1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd, St Louis, MO 63104, USA
2 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd, St Louis, MO 63104, USA
Correspondence
Jennifer K. Lodge
lodgejk{at}slu.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glutathione peroxidases have been shown to exhibit a broad substrate range, including hydrogen peroxide, organic peroxides and peroxynitrite (Arteel et al., 1999; Arthur, 2000
). All three glutathione peroxidases from Saccharomyces cerevisiae express phospholipid hydroperoxidase activity as well (Avery & Avery, 2001
). Glutathione peroxidase gene expression has been studied in S. cerevisiae and induction of these genes is observed in response to oxidative stress or glucose repression (Inoue et al., 1999
).
The glutathione system has not been studied in any fungal pathogen to date, but enzymes important for resistance to oxidative and nitrosative stress have been linked to both virulence and viability of Cryptococcus neoformans (de Jesus-Berrios et al., 2003; Cox et al., 2003
; Missall et al., 2004b
, 2005
; Missall & Lodge, 2005
). For example, flavohaemoglobin denitrosylase, Fhb1, which is necessary for nitric oxide resistance (de Jesus-Berrios et al., 2003
), and the thiol peroxidase, Tsa1, which is important for both oxidative and nitrosative stress resistance (Missall et al., 2004b
), contribute significantly to virulence in C. neoformans. In addition, thioredoxin reductase, upon which Tsa1 is thought ultimately to be dependent for reduction, has been shown to be induced during oxidative and nitrosative stress and to be necessary for viability in C. neoformans (Missall & Lodge, 2005
). While some enzymes important for resistance to stress, including a superoxide dismutase, are dispensible for virulence (Giles et al., 2005
), other enzymes have been shown to be compensatory, their action only necessary for resistance in the absence of another stress-related enzyme. For example, only in the absence of Tsa1 is the laccase Lac2 induced in response to nitrosative stress and therefore important for resistance (Missall et al., 2005
).
The importance of glutathione peroxidases to the virulence of a pathogen has only been studied in the bacterium Streptococcus pyogenes, in which its GpoA has been shown to be necessary for pathogenicity (Brenot et al., 2004). Glutathione peroxidases have not been studied in any fungal pathogen (Missall et al., 2004a
), though a glutathione peroxidase homologue has been shown to be transcriptionally abundant during experimental cryptococcosis (Steen et al., 2003
). Here, we determine the expression patterns and localization of the two glutathione peroxidases and test their importance to stress resistance, macrophage survival and mammalian virulence of the fungal pathogen C. neoformans.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Localization using green fluorescent protein (GFP).
The plasmid containing the coding sequence for GFP (accession U73901) was generously provided by John Perfect. Primers used to amplify this coding region annealed to bases 524 and 710730. Overlap PCR technology (Davidson et al., 2002) was used to generate the 3' GFP fusion constructs, which included the entire genomic sequence of the desired gene and
1 kb of the upstream sequence, as well as the G418 resistance marker.
RNA extraction and cDNA synthesis.
Following the appropriate treatments, 50 ml C. neoformans cells was collected by centrifugation at 1800 g for 5 min, washed once with distilled water, and lyophilized overnight. The lyophilized pellet was then vortexed with 3 ml glass beads (1 mm, Biospec) and resuspended in 4 ml TRIzol Reagent (Invitrogen). After sitting at room temperature for 5 min, 800 µl chloroform was added and the mixture was shaken for 30 s. This cell lysate was then centrifuged at 4000 r.p.m. for 10 min, and the supernatant was transferred to a new tube. 2-Propanol (2 ml) was added, followed by incubation for 10 min at room temperature and centrifugation at 4000 r.p.m. for 10 min. After washing with 75 % ethanol, the pellet was resuspended in water and incubated with DNase I at 37 °C for 1 h. The RNA was extracted again with TRIzol and chloroform and precipitated with 2-propanol, as above. The dried pellet was resuspended in 300 µl RNase-free water (Gibco) and stored at 80 °C. First strand cDNA was made using the First Strand cDNA synthesis kit for RT-PCR (Roche).
Real-time PCR.
C. neoformans H99 was grown in minimal media at 30 °C overnight with shaking. Exponentially growing cells were treated with t-butylhydroperoxide, cumene hydroperoxide, hydrogen peroxide or sodium nitrite and allowed to grow at 30 °C with shaking for 2 h. RNA was extracted and first strand cDNA made as described above. This cDNA was used as template in a real-time PCR reaction using SYBR Green PCR reagents (Sigma) according to the manufacturer's recommendations. The GPX1 primers annealed to bases 210231 and 857877 of the 838 bp gene, and the GPX2 primers annealed to bases 102122 and 847867 of the 859 bp gene. Base numbering relates to the genomic sequence starting with the start codon ATG. The DNA Engine Opticon (MJ Research) was used as the fluorescence detector with the following protocol for the PCR reaction: 35 s at 94 °C, 50 s at 53 °C, 50 s at 72 °C, and a plate reading was repeated for a total of 40 cycles after a hot start of 4 min at 94 °C. A melting curve was performed at the end of the reaction to confirm a single product. A series of 10-fold dilutions of the cDNA was used in both the control and the experimental reactions. The induction data were taken from dilutions that came up 3·3 cycles apart, indicating that the reaction was in the linear range. The data were normalized to actin cDNA expression amplified in the set of PCR reactions.
Generation of deletion constructs.
An overlap PCR gene deletion technology (Davidson et al., 2002) was used to generate gene-specific deletion cassettes of GPX1 (GenBank accession no. XM 570772) and GPX2 (GenBank accession no. XM 568531) that included a nourseothricin (McDade & Cox, 2001
) or hygromycin (Hua et al., 2000
) cassette and resulted in the deletion of the entire coding regions of the appropriate genes. The double gpx1
gpx2
mutant was generated by deleting the GPX2 coding sequence from the gpx1
mutant. Mutant strains were reconstituted by fusing a
3 kb fragment that contained the GPX1 or GPX2 coding sequence and
1 kb of the putative promoter sequence to a G418 (Hua et al., 2000
) or nourseothricin (McDade & Cox, 2001
) selectable marker, and the entire construct was transformed into the appropriate mutant strains.
Transformation of C. neoformans.
H99 and mutant strains were transformed using biolistic techniques (Toffaletti et al., 1993; Hua et al., 2000
). Cells were grown in YPD to late exponential phase, concentrated, and plated onto YPD agar for transformation. The cells were bombarded with 0·6 µm gold beads (Bio-Rad) which were coated with DNA of the target construct according to the manufacturer's recommendations. Following the transformation, the cells were incubated at 30 °C for 4 h on non-selective media to allow for recovery, and then transferred with 0·8 ml sterile PBS to the appropriate selective media. Transformants were observed in 35 days.
Analysis of transformants.
To isolate stable transformants, all transformants were passaged five times on non-selective YPD medium and then tested for resistance to the appropriate selective marker. Only those transformants that grew as well on the selective media as on non-selective media were used as stable transformants. A three-primer PCR screen was used to prove homologous integration on both the 5' and 3' ends of the deletion construct (Nelson et al., 2003). In this manner, homologous recombinants can be distinguished from wild-type. A PCR screen using primers outside the deletion construct will amplify the entire gene region, demonstrating that a single copy of the transforming DNA has been inserted at the desired locus. Southern blots were performed to screen for single integration in the genome. Single bands were observed on all Southern blots when probed with a selectable marker-specific probe. All deletion strains generated for this work had a single deletion construct homologously integrated at the appropriate locus, and no other insertions in the genome (data not shown).
Genomic DNA preparation.
Genomic DNA was prepared by a modification of the glass-bead DNA extraction protocol described by Fujimura & Sakuma (1993). C. neoformans cells were suspended in a microfuge tube in 500 µl lysis buffer (50 mM Tris, pH 7·5, 20 mM EDTA, 1 % SDS), with 400 mg glass beads (425600 µm, Sigma G-9268). Cells were disrupted by vortexing for 5 min, followed by a 10 min incubation at 70 °C. After brief vortexing, 200 µl 5 M potassium acetate and 150 µl 5 M sodium chloride were added. The tubes were placed on ice for 20 min and centrifuged at 14 000 r.p.m. for 20 min. The supernatant was mixed with 500 µl phenol/chloroform and spun for 2 min at 14 000 r.p.m. The aqueous phase was then mixed with 450 µl chloroform and spun for 2 min at 14 000 r.p.m. The DNA was then precipitated by the addition of 200 µl ethanol, washed with 70 % ethanol, dried, and resuspended in 50 µl deionized water.
Southern hybridizations.
Approximately 10 µg of genomic DNA from each strain was digested with various restriction endonucleases according to the manufacturer's recommendations. Restriction fragments were separated on a 1 % agarose gel and transferred to nylon membranes using a Turbo-Blot apparatus (Schleicher & Schuell) and 10x SSC as transfer buffer. Probes for Southern analysis were prepared by random priming (random priming kit; Roche) using 50 µCi (1·9 MBq) [-32P]dCTP (Amersham AA0005) according to the manufacturer's instructions. The blots were incubated in 10 ml of a 6x SSC, 0·1 % SDS and 5 % non-fat dry milk (Carnation) solution for 1 h at 65 °C, then probe was added to this solution, and the blots were hybridized at 65 °C overnight. The blots were washed twice in 2x SSC, 0·1 % SDS at room temperature for 10 min and once for 10 min in 0·2x SSC, 0·1 % SDS that had been prewarmed to 65 °C.
Oxidative and nitrosative stress plates.
Solid minimal media were made with designated amounts of hydrogen peroxide, t-butylhydroperoxide, cumene hydroperoxide or sodium nitrite. C. neoformans strains were grown to mid-exponential phase in YNB, pH 4·0, and 10-fold dilutions were made. Aliquots (5 µl) of the undiluted and diluted cultures for each strain were spotted onto the solid minimal media and grown at 30 °C for two nights.
Protein lysate preparation.
C. neoformans cells were grown to mid-exponential phase in YPD at 30 °C. The cells were collected by centrifugation, washed three times in sterile PBS and resuspended in chilled lysis buffer [40 mM Tris/HCl, pH 9·0, 4 % CHAPS, 1x complete protease inhibitor cocktail (Roche)] at a concentration of 2x109 cells ml1. One millilitre of cells and 2·2 g of 0·5 mm zirconium/silica beads were added together in a 2 ml tube, and the cells were disrupted on a Biospec BeadBeater for 30 s at 50 000 r.p.m., repeated seven times, alternated with 2 min on ice. The cell debris was removed by centrifugation (10 000 r.p.m. for 20 min) and syringe filtered (0·45 µm). The supernatant was assayed using a Bio-Rad RC-DC protein assay. Typical lysates resulted in 57 mg protein ml1.
Glutathione peroxidase activity.
Total protein lysates were assayed for glutathione peroxidase activity using the BIOXYTECH GPx-340 colorimetric assay (OxisResearch, Portland, OR). Briefly, the lysate is added to a solution containing glutathione, glutathione reductase and NADPH. The enzymic reaction is initiated by adding t-butylhydroperoxide as a substrate and the A340 is recorded for 5 min. The oxidation of NADPH to NADP+, which is directly proportional to the Gpx activity, results in a decrease in A340. Controls lacking lysate or glutathione solution were performed to ensure specificity of glutathione-dependent Gpx activity. Twofold dilutions of total-protein lysates were used to ensure that the activity measurements observed were in the linear range.
Macrophage assay.
RAW 264.7 macrophages were diluted to 105 cells ml1 in Dulbecco's Modified Eagle Medium (DMEM). A 100 µl volume (104 cells) of macrophages was plated into each well of a pre-treated microtitre dish. C. neoformans cells grown in YNB, pH 4·0, overnight were diluted in DMEM to 105 cells ml1. The cryptococcal cells were added to the macrophages at an m.o.i. of 1 and incubated at 37 °C and 5 % CO2 for 24 h. One hundred microlitres of 5 % SDS was added to each well and the mixture was incubated at room temperature for 5 min to lyse the macrophages. Serial dilutions were plated on YPD agar and incubated at 30 °C for 2 days. Control wells without macrophages were done for each strain to control for growth of cryptococcal cells in DMEM media. Data presented are representative of three independent experiments.
Inhalation mouse model.
C. neoformans strains were grown at 30 °C with shaking for two nights in YPD. The cells were centrifuged, washed in endotoxin-free PBS and resuspended in endotoxin-free PBS. The cells were counted on a haemocytometer and diluted to 1x107 cells ml1. CBA/J female mice (Jackson Laboratories) were anaesthetized and allowed to inhale 5x105 (50 µl) cells, which were dripped into the nares (Cox et al., 2000). Mice were weighed before and during the course of infection. Mice were sacrificed by CO2 asphyxiation once they reached 80 % of their original body weight. At this point, the mice showed signs of being morbidly ill, including a ruffled coat, lethargy, a hunched posture, unstable gait and loss of appetite.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Glutathione peroxidase mutants are sensitive to various peroxides
To understand the importance of the glutathione peroxidase enzymes to the stress resistance of C. neoformans, we generated deletion mutants of each and both of these enzymes. While the gpx mutants are both morphologically similar to wild-type and show normal melanin and capsule production, they respond differently in the presence of peroxide stress. Both the gpx1 and gpx2
mutants are hypersensitive to cumene hydroperoxide (an aromatic hydroperoxide), but at high concentrations, the gpx2
mutant is slightly more sensitive than the gpx1
mutant (Fig. 3
). Conversely, the gpx1
mutant, but not the gpx2
mutant, is hypersensitive to t-butylhydroperoxide (an alkyl hydroperoxide) (Fig. 3
). All phenotypes observed were complemented by reintroduction of the appropriate GPX gene, as shown (Fig. 3
). Since we show that both enzymes are induced during various peroxide stresses, these results may suggest a difference in the localization of the two Gpx enzymes, a difference in their activity, or an alternative regulation in the deletion mutants.
|
Gpx1 and Gpx2 are both localized to the cytoplasm
Since the gpx mutants show specific phenotypes to either t-butylhydroperoxide or cumene hydroperoxide, one hypothesis that would explain this difference is that the glutathione peroxidases are differentially localized. To determine the cellular localization of the glutathione peroxidases in C. neoformans, we fused the GFP coding sequence to the 3' end of the GPX genes and expressed these fusion constructs in the gpx mutants. By expressing GpxGFP in the gpx mutants, we were able to confirm the functionality of the resulting fusion proteins with in vitro peroxide sensitivity tests. Our localization studies show that both the functional Gpx1GFP and Gpx2GFP fusion proteins are localized in a similar manner in the cytoplasm throughout the cryptococcal cell (Fig. 4).
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Based on our deletion studies, Gpx1 appears to be necessary for defence against t-butylhydroperoxide, while Gpx2 appears to be more important for cumene hydroperoxide stress. Neither gpx mutant shows sensitivity towards hydrogen peroxide stress, though we show, based on gene expression, that there is a potential for compensation in the gpx2 mutant by induction of GPX1 mRNA, possibly due to an increased effective concentration of peroxide seen by this mutant compared to wild-type. But since the double mutant is also not sensitive to hydrogen peroxide, this suggests that another peroxide defence system in C. neoformans, such as catalases, glutaredoxins or other peroxidases, is more efficient and specific for the stress induced by hydrogen peroxide compared to more complex hydroperoxides. In previous work, we have shown the importance of the thiol peroxidase Tsa1 to the hydrogen peroxide stress resistance of C. neoformans (Missall et al., 2004b
).
We observe GPX gene induction in the gpx mutants during cumene hydroperoxide stress similar to that of the GPX genes observed in wild-type, which is consistent with our functional analysis showing additive sensitivity to this peroxide in the double gpx1gpx2
mutant compared to the single mutants. Interestingly, while we see wild-type induction of GPX1 in the gpx2
mutant during t-butylhydroperoxide stress, we observe no induction of expression of GPX2 in the gpx1
mutant. These data suggest that there may not be any substrate specificity, but simply changes in expression that affect the sensitivities of the mutants to peroxides. It is possible that Gpx1 is important for the regulation of GPX2 during this specific stress, as glutathione peroxidases have been implicated in the regulation of stress responses in S. cerevisiae by acting as a sensor and transducer of a hydroperoxide signal to a transcriptional activator (Delaunay et al., 2002
).
It is possible that in addition to these changes in expression in the glutathione peroxidase mutants, there is a difference in substrate specificity between the two enzymes. Substrate specificity has been observed among similar glutathione-type peroxidases of the parasite Trypanosoma brucei (Schlecker et al., 2005). This would help explain the increased sensitivity to high concentrations of cumene hydroperoxide stress that we observe in the gpx2
mutant. Since we observe additional Gpx-independent glutathione peroxidase activity in C. neoformans, we are unable to determine a substrate specificity in the whole-cell-lysate activity assay. Future studies may identify this potential substrate specificity by purifying the two glutathione peroxidase enzymes.
Our functional analysis shows that both glutathione peroxidases are important for defence against the oxidants encountered in the macrophage environment of the host, while they are dispensable for virulence in mice. This points out the complexity of a mammalian infection involving more than the macrophage stress. There are many factors that contribute to the ability of C. neoformans to evade the innate immune system and many more that contribute to the ability to cause disease. Our data may simply indicate that another antioxidant system is more important for defence against the oxidative and nitrosative stress to which C. neoformans is exposed during infection. Since mutation of the GPX genes does not affect virulence or resistance to hydrogen peroxide and nitric oxide, it is plausible that these two compounds, and not the more complex alkyl or aromatic peroxides to which the gpx mutants are sensitive, are more important to the host defence against C. neoformans. Both the thiol peroxidase Tsa1, which is important for hydrogen peroxide, t-butylhydroperoxide and nitric oxide resistance, and the flavohaemoglobin denitrosylase Fhb1, which is important specifically for nitric oxide resistance, are important to the virulence of C. neoformans (de Jesus-Berrios et al., 2003; Missall et al., 2004b
). Another possibility is that in the absence of a GPX, other antioxidant enzyme(s) are upregulated in vivo to compensate for the lack of Gpx activity. For example, in S. cerevisiae, 1-Cys peroxiredoxins have been shown to possess glutathione peroxidase activity (Chen et al., 2000
). In addition, it has been reported that glutaredoxins, which are small glutathione-dependent oxidoreductases, can act as glutathione peroxidases and reduce peroxides in S. cerevisiae (Collinson et al., 2002
). It has also been observed that during oxidative stress, there is an overexpression of two of these glutaredoxins (Collinson et al., 2002
). We also show that C. neoformans has additional enzymes with glutathione peroxidase activity, which may function in vivo, compensating for the absence of the two glutathione peroxidases. After the source of the additional glutathione peroxidase activity is identified, the challenge will be to measure this potentially compensatory activity in vivo. These and other future studies may reveal that a similar mechanism of compensation within the glutathione system also exists in C. neoformans. In conclusion, while both glutathione peroxidases of C. neoformans are dispensable for virulence, these antioxidant enzymes are necessary for resistance to specific peroxides and survival of this fungal pathogen in macrophages.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arthur, J. R. (2000). The glutathione peroxidases. Cell Mol Life Sci 57, 18251835.[Medline]
Avery, A. M. & Avery, S. V. (2001). Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases. J Biol Chem 276, 3373033735.
Brenot, A., King, K. Y., Janowiak, B., Griffith, O. & Caparon, M. G. (2004). Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes. Infect Immun 72, 408413.
Chen, J. W., Dodia, C., Feinstein, S. I., Jain, M. K. & Fisher, A. B. (2000). 1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities. J Biol Chem 275, 2842128427.
Collinson, E. J., Wheeler, G. L., Garrido, E. O., Avery, A. M., Avery, S. V. & Grant, C. M. (2002). The yeast glutaredoxins are active as glutathione peroxidases. J Biol Chem 277, 1671216717.
Cox, G. M., Mukherjee, J., Cole, G. T., Casadevall, A. & Perfect, J. R. (2000). Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68, 443448.
Cox, G. M., Harrison, T. S., McDade, H. C., Taborda, C. P., Heinrich, G., Casadevall, A. & Perfect, J. R. (2003). Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages. Infect Immun 71, 173180.
Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus Berrios, M., Hull, C. M., D'Souza, C., Wang, P. & Heitman, J. (2002). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 26072615.[Medline]
de Jesus-Berrios, M., Liu, L., Nussbaum, J. C., Cox, G. M., Stamler, J. S. & Heitman, J. (2003). Enzymes that counteract nitrosative stress promote fungal virulence. Curr Biol 13, 19631968.[CrossRef][Medline]
Delaunay, A., Pflieger, D., Barrault, M. B., Vinh, J. & Toledano, M. B. (2002). A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111, 471481.[CrossRef][Medline]
Dickinson, D. A. & Forman, H. J. (2002). Cellular glutathione and thiols metabolism. Biochem Pharm 64, 10191026.[CrossRef][Medline]
Fujimura, H. & Sakuma, Y. (1993). Simplified isolation of chromosomal and plasmid DNA from yeasts. Biotechniques 14, 538540.[Medline]
Giles, S. S., Perfect, J. R. & Cox, G. M. (2005). Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans. Fungal Genet Biol 42, 2029.[CrossRef][Medline]
Hua, J. H., Meyer, J. D. & Lodge, J. K. (2000). Development of positive selectable markers for the fungal pathogen, Cryptococcus neoformans. Clin Diagn Lab Immunol 7, 125128.
Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S. & Kimura, A. (1999). Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae. J Biol Chem 274, 2700227009.
Loftus, B. J., Fung, E., Roncaglia, P. & 51 other authors (2005). The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307, 13211324.
McDade, H. C. & Cox, G. M. (2001). A new dominant selectable marker for use in Cryptococcus neoformans. Med Mycol 39, 151154.[Medline]
Missall, T. A. & Lodge, J. K. (2005). Thioredoxin reductase is essential for viability in the fungal pathogen, Cryptococcus neoformans. Eukaryot Cell 4, 487489.
Missall, T. A., Lodge, J. K. & McEwen, J. E. (2004a). Mechanisms of resistance to oxidative and nitrosative stress: implications for fungal survival in mammalian hosts. Eukaryot Cell 3, 835846.
Missall, T. A., Pusateri, M. E. & Lodge, J. K. (2004b). Thiol peroxidase is critical for virulence and resistance to nitric oxide and peroxide in the fungal pathogen, Cryptococcus neoformans. Mol Microbiol 51, 14471458.[CrossRef][Medline]
Missall, T. A., Moran, J. M., Corbett, J. A. & Lodge, J. K. (2005). Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant, Tsa1. Eukaryot Cell 4, 202208.
Nelson, R. T., Pryor, B. A. & Lodge, J. K. (2003). Sequence length required for homologous recombination in Cryptococcus neoformans. Fungal Genet Biol 38, 19.[CrossRef][Medline]
Schlecker, T., Schmidt, A., Dirdjaja, N., Voncken, F., Clayton, C. & Krauth-Siegel, R. L. (2005). Substrate specificity, localisation and essential role of the glutathione peroxidase-type tryparedoxin peroxidases in Trypanosoma brucei. J Biol Chem 280, 1438514394.
Steen, B. R., Zuyderduyn, S., Toffaletti, D. L., Marra, M., Jones, S. J. M., Perfect, J. R. & Kronstad, J. (2003). Cryptococcus neoformans gene expression during experimental cryptococcal meningitis. Eukaryot Cell 2, 13361349.
Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T. & Perfect, J. R. (1993). Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175, 14051411.[Abstract]
Trotter, E. W. & Grant, C. M. (2003). Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems. EMBO Rep 4, 184188.
Tsuzi, D., Maeta, K., Takatsume, Y., Izawa, S. & Inoue, Y. (2004). Regulation of the yeast phospholipid hydroperoxide glutathione peroxidase GPX2 by oxidative stress is mediated by Yap1 and Skn7. FEBS Lett 565, 148154.[CrossRef][Medline]
Received 18 April 2005;
revised 12 May 2005;
accepted 15 May 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |