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Effect of pH on gene expression in two glucosyl ceramide (GlcCer) mutant strains of Cryptococcus neoformans H99

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 (null mutants’ delta-gcs1 and delta-smt1 lacking glucosylceramide synthase 1 gene and C9 sphingolipid methyltransferase 1 gene respectively) 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. The 6 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), all of which are membrane-localized and have significantly altered gene expression levels. Therefore, we speculate 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 trans membrane signaling complex, which in turn contributes to Cryptococcal osmotic, pH, ion homeostasis and its pathobiology. Conclusion Gene expression microarrays identified 6 genes by gene set enrichment analysis and validated by qRT-PCR, which are involved in the trans membrane signaling network in Cryptococcus neoformans, controlling the rigidity of the membrane in neutral-alkaline pH and therefore the pathobiology of the fungus in these conditions.

ORGANISM(S): Cryptococcus neoformans var. grubii H99

PROVIDER: GSE69361 | GEO | 2015/05/30

SECONDARY ACCESSION(S): PRJNA285298

REPOSITORIES: GEO

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Project description:Every cyanobacterial species contains gene encoding site-2-protease (S2P) homolog. The studied prokaryotic S2P homologs play essential roles in regulating stress response through intramembrane proteolysis of membrane-bound anti-sigma factors. Here, gene of Slr0643, one of four S2P homologs in Synechocystis sp. PCC 6803, was insertionally disrupted to explore its physiological role. Only a partially segregated mutant was obtained, indicating its indispensability for growth. The partially disrupted mutant could not grow at pH 6.5, while wild type acclimated to pH 6.5 quickly. The slr0643 gene expression was transiently induced after pH transfer from 7.5 to 6.5. Both evidences demonstrated the pivotal role of fully functional Slr0643 in acid acclimation. DNA microarray and quantitative RT-PCR analyses decoded genes involved in early acid acclimation and revealed differentially expressed genes due to slr0643 disruption at both pH conditions. Early acid acclimation to pH 6.5 included upregulation of sigH, hik16 and hik35, and downregulation of pcrR and sigG; as well as downregulation of porins and upregulation of inorganic carbon and nitrogen transporters. Defective photosynthesis and excess expression of NADH dehydrogenase, together with over upregulation of carbon transporter and repression of nitrogen transporter and metabolism gene contributed to the acid lethality of mutant at pH 6.5. Most interestingly, analysis of microarray data revealed the close relationship between slr0643 disruption and expression of sigH operon. Therefore it was implied that Slr0643/Sll0857/SigH might acts through S2P/anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation. M-bM-^@M-^C Loop design was used to compare differential expression of wild type and mutant at pH 7.5 and pH 6.5 respectively, including dye-swape. Biological variation was sampled by extracting RNA from three independent experiments and pooling them together before hybridizations. Two or four replicate chips for each comparison and two replicate printings per chip represent technical repeats.

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Effect of pH on gene expression in two glucosyl ceramide (GlcCer) mutant strains of Cryptococcus neoformans H99

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