title: Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.
abstract: BACKGROUND
  The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression.
  
  
  RESULTS
  By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology.
  
  
  CONCLUSIONS
  Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. arpitamanu@gmail.com.|Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, 345 Crispell Dr, Carter Harrison Building, Charlottesville, VA, 22908, USA. arpitamanu@gmail.com.
authors.forename: Arpita
authors.initials: A
authors.lastname: Singh
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. antonella.rella@stonybrook.edu.|Department of Molecular Genetics and Microbiology, Stony Brook University, 150 Life Science Building, Stony Brook, NY, 11794, USA. antonella.rella@stonybrook.edu.
authors.forename: Antonella
authors.initials: A
authors.lastname: Rella
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. jschwack@gmail.com.|Integrated Systems and Solutions Division, Scientific Research Corporation, Remount Road, North Charleston, SC, 29406, USA. jschwack@gmail.com.
authors.forename: John
authors.initials: J
authors.lastname: Schwacke
authors.affiliation: Department of Preventive Medicine, University of Stony Brook, Stony Brook, NY, 11794, USA. caterina.vacchi-suzzi@stonybrookmedicine.edu.
authors.forename: Caterina
authors.initials: C
authors.lastname: Vacchi-Suzzi
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. chiara.luberto@stonybrook.edu.|Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY, 11794, USA. chiara.luberto@stonybrook.edu.
authors.forename: Chiara
authors.initials: C
authors.lastname: Luberto
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. maurizio.delpoeta@stonybrook.edu.|Department of Molecular Genetics and Microbiology, Stony Brook University, 150 Life Science Building, Stony Brook, NY, 11794, USA. maurizio.delpoeta@stonybrook.edu.|Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, 29425, USA. maurizio.delpoeta@stonybrook.edu.|Department of Craniofacial Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. maurizio.delpoeta@stonybrook.edu.|Division of Infectious Diseases, Medical University of South Carolina, Charleston, SC, 29425, USA. maurizio.delpoeta@stonybrook.edu.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Del Poeta
mesh_terms: D003455:Cryptococcus neoformans / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D005963:Glucosylceramides / Q000378:metabolism*; D046228:Microarray Analysis; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D005963:Glucosylceramides