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Cross talk between the cell wall integrity and cAMP/protein kinase A pathways in Cryptococcus neoformans

ABSTRACT: Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work by us and others have shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely PKC1, BCK1, MKK2 and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors and, for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional response of gene deletions of BCK1, MKK2 and MPK1 compared to wild-type cells. We found over 1000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cAMP/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion stains revealed significantly less cAMP in all of the deletion strains compared to wild-type cells. The deletion strains also produced significantly smaller capsules than wild type KN99 under capsule inducing conditions, although they shed similar levels of capsule as wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to near wild-type levels. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.

ORGANISM(S): Cryptococcus neoformans

PROVIDER: GSE57217 | GEO | 2014/07/01

SECONDARY ACCESSION(S): PRJNA245919

REPOSITORIES: GEO

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Project description:The cAMP-pathway plays a central role in regulation of growth, differentiation, and virulence of human pathogenic fungi, including Cryptococcus neoformans. Three major upstream signaling regulators of the adenylyl cyclase (Cac1), Ras, Aca1 (Adenylyl cyclase-associated protein 1) and G-alpha subunit protein (Gpa1), have been identified to control the cAMP-pathway in C. neoformans, but their functional relationship remains elusive. Here we performed genome-wide transcriptome analysis with C. neoformans ras1, gpa1, cac1, aca1, and pka1 pka2 mutants by DNA microarray. The aca1, gpa1, cac1, and pka1 pka2 mutants displayed similar transcriptome patterns to each other whereas the ras1 mutant exhibited distinctive transcriptome patterns compared to WT and the cAMP mutants. Interestingly, a number of environmental stress response genes are differentially modulated in the ras1 and cAMP mutants. In fact, the Ras1-signaling pathway was found to be involved in osmotic and genotoxic stress response, and maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Through this microarray analysis, we have identified a number of cAMP-dependent genes, including GRE2, HSP12, ENA1, TCO2, PKP2, CAT1, in C. neoformans. Notably, a majority of ergosterol biosynthesis genes were found to be upregulated in the cAMP mutants. Interestingly, the gpa1, cac1, and pka1 mutants, but not the aca1 and pka2 mutants were hypersensitive to amphotericin B, but resistant to fluconazole. In conclusion, we demonstrated in this study that the Ras1- and cAMP-signaling pathways are involved in stress response and sterol biosynthesis of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 100 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted with 6 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), ras1Î, aca1Î, gpa1Î, cac1Î, pka1Îpka2Î), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. several sample are dye swaped.

Project description:Caffeine is a natural purine analog that elicits pleiotropic effects, which ultimately lead to cell death by a mechanism that is still largely uncharacterized. This drug can activate the PKC1-MAPK cell integrity pathway, as shown by phosphorylation of Mpk1 kinase. However, and contrary to expectation, the caffeine-induced hyperphosphorylation of Mpk1 was accompanied by a negligible activation of its downstream targets Rlm1 and SBF transcription factors, which suggested that the fortification of the cell wall induced by caffeine was independent on the MAP kinase activation. This result was consistent with the finding that the loss of RLM1 had no consequence on the increased resistance of caffeine-treated cells to zymolyase, and was further consolidated by a genome-wide microarray analysis showing that, contrary to the cell wall drugs Congo Red and Calcofluor white, caffeine did not cause upregulation of Rlm1-dependent genes encoding cell wall remodeling enzymes. Interestingly, this global expression analysis revealed a striking resemblance of the transcriptomic responses to caffeine with those of rapamycin, a potent inhibitor of the TOR1 and TOR2 kinases. Consistent with this analysis, we found that the caffeine-induced phosphorylation of Mpk1 was lost in a tor1(delta) mutant but it was conserved in a TOR1-1 strain bearing a rapamycin-insensitive Tor1 kinase. Also, and contrary to the mechanism by which rapamycin led to activation of the PKC pathway, the caffeine-dependent process did not necessitate cell wall sensors and was completely abolished upon deletion of ROM2 encoding a GDP/GTP exchange factor of the Rho1-PKC pathway. Moreover, addition of caffeine to yeast cells resulted in a transient drop in intracellular cAMP, and this effect was not observed in a rom2(delta) mutant. In summary, our results revealed that Tor1 is a potential direct target of caffeine in yeast, whose inhibition leads to activation of the Pkc1-Mpk1 kinase cascade and inhibition of the Ras/cAMP pathway, and that for this specific case, the cross-talk between these signaling pathways is mediated by Rom2. These results may have broad implication for our understanding of caffeine effects in analogous regulatory networks in mammalian cells. Key words: caffeine, antifungal drugs, cell wall, transcript profiling, TOR, PKC1, RAS/cAMP. Keywords: time course, dose response

Project description:The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that is activated by varying nutrient and environmental signals and governs a plethora of eukaryotic biological processes. Nevertheless, its role in the human fungal pathogen Cryptococcus neoformans remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in C. neoformans. We successfully deleted TLK1, but not TOR1. TLK1 deletion did not result in any evident in vitro phenotypes, except for a minor role in diamide and polyene resistance, suggesting that Tlk1 is mainly dispensable for the growth of C. neoformans. We further demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To analyze the functions of Tor1 in more detail, we constructed constitutive TOR1 overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of C. neoformans. We also found that TOR1 overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, autophagy, and stress response. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of signaling pathways, including the Hog1 pathway. In conclusion, our study demonstrates that a single Tor1 kinase plays a variety of roles in the fungal meningitis pathogen C. neoformans.

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Cross talk between the cell wall integrity and cAMP/protein kinase A pathways in Cryptococcus neoformans

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