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1.  The Emerging Role of Urease as a General Microbial Virulence Factor 
PLoS Pathogens  2014;10(5):e1004062.
PMCID: PMC4022740  PMID: 24831297
2.  The Yeast Copper Response Is Regulated by DNA Damage 
Molecular and Cellular Biology  2013;33(20):4041-4050.
Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast.
PMCID: PMC3811678  PMID: 23959798
3.  Factors Required for Activation of Urease as a Virulence Determinant in Cryptococcus neoformans 
mBio  2013;4(3):e00220-13.
Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria.
Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans
PMCID: PMC3663189  PMID: 23653445
4.  Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci 
PLoS ONE  2010;5(12):e15968.
The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the macrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed.
PMCID: PMC3013146  PMID: 21209844
5.  A Mep2-dependent Transcriptional Profile Links Permease Function to Gene Expression during Pseudohyphal Growth in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2008;19(7):3028-3039.
The ammonium permease Mep2 is required for the induction of pseudohyphal growth, a process in Saccharomyces cerevisiae that occurs in response to nutrient limitation. Mep2 has both a transport and a regulatory function, supporting models in which Mep2 acts as a sensor of ammonium availability. Potentially similar ammonium permease-dependent regulatory cascades operate in other fungi, and they may also function in animals via the homologous Rh proteins; however, little is known about the molecular mechanisms that mediate ammonium sensing. We show that Mep2 is localized to the cell surface during pseudohyphal growth, and it is required for both filamentous and invasive growth. Analysis of site-directed Mep2 mutants in residues lining the ammonia-conducting channel reveal separation of function alleles (transport and signaling defective; transport-proficient/signaling defective), indicating transport is necessary but not sufficient to sense ammonia. Furthermore, Mep2 overexpression enhances differentiation under normally repressive conditions and induces a transcriptional profile that is consistent with activation of the mitogen-activated protein (MAP) kinase pathway. This finding is supported by epistasis analysis establishing that the known role of the MAP kinase pathway in pseudohyphal growth is linked to Mep2 function. Together, these data strengthen the model that Mep2-like proteins are nutrient sensing transceptors that govern cellular differentiation.
PMCID: PMC2441671  PMID: 18434596
6.  Amt2 Permease Is Required To Induce Ammonium-Responsive Invasive Growth and Mating in Cryptococcus neoformans▿  
Eukaryotic Cell  2007;7(2):237-246.
The conserved AmtB/Mep/Rh family of proteins mediate the transport of ammonium across cellular membranes in a wide range of organisms. Certain fungal members of this group are required to initiate filamentous growth. We have investigated the functions of two members of the AmtB/Mep/Rh family from the pathogenic basidiomycete Cryptococcus neoformans. Amt1 and Amt2 are low- and high-affinity ammonium permeases, respectively, and a mutant lacking both permeases is unable to grow under ammonium-limiting conditions. AMT2 is transcriptionally induced in response to nitrogen limitation, whereas AMT1 is constitutively expressed. Single and double amt mutants exhibit wild-type virulence in two models of cryptococcosis. Consistent with this, the formation of two C. neoformans virulence factors, cell wall melanin and the extracellular polysaccharide capsule, is not impaired in cells lacking either or both of the Amt1 and Amt2 permeases. Amt2 is, however, required for the initiation of invasive growth of haploid cells under low-nitrogen conditions and for the mating of wild-type cells under the same conditions. We propose that Amt2 may be a new fungal ammonium sensor and an element of the signaling cascades that govern the mating of C. neoformans in response to environmental nutritional cues.
PMCID: PMC2238157  PMID: 18055915
7.  Impact of Ammonium Permeases MepA, MepB, and MepC on Nitrogen-Regulated Secondary Metabolism in Fusarium fujikuroi▿  
Eukaryotic Cell  2007;7(2):187-201.
In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of ΔmepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In ΔmepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as ΔmepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the ΔmepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi.
PMCID: PMC2238153  PMID: 18083831
9.  A chemical potentiator of copper-accumulation used to investigate the iron-regulons of Saccharomyces cerevisiae 
Molecular Microbiology  2014;93(2):317-330.
The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron–sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron–sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation.
PMCID: PMC4149784  PMID: 24895027

Results 1-9 (9)