Dataset Information

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

ABSTRACT: Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.

SUBMITTER: Mogensen EG

PROVIDER: S-EPMC1360268 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

Mogensen Estelle Geweiss EG Janbon Guilhem G Chaloupka James J Steegborn Clemens C Fu Man Shun MS Moyrand Frédérique F Klengel Torsten T Pearson David S DS Geeves Michael A MA Buck Jochen J Levin Lonny R LR Mühlschlegel Fritz A FA

Eukaryotic cell 20060101 1

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells ...[more]

PMID: 16400172

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Project description:Carbon dioxide (CO(2)) 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 CO(2) 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 P(CTR4)CAN2 strain showed growth defects in a CO(2)-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 beta 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 CO(2)-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/CO(2)-sensing pathways in pathogenic fungi.

Project description:Carbon dioxide (CO2) sensing, transport, and metabolism play a pivotal role in survival and proliferation of pathogenic microbes infecting human host from natural environments due to the drastic difference of CO2 levels. Carbonic anhydrases (CAs) are not only key CO2-metabolic enzymes catalyzing reversible interconversion between CO2 and bicarbonate (HCO3-), but also important CO2-signaling modulators. Cryptococcus neoformans that causes fatal fungal meningitis contains two functional CAs, Can1 and Can2, among which Can2 plays essential roles for growth and sexual differentiation of the pathogen. However, downstream genes and signaling network regulated by CAs have not been studied thus far. In this study, we constructed a C. neoformans strain where CAN2 expression is controlled by the CTR4 (copper transporter) promoter and elucidated its transcriptome patterns by using DNA microarray analysis to elucidate downstream target genes of Can2. Expectedly, the CAN2 promoter replacement strain showed growth defects in a CO2-dependent manner when CAN2 is repressed. The CA-transcriptome analysis discovered a number of Can2-dependent genes, including those involved in fatty acid biosynthesis (FAS1), sexual differentiation (GPB1), capsule structure organization (CAS3), iron-metabolism (CFO2), and a number of stress-regulated genes, including an oxidative stress-responsive Atf1 transcription factor, although a majority of them do not have any orthologs in other fungi. The present study revealed other roles of Atf1 in capsule and melanin production and diverse stress responses, including thermotolerance and antifungal drug resistance, of C. neoformans. This study provides further insights into the signaling network of CA/CO2-sensing pathway in pathogenic fungi. 17 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 2 strains (H99 Wild type strain, CTR4::CAN2) at 2 time points (0hr time, 12hr time) in 2 conditions (BCS, CuSO4). 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 experiment samples are dye swaped.

Project description:Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

Dataset Information

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

Publications

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

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