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1.
J Infect Dis ; 212(11): 1827-34, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26014799

ABSTRACT

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography-tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients' sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction-based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.


Subject(s)
Biomarkers/analysis , Mycobacterium tuberculosis/chemistry , Oxazoles/analysis , Tuberculosis/diagnosis , Adenosine/analogs & derivatives , Adenosine/analysis , Animals , Bacterial Typing Techniques , Chromatography, Liquid , Humans , Lung/microbiology , Mice , Mycobacterium tuberculosis/isolation & purification , Sputum/microbiology , Tandem Mass Spectrometry
2.
Chem Biol ; 22(4): 516-526, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-25910243

ABSTRACT

Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise >1% of all Mycobacterium tuberculosis lipid. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and nuclear magnetic resonance, we established the structure of the previously unknown molecule, N(6)-tuberculosinyladenosine (N(6)-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N(6)-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M. tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria, and vaccine strains. With no substantially similar known molecules in nature, the discovery and in vivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.


Subject(s)
Lipids/biosynthesis , Mycobacterium tuberculosis/metabolism , Nucleosides/analysis , Terpenes/chemistry , Tuberculosis/diagnosis , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatography, High Pressure Liquid , Isomerism , Lipids/analysis , Lipids/isolation & purification , Lung/metabolism , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/genetics , Nucleosides/biosynthesis , Nucleosides/chemistry , Polymorphism, Single Nucleotide , Spectrometry, Mass, Electrospray Ionization
3.
G3 (Bethesda) ; 3(10): 1675-86, 2013 Oct 03.
Article in English | MEDLINE | ID: mdl-23934995

ABSTRACT

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.


Subject(s)
Candida glabrata/genetics , DNA, Recombinant/genetics , Genetic Vectors/genetics , Plasmids/genetics , Candida glabrata/metabolism , DNA, Recombinant/metabolism , Genes, Reporter , Genetic Markers , Genetic Vectors/metabolism , Plasmids/metabolism , Promoter Regions, Genetic
4.
Eukaryot Cell ; 11(12): 1512-9, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23087370

ABSTRACT

Many fungal species use glycerol as a compatible solute with which to maintain osmotic homeostasis in response to changes in external osmolarity. In Saccharomyces cerevisiae, intracellular glycerol concentrations are regulated largely by the high osmolarity glycerol (HOG) response pathway, both through induction of glycerol biosynthesis and control of its flux through the plasma membrane Fps1 glycerol channel. The channel activity of Fps1 is also controlled by a pair of positive regulators, Rgc1 and Rgc2. In this study, we demonstrate that Candida glabrata, a fungal pathogen that possesses two Fps1 orthologs and two Rgc1/-2 orthologs, accumulates glycerol in response to hyperosmotic stress. We present an initial characterization of mutants with deletions in the C. glabrata FPS1 (CAGL0C03267 [www.candidagenome.org]) and FPS2 (CAGL0E03894) genes and find that a double mutant accumulates glycerol, experiences constitutive cell wall stress, and is hypersensitive to treatment by caspofungin, an antifungal agent that targets the cell wall. This mutant is cleared more efficiently in mouse infections than is wild-type C. glabrata by caspofungin treatment. Finally, we demonstrate that one of the C. glabrata RGC orthologs complements an S. cerevisiae rgc1 rgc2 null mutant, supporting the conclusion that this regulatory assembly is conserved between these species.


Subject(s)
Candida glabrata/metabolism , Fungal Proteins/metabolism , Glycerol/metabolism , Porins/metabolism , Stress, Physiological , Animals , Antifungal Agents/pharmacology , Candida glabrata/genetics , Candida glabrata/pathogenicity , Caspofungin , Cell Wall/drug effects , Cell Wall/metabolism , Echinocandins/pharmacology , Fungal Proteins/genetics , Lipopeptides , Mice/microbiology , Mutation , Osmolar Concentration , Porins/genetics
5.
Genetics ; 190(4): 1285-97, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22234857

ABSTRACT

Candida glabrata, an opportunistic fungal pathogen, adheres to mammalian epithelial cells; adherence is mediated primarily by the Epa1 adhesin. EPA1 is a member of a large gene family of ≈ 23 paralogues, which encode putative adhesins. In this study, we address how EPA1 transcription is regulated. Our data show that EPA1 expression is subject to two distinct negative regulatory mechanisms. EPA1 transcription is repressed by subtelomeric silencing: the Sir complex (Sir2-Sir4), Rap1, Rif1, yKu70, and yKu80 are required for full repression. Activation of EPA1 occurs immediately after dilution of stationary phase (SP) cells into fresh media; however, transcription is rapidly repressed again, limiting expression to lag phase, just as the cells exit stationary phase. This repression following lag phase requires a cis-acting regulatory negative element (NE) located in the EPA1 3'-intergenic region and is independent of telomere proximity. Bioinformatic analysis shows that there are 10 copies of the NE-like sequence in the C. glabrata genome associated with other EPA genes as well as non-EPA genes.


Subject(s)
Candida glabrata/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Lectins/metabolism , Regulatory Elements, Transcriptional , Candida glabrata/growth & development , Candida glabrata/metabolism , Cell Adhesion , Cell Division , Cells, Cultured , Chromosome Mapping , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Culture Media/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Fungal Proteins/genetics , Gene Silencing , Genes, Fungal , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Lectins/genetics , Microbiological Techniques , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Telomere/genetics , Telomere/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional Activation
6.
Mol Cell Biol ; 29(15): 4067-79, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19451228

ABSTRACT

The yeast Candida glabrata is an opportunistic pathogen of humans. C. glabrata is a NAD(+) auxotroph, and its growth depends on the availability of niacin (environmental vitamin precursors of NAD(+)). We have previously shown that a virulence-associated adhesin, encoded by EPA6, is transcriptionally induced in response to niacin limitation. Here we used transcript profiling to characterize the transcriptional response to niacin limitation and the roles of the sirtuins Hst1, Hst2, and Sir2 in mediating this response. The majority of genes transcriptionally induced by niacin limitation are regulated by Hst1, suggesting that it is the primary sensor of niacin limitation in C. glabrata. We show that three highly induced genes, TNA1, TNR1, and TNR2, encode transporters which are necessary and sufficient for high-affinity uptake of NAD(+) precursors. Strikingly, if a tna1 tnr1 tnr2 mutant is starved for niacin, it exhibits an extended lag phase, suggesting a central role for the transporters in restoring NAD(+) homeostasis after niacin limitation. Lastly, we had previously shown that the adhesin encoded by EPA6 is induced during experimental urinary tract infection (UTI); we show here that EPA6 transcriptional induction during UTI is strongly enhanced in the tna1 tnr1 tnr2 mutant strain, implicating the transporters in the growth of C. glabrata during infection.


Subject(s)
Candida glabrata/metabolism , Fungal Proteins/metabolism , Histone Deacetylases/metabolism , Membrane Transport Proteins/metabolism , NAD/metabolism , Sirtuins/metabolism , Animals , Binding, Competitive/drug effects , Candida glabrata/genetics , Candida glabrata/pathogenicity , Candidiasis/microbiology , Fungal Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Fungal/drug effects , Histone Deacetylases/genetics , Membrane Transport Proteins/genetics , Mice , Mice, Inbred BALB C , Mutation , Niacin/metabolism , Niacin/pharmacology , Oligonucleotide Array Sequence Analysis , Open Reading Frames/genetics , Protein Binding/drug effects , Sirtuins/genetics , Vitamin B Complex/metabolism , Vitamin B Complex/pharmacology
7.
Eukaryot Cell ; 7(12): 2168-78, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18836091

ABSTRACT

Candida glabrata, a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the EPA genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the EPA family, we have assessed the importance of C. glabrata orthologues of known regulators of subtelomeric silencing in Saccharomyces cerevisiae. To this end, we used a series of strains containing insertions of the reporter URA3 gene within different intergenic regions throughout four telomeres of C. glabrata. Using these reporter strains, we have assessed the roles of SIR2, SIR3, SIR4, HDF1 (yKu70), HDF2 (yKu80), and RIF1 in mediating silencing at four C. glabrata telomeres. We found that, whereas the SIR proteins are absolutely required for silencing of the reporter genes and the native subtelomeric EPA genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a cis element adjacent to the EPA3 locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the C. glabrata telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements.


Subject(s)
Candida glabrata/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Gene Silencing , Telomere-Binding Proteins/metabolism , Telomere/genetics , Candida glabrata/metabolism , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Fungal Proteins/genetics , Telomere/metabolism , Telomere-Binding Proteins/genetics
8.
Nature ; 452(7187): 604-9, 2008 Apr 03.
Article in English | MEDLINE | ID: mdl-18385733

ABSTRACT

Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.


Subject(s)
Candida glabrata/metabolism , Drug Resistance, Fungal , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Receptors, Steroid/metabolism , Saccharomyces cerevisiae/metabolism , Animals , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Candida glabrata/drug effects , Candida glabrata/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Resistance, Fungal/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/genetics , Genes, Fungal/genetics , Mediator Complex , Multigene Family , Pregnane X Receptor , Protein Structure, Tertiary , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, Genetic/genetics , Xenobiotics/metabolism
9.
Mol Microbiol ; 66(1): 14-25, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17725566

ABSTRACT

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.


Subject(s)
Candida glabrata/metabolism , Metabolic Networks and Pathways , NAD/biosynthesis , Animals , Candida glabrata/genetics , Candida glabrata/growth & development , Gene Deletion , Mice , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Niacin/metabolism , Niacinamide/analogs & derivatives , Niacinamide/metabolism , Nicotinamidase/genetics , Nicotinamidase/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Pyridinium Compounds
10.
Mol Microbiol ; 55(4): 1246-58, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15686568

ABSTRACT

The pathogenic yeast Candida glabrata is able to bind in vitro to human epithelial cells. This interaction depends on expression of the adhesin Epa1p. The genome contains a number of EPA1 paralogues which localize to the subtelomeric regions of the C. glabrata. We have identified three hyperadherent mutants of C. glabrata. The first has an insertion adjacent to EPA7, an EPA1-related adhesin. The others disrupt the SIR3 and RIF1 genes of C. glabrata. We show that SIR3 and RIF1 are required for subtelomeric silencing in C. glabrata and that RIF1 regulates telomere length in C. glabrata. We show that the hyperadherent phenotype of the sir3Delta and rif1Delta deletion strains depends primarily on derepression of two novel members of the EPA gene family -EPA6 and EPA7. The sir3Delta and rif1Delta mutants show increased colonization of the kidney in a murine model of disseminated infection and this hypercolonization depends, at least in part, on derepression of EPA6 and EPA7. The analysis here is the first evidence that multiple EPA genes encode adhesins and demonstrates that transcription of at least two of these adhesins is regulated by subtelomeric silencing.


Subject(s)
Candida glabrata/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/genetics , Lectins/genetics , Telomere/genetics , Telomere/ultrastructure , Animals , Base Sequence , CHO Cells , Cell Line , Cricetinae , DNA Primers , Genotype , Humans , Kidney , Molecular Sequence Data , Mutation , Plasmids/genetics , Transfection
11.
Mol Cell Neurosci ; 27(1): 44-58, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15345242

ABSTRACT

Rett syndrome (RTT) is a severe neurodevelopmental disorder with features of autism that results from mutation of the gene encoding the transcriptional repressor methyl-CpG binding protein (MECP2). The consequences of loss of a transcription factor may be complex, affecting the expression of many proteins, thus limiting understanding of this class of diseases and impeding therapeutic strategies. This is true for RTT. Neither the cell biological mechanism(s) nor the developmental stage affected by MECP2 deficiency is known. In vivo analysis of the olfactory system demonstrates that Mecp2 deficiency leads to a transient delay in the terminal differentiation of olfactory neurons. This delay in maturation disrupts axonal targeting in the olfactory bulb, resulting in abnormal axonal projections, subglomerular disorganization, and a persistent reduction in glomerular size. These results indicate a critical cell biological function for Mecp2 in mediating the final stages of neuronal development.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Neurons/metabolism , Olfactory Bulb/abnormalities , Olfactory Pathways/abnormalities , Olfactory Receptor Neurons/abnormalities , Repressor Proteins/genetics , Animals , Biomarkers , Cell Differentiation/genetics , Disease Models, Animal , GAP-43 Protein/metabolism , Gene Expression Regulation, Developmental/genetics , Growth Cones/metabolism , Growth Cones/ultrastructure , Methyl-CpG-Binding Protein 2 , Mice , Mice, Knockout , Nerve Tissue Proteins/metabolism , Neurons/ultrastructure , Neuropil/cytology , Neuropil/metabolism , Olfactory Bulb/cytology , Olfactory Bulb/metabolism , Olfactory Marker Protein , Olfactory Pathways/cytology , Olfactory Pathways/metabolism , Olfactory Receptor Neurons/cytology , Olfactory Receptor Neurons/metabolism , Rett Syndrome/genetics , Rett Syndrome/metabolism , Synapses/genetics , Synapses/metabolism
12.
Genes Dev ; 17(18): 2245-58, 2003 Sep 15.
Article in English | MEDLINE | ID: mdl-12952896

ABSTRACT

Candida glabrata is an important opportunistic pathogen causing both mucosal and bloodstream infections. C. glabrata is able to adhere avidly to mammalian cells, an interaction that depends on the Epa1p lectin. EPA1 is shown here to be a member of a larger family of highly related genes encoded in subtelomeric clusters. Subtelomeric clustering of large families of surface glycoprotein-encoding genes is a hallmark of several pathogens, including Plasmodium, Trypanosoma, and Pneumocystis. In these other pathogens, a single surface glycoprotein is expressed, whereas other genes in the family are transcriptionally silent. Similarly, whereas EPA1 is expressed in vitro, EPA2-5 are transcriptionally repressed. This repression is shown to be due to regional silencing of the subtelomeric loci. In Saccharomyces cerevisiae, subtelomeric silencing is initiated by Rap1p binding to the telomeric repeats and subsequent recruitment of the Sir complex by protein-protein interaction. We demonstrate here that silencing of the subtelomeric EPA loci also depends on functional Sir3p and Rap1p. This identification and analysis of the EPA gene family provides a compelling example in an ascomycete of chromatin-based silencing of natural subtelomeric genes and provides for the first time in a pathogen, molecular insight into the transcriptional silencing of large subtelomeric gene families.


Subject(s)
Candida glabrata/genetics , Fungal Proteins/genetics , Gene Silencing , Lectins/genetics , Transcription Factors/genetics , Candida glabrata/pathogenicity , Fungal Proteins/metabolism , Lectins/metabolism , Multigene Family , Transcription Factors/metabolism , Transcription, Genetic , Virulence/genetics , Virulence/physiology
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