Dataset Information

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

ABSTRACT: In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

SUBMITTER: Giles SS

PROVIDER: S-EPMC1563583 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

Giles Steven S SS Stajich Jason E JE Nichols Connie C Gerrald Quincy D QD Alspaugh J Andrew JA Dietrich Fred F Perfect John R JR

Eukaryotic cell 20060901 9

In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 ...[more]

PMID: 16963629

Similar Datasets

Project description:UNLABELLED:While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-length CTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function of CTR4 in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role for CTR4 in copper-related homeostasis and subsequently in fungal virulence. IMPORTANCE:Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a ?ctr4 mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4 during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.

Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Project description:BackgroundBemisia tabaci MEAM1 (Middle East-Asia Minor 1) feeding alters antioxidative enzyme activity in some plant species. Infestation of B. tabaci nymphs decreases Myzus persicae performance on systemic, but not local leaves of tobacco plants. However, it is unclear if B. tabaci nymphs induced antioxidant activities contributing to the aphid resistance.Methodology/principal findingsWe investigated the relationship between antioxidants induced by nymphs of B. tabaci feeding on tobacco and aphid resistance. The activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and the concentration of hydrogen peroxide (H2O2) were assayed in tobacco leaves at different feeding times following infestation of B. tabaci nymphs. The infestation altered the activities of CAT and POD, but had no significant effect on SOD activity. The highest CAT activity was observed at 15 d after infestation. This was 98.2% greater than control systemic leaves, but 32.6% lower than the control in local leaves. Higher POD activity was recorded in local vs. systemic leaves after 15 d of infestation. POD activity was 71.0% and 112.9% higher in local and systemic leaves, respectively, than in the controls. The changes of CAT, but not POD or SOD activity were correlated to levels of aphid resistance. H2O2 levels were higher in local than in systemic leaves in contrast to CAT activity. Tobacco curly shoot virus mediated virus-induced gene silencing was employed to determine if CAT activation was involved in the aphid resistance induced by B. tabaci nymphs. B. tabaci induced CAT activity decreased when the Cat1 expression was silenced. The performance assay indicated that Cat1 silencing made B. tabaci infested plants a more suitable host for aphids than infested control plants. The aphid survival rate was reduced by 40.4% in infested control plants, but reduced by only 26.1% in Cat1-silenced plants compared to uninfested controls. Also, qPCR results showed that silencing of Cat1 led to the suppression of the B. tabaci mediated PR-2a expression.Conclusions/significanceAphid resistance in plants infested with B. tabaci nymphs is associated with enhanced antioxidant activities in which CAT may play a dominant role. This resistance probably acted via interactions with SA-mediated defense responses.

Dataset Information

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

Publications

The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets