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Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

ABSTRACT: A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.

SUBMITTER: Hu G

PROVIDER: S-EPMC1828699 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.

Project description:The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome.The proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice.We identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis.Overall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.

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Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

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