Comparative Transcriptome Analysis of the CO2 Sensing Pathway Via Differential Expression of Carbonic Anhydrase in Cryptococcus neoformans

Abstract

Carbon dioxide (CO2) sensing and metabolism via carbonic anhydrases (CAs) play pivotal roles in survival and proliferation of pathogenic fungi infecting human hosts from natural environments due to the drastic difference in CO2 levels. In Cryptococcus neoformans, which causes fatal fungal meningoencephalitis, the Can2 CA plays essential roles during both cellular growth in air and sexual differentiation of the pathogen. However the signaling networks downstream of Can2 are largely unknown. To address this question, the present study employed comparative transcriptome DNA microarray analysis of a C. neoformans strain in which CAN2 expression is artificially controlled by the CTR4 (copper transporter) promoter. The PCTR4∷CAN2 strain showed growth defects in a CO2-dependent manner when CAN2 was repressed but resumed normal growth when CAN2 was overexpressed. The Can2-dependent genes identified by the transcriptome analysis include FAS1 (fatty acid synthase 1) and GPB1 (G-protein β subunit), supporting the roles of Can2 in fatty acid biosynthesis and sexual differentiation. Cas3, a capsular structure designer protein, was also discovered to be Can2-dependent and yet was not involved in CO2-mediated capsule induction. Most notably, a majority of Can2-dependent genes were environmental stress-regulated (ESR) genes. Supporting this, the CAN2 overexpression strain was hypersensitive to oxidative and genotoxic stress as well as antifungal drugs, such as polyene and azole drugs, potentially due to defective membrane integrity. Finally, an oxidative stress-responsive Atf1 transcription factor was also found to be Can2-dependent. Atf1 not only plays an important role in diverse stress responses, including thermotolerance and antifungal drug resistance, but also represses melanin and capsule production in C. neoformans. In conclusion, this study provides insights into the comprehensive signaling networks orchestrated by CA/CO2-sensing pathways in pathogenic fungi.

Distinct and Redundant Roles of Protein Tyrosine Phosphatases Ptp1 and Ptp2 in Governing the Differentiation and Pathogenicity of Cryptococcus neoformans

“…1B). These data corroborated previous findings that Atf1 is involved mainly in the oxidative stress response, not the osmotic stress response (32,33). Taken together, our data supported that PTP1 and PTP2 were stressinducible PTPs, whose basal and induced expressions were both primarily regulated by Hog1, partly through the transcription factor Atf1 in C. neoformans.…”

“…Cell fusion efficiency and pheromone gene expression were measured as previously described (25,27). Adherence and invasion were assessed as previously described (32).…”

A Flucytosine-Responsive Mbp1/Swi4-Like Protein, Mbs1, Plays Pleiotropic Roles in Antifungal Drug Resistance, Stress Response, and Virulence of Cryptococcus neoformans

“…These data could be related to our previous finding showing that Ras1 promotes flucytosine resistance in C. neoformans (28). Ras1 undergoes two essential posttranslational modifications, palmitoylation and prenylation, which are required for proper cellular localization and function of the small GTPase protein (44).…”

“…Qualitative visualization and quantitative measurement of capsule and melanin production were performed as described previously (26,28). Ergosterol extraction and calculation of ergosterol content were done following the method outlined by Ko et al (29).…”

The Cryptococcus neoformans Capsule: a Sword and a Shield

“…The two-component sensor kinases Tco1 and Tco2 respond to environmental conditions and signal through the Ypd1 histidine kinase relay protein to phosphorylate the Ssk1 response regulator (13,41,109,125). Ssk1 then phosphorylates Pbs2, which phosphorylates Hog1 (10,13).…”