difficult to cure, since they are often produced in debilitated patients, which spread as a hotbed infectious to other visceral organs. C.albicans is also a model system in research for their ability to behave like a fungus dimórfico, ie exists in two main ways: as cell levaduriformes (blastocoidios) and structures miceliares (hyphae and pseudohifas). It has been shown that there is a direct correlation between the process of transition morphological levadura-micelio and acquisition of pathogenicity during the colonization of the host tissues (Odds, 1988, 19974; KÃ ¶ hler et al., 1996; Lo et al. , 1997). The transition morphological levadura-micelio is a complex process in which intevienen various external factors that produce signals trigger the morphological changes (Brown and Gow, 1999; Ernst, 2000). There is conflicting evidence about the mobilization of trehalose, as a requirement of energy needed for such change (Sullivan et al. 1983; Ram et al. 1984), this has been one of the key objectives set out in this project: analyzing fluctuations in the during trehalose metabolism change dimórfico levadura-micelio of C.albicans induced specifically induced by human serum. To this end have segudio two approaches experimental basic: 1-Measuring levels of trehalose along with variations of hydrolytic enzymes and biosintéticas in different conditions induction of progrma dimórfico. 2-Analyzing the behavior of the double mutant tps1/tps1, deficient in both alleles of the gene encoding the tehalosa-6P synthetase and therefore unable to accumulate trehalose. Taken together, our observations are consistent with the idea that the hydrolysis of disaccharide is not a preferred energy source for the viability of this dimorphism in yeast. It also allows us to discard left by lomenos- the trehalasa external play a vital role in this process morphology. We disc 8 utido am 1869 pliamente this proposal, which openly disagreed with the suggestion made in two previous studies (Sullivan et al., 1983; Ram et al., 1984), whose authors suggest active participation of the hydrolase in mobilizing trehalose. We have deepened in other relevant physiological aspects on the role of trehalose in this levdura opportunistic. The data provided in our work confirm the involvement of the disaccharide in processes related to the recovery of active cell growth amid fresh stationary, as well as the rapid metabolic response to sudden changes in environmental conditions (temperature variations or perhaps including serum ) C.albicans is extremely sensitive to heat shock treatment of severe (52.5 Â ° C for 5 min), where it grows exponentially in medium containing glucose. Rather these cells have the phenomenon conoci8do as "adaptive response", which enables them to achieve a state of thermal tolerance, provided that previously were incubated at a temperature mild (42Â ° C). This protective effect, extrapolates to other types of stress, is due to the activation of the trehalosa-6P synthetase. The double mutant tps1/tps1 has been a useful genetic tool in our studies. While blastoconidios of mutant tps1/tps1 are competent to enter the morphogenetic program, their level of miceliación was less than that observed in wild-type cells, collected at the same stage of growth. This defect could be attributable to the profound alteration of the way glucolítica in that mutant when growing sugar as a source of carbon, a defect which manifests itself as dependent on the temperature. Accordingly, the formation of hyphae is virtually zero in cells tps1/tps1 exponential preincubadas above average YP YP + + glucose or fructose, while induction into YP alone (yeast extract 1% peptone 2%) produces a fraction very similar germinativos tubes. Also, the replacement of glucose and fructose by galactos or glycerol increases the level of dimorphism in an equivalent degree, while acetate promotes a slight decline. The presence of ethanol is very damaging for change dimórfico particularly in the form of mutant. In the course of work, we make a surprising observation, the extreme sensitivity of mutant tps1/tps1 to oxidative agents (such as H2O2 specifically). Consequently, we have devoted a chapter to the investigation of this process. The cells hospedadoreas have response mechanisms estrás acting in a coordinated manner (Estruch, 2000). One is the system dependent on oxygen, and when the fungus interacts with the membrane of nuetróflilo boosts activation oxidasas, with the subsequent reduction of O2 molecular superoxide anion (O2). In vacuolas fagocíticas, O2 becomes either spontaneously because of the low pH or catabólicamente by the action of superoxide dismutase in hydrogen peroxide (H2O2). Both O2 as H2O2 are agents microbicides. Some studies indicate that in S.cerevisiae and to a greater degree in C.albicans, if crops are subject to low concentrations of reactive oxygen species (ROS) as H2O2, OH and O2, are protected against further exposure to concentrations most are capable of withstanding high. From midmo way, when S.cerevisiae is subjected to a mild thermal exposure, acquires protection against H2O2, (cross tolerance), but this protection is lower when the process occurs in reverse. Until now, these effects are unknown described in C.albicans. Based on our work, we can conclude that the trehalose protects cells levaduriformes face severe oxidative exhibitions. However, the accumulation of disaccharide not seem to be required during the adaptive response to oxidative stress, a process that we believe certainly relevant in the course of infection in vivo by this opportunistic pathogen. Moreover, the protective role of trehalose appears to adhere exclusively to oxidizing agents, resulting in less apparent compared to other types of stress. In addition, we propose that the response to ROS involves two distinct stages: an initial defense immediately be induced directly by the agent stressful and requires the accumulation of trehalose, while the second is manifested after a meter waiting before being operational and is dependent uan route still unidentified. In the absence of trehalose synthesis of this second mechanism would become operational after a previous contact with a moderate exposure and non-lethal agent stressful. The absence of expression of mRNA in the presence of H2O2, in conjunction with the negligible effect of cicloheximida about business Tsp1 leads us to suggest that the activation of the trehalose synthetase dependent H2O2 is caused by a change postraduccional of the enzyme. A highlight of this report is the great repertoire methodology used, which include: determinations conventional microbiological testing ezimáticos conventional techniques electroforéticas procedures and Molecular Biology (RNA analysis). At the direction of my Director, I did a postdoctoral stay short (one month) in the laboratory led by Dr. Carlos Gancedo, which enabled me to not only faster learning methods not available in the department of Murcia, but also to obtain some valuable results. Finally, emphasize that the validity of my work is supported by the publication of three specialized monographs, whose reference follows. She is in the editing stage, fourth scientific article.

MICROBIAL METABOLISM