SummaryCryptococcus neoformans is a fungal pathogen most commonly causing meningitis in immunocompromised patients. Current therapies are inadequate, and novel antifungal targets are needed. We have identified by proteomics two thiol peroxidases that are differentially expressed at 37 ∞ ∞ ∞ ∞ C, the temperature of the mammalian host. Consistent with their antioxidant role, we show that the genes encoding these thiolspecific antioxidants, TSA1 and TSA3 , are transcriptionally induced when C. neoformans is exposed to hydrogen peroxide. Genome sequence analysis of C. neoformans revealed a third thiol peroxidase, TSA4 .
The ability of the fungal pathogen Cryptococcus neoformans to evade the mammalian innate immune response and cause disease is partially due to its ability to respond to and survive nitrosative stress. In this study, we use proteomic and genomic approaches to elucidate the response of C. neoformans to nitric oxide stress. This nitrosative stress response involves both transcriptional, translational, and posttranslational regulation. Proteomic and genomic analyses reveal changes in expression of stress response genes. In addition, genes involved in cell wall organization, respiration, signal transduction, transport, transcriptional control, and metabolism show altered expression under nitrosative conditions. Posttranslational modifications of transaldolase (Tal1), aconitase (Aco1), and the thiol peroxidase, Tsa1, are regulated during nitrosative stress. One stress-related protein up-regulated in the presence of nitric oxide stress is glutathione reductase (Glr1). To further investigate its functional role during nitrosative stress, a deletion mutant was generated. We show that this glr1⌬ mutant is sensitive to nitrosative stress and macrophage killing in addition to being avirulent in mice. These studies define the response to nitrosative stress in this important fungal pathogen.To survive the oxidative and nitrosative attack initiated by phagocytic cells of the host, pathogens must respond appropriately (reviewed in reference 35). This antimicrobial attack is established by two main systems including the inducible nitric oxide synthase pathway and the NADPH oxidase pathway (14). These two pathways generate either reactive nitrogen species (RNS) or reactive oxygen species. In the absence of either of these two pathways, mammalian hosts are more susceptible to both bacterial and fungal infections (18, 41). To cause infection, pathogens must evade the immune system by initiating a response to the stresses encountered.Previously, transcriptional responses to temperature, osmotic, and hydrogen peroxide stress as well as the stresses encountered in macrophages have been studied in fungi, including Saccharomyces cerevisiae and Candida albicans (13,25,29). A proteomic response to stress has only been determined in S. cerevisiae during hydrogen peroxide exposure (17). Proteomic analysis of the nitrosative stress response has not been studied in fungi, though transcriptional responses to RNS have been recently described in S. cerevisiae, C. albicans, and Histoplasma capsulatum (21,39,46). Though the response to nitrosative stress has not been studied in Cryptococcus neoformans, it has been implicated in both stress resistance and virulence of this fungal pathogen (10,33,36). It has been shown that macrophages produce nitric oxide in response to cryptococcal cells (20) and that the anticryptococcal activity of macrophages is mostly dependent on RNS (48). Recently, it was determined that during experimental cryptococcosis, the inducible form of nitric oxide synthase (iNOS) is expressed at increasing levels during infection (...
Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.
A method is presented for measuring rapid changes in the rate of glucose phosphorylation in mouse brain with nonradioactive 2-deoxyglucose (DG). After times as short as 1 min after DG injection, the mouse is frozen rapidly, and selected brain regions are analyzed enzymatically for DG, 2-deoxyglucose 6-phosphate (DG6P), and glucose. The rate of glucose phosphorylation can be directly calculated from the rate of change in DG6P, the average levels of DG and glucose, and a constant derived from direct comparison of the rate of changes in glucose and DG6P after decapitation. Experiments with large brain samples provided evidence for a 2% per min loss of DG6P and at least two compartments differing in their rates of glucose metabolism, one rapidly entered by DG with glucose phosphorylation almost double that of average brain and another more slowly entered with a much lower phosphorylation rate. The method is illustrated by changes in phosphorylation within 2 min after injection of a convulsant or an anesthetic and over a 48-min time course with and without anesthesia. The sensitivity of the analytical methods can be amplified as much as desired by enzymatic cycling. Consequently, the method is applicable to very small brain samrles. (1) to map patterns of neural activity in a wide variety of physiological and drug-induced states. The number of studies that have used this method shows the great demand for information about regional brain activity. Nevertheless, as useful as the method has proven to be, it has one basic limitation, i.e., the necessary 30-to 45-min lag period between DG injection and brain fixation. The lag is needed to allow free DG to largely dissipate, since it is the 2-deoxyglucose 6-phosphate (DG6P) accumulation that is the index of glucose phosphorylation and hence of its metabolism. Many investigators are interested in brain events that take place in a much shorter time frame. This paper presents a DG procedure with temporal resolution of a minute or less. The method depends upon direct measurement of DG, DG6P, and glucose without physical separation (2). The sensitivity is sufficient to assay samples as small as, or smaller than, the areas resolved in the usual radioautographs. Although a larger than tracer dose of DG is required, this is kept low enough not to significantly distort glucose metabolism. The assessment of glucose phosphorylation from directly observed levels of the primary metabolites concerned (DG, DG6P, and glucose) avoids many of the uncertainties that exist when these metabolites are calculated from plasma DG levels. In working out the method, some features of brain glucose metabolism have become evident that probably must be considered in any study of this kind, whether the time scale is short or long. Analytical Procedures. Measurement of DG, DG6P, and glucose depends on the facts (i) that glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) reacts with DG6P, but at a 2000-fold slower rate than with glucose 6-phosphate and (ii) that hexokinase reacts rapidly with both ...
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