Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

Guanggan Hu,Anita P Sham,Kristin L Tangen,Barbara R Steen,Tianshun Lian,Nicola Tam,James W Kronstad,Joseph Heitman

doi:10.1371/journal.ppat.0030042

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.

Highlights

Cryptococcus neoformans is a basidiomycete fungal pathogen that infects both immunocompromised and immunocompetent individuals to cause meningioencephalitis [1]
The cyclic adenosine 59-monophosphate signaling pathway in C. neoformans plays a key role in sensing conditions such as nutrient availability to control expression of virulence factors, and defects in the pathway lead to attenuated or accentuated disease
Transcriptional profiling identified a regulatory link between the cyclic adenosine 59-monophosphate (cAMP) pathway and components of the machinery for transport to the cell surface

Summary

Introduction

Cryptococcus neoformans is a basidiomycete fungal pathogen that infects both immunocompromised and immunocompetent individuals to cause meningioencephalitis [1]. A common theme is that many of the virulence factors require transport to the plasma membrane or cell wall, or secretion to the extracellular environment This is true for the capsule polysaccharide that is considered to be the major virulence factor of the fungus [1,3,4]. The capsule is known to have a variety of immunomodulatory effects, and acapsular mutants are attenuated for virulence in animal models [3,4,5,6,7] Many factors, such as iron starvation, serum, carbon dioxide levels, and pH and glucose levels, influence the size of the capsule in C. neoformans [4,8]. Melanization in C. neoformans contributes to survival within alveolar macrophages, resistance to oxidative stress, and extra-pulmonary dissemination; melanin may protect the fungus from environmental predators such as amoebae or from UV irradiation [10,11,12,13,14,15]

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