Functional Characterization of cAMP-Regulated Gene, CAR1, in Cryptococcus neoformans.

The cyclic AMP (cAMP) pathway plays a major role in growth, sexual differentiation, and virulence factor synthesis of pathogenic fungi. In Cryptococcus neoformans, perturbation of the cAMP pathway, such as a deletion in the gene encoding adenylyl cyclase (CAC1), causes defects in the production of virulence factors, including capsule and melanin production, as well as mating. Previously, we performed a comparative transcriptome analysis of the Ras- and cAMP-pathway mutants, which revealed 163 potential cAMP-regulated genes (38 genes at a 2-fold cutoff). The present study characterized the role of one of the cAMP pathway-dependent genes (serotype A identification number CNAG_ 06576.2). The expression patterns were confirmed by Northern blot analysis and the gene was designated cAMP-regulated gene 1 (CAR1). Interestingly, deletion of CAR1 did not affect biosynthesis of any virulence factors and the mating process, unlike the cAMP-signaling deficient cac1Δ mutant. Furthermore, the cac1Δ mutant exhibited wild-type levels of the stress-response phenotype against diverse environmental cues, indicating that Car1, albeit regulated by the cAMP-pathway, is not essential to confer a cAMP-dependent phenotype in C. neoformans.

The ability to sense and respond to diverse environmental stresses is indispensable for all living organisms, and is particularly critical for pathogenic fungi that have to survive in harsh host conditions. This adaptation process is mediated by multiple signaling pathways. Among these, the cyclic AMP (cAMP) pathway plays an important role in maintenance of cellular homeostasis, sexual differentiation, and virulence factor production in pathogenic fungi [1, 2]. The general signal transduction mechanism of the cAMP-signaling pathway that has been studied in fungi can be summarized as follows. Plasma membrane-associated adenylyl cyclase converts ATP to cAMP, which is a key second messenger, in response to extracellular signal via small or heterotrimeric GTP-binding proteins coupled with G-protein coupled receptor (GPCR). The resulting high concentrations of cAMP lead to activation of protein kinase A (PKA), which subsequently triggers diverse cellular responses. To maintain the cAMP levels under a lethal threshold, the cAMP-signaling pathway is negatively controlled by phosphodiesterases, which degrade cAMP to AMP.

In Saccharomyces cerevisiae, activated adenylyl cyclase complex composed of Cyr1 and the cyclase-associated protein increases the intracellular levels of cAMP, which activate PKA by dissociating the PKA catalytic subunit (Tpk1/Tpk2/Tpk3) from PKA regulatory subunits (Bcy1) that repress the PKA catalytic subunit under normal conditions [3, 4]. Upon removal or adaptation to external signals, cAMP is degraded to AMP by low affinity phosphodiesterase (PDE) 1 and high affinity PDE2 [5, 6]. The cAMP-signaling pathway regulates diverse cellular functions including cellular growth, stress responses, and morphological differentiation in S. cerevisiae [7].

With the advent of acquired immunodeficiency syndrome, treatment of the opportunistic and life-threatening fungal infections and diseases caused by pathogens such as Cryptococcus neoformans has become urgent. The basidiomycetous C. neoformans infects the central nervous system in immunocompromised individuals through the respiratory system, which ultimately results in fungal meningitis [8]. Recently, C. gattii atypically appeared in British Columbia in Canada, causing disease in a number of immunocompetent individuals [9]. Two well-known virulence factors in C. neoformans are antiphagocytic polysaccharide capsule and antioxidant melanin [10, 11]. The capsule enhances the intracellular survival by preventing C. neoformans from being phagocytosed by host immune cells and from being dehydrated [12]. Melanin allows C. neoformans to strongly resist oxidative damages, ultraviolet irradiation, and high temperature exposure [13, 14]. Mutants defective in capsule or melanin production are avirulent [15, 16]. These two virulence factors are controlled by the cAMP-pathway in C. neoformans [17, 18]. Furthermore, the mating process is also governed by the cAMP-pathway in C. neoformans [17, 18]. The cAMP-pathway in C. neoformans comprises Cac1 (adenylyl cyclase), Aca1 (adenylyl cyclase-associated protein), Gpa1 (Gα subunit) GPCR Gpr4, catalytic subunits (Pka1 and Pka2) and regulatory subunit (Pkr1) of PKA, and phosphodiesterases (PDE1 and PDE2) [19, 20].

Recently, we identified a number of cAMP-regulated genes in C. neoformans through a comparative transcriptome analysis of cAMP pathway-dependent mutants [21]. DNA microarray analysis was utilized to compare the genome-wide transcriptome patterns of the C. neoformans mutants deleted of a major Ras/cAMP-signaling component, including Cac1, Aca1, Gpa1, Pka1/2 (catalytic subunits of protein kinase A), and Ras1 with those of the serotype A H99 wild-type strain [21]. Through this analysis, the cAMP-pathway was also found to control expression of diverse stress response genes and was linked to certain environmental stress responses [21]. Notably, it has been reported that the cAMP-pathway is required for promoting resistance to polyene drugs including amphotericin B [21], suggesting that a potential inhibitor of the cAMP-pathway will confer a synergistic antifungal activity along with amphotericin B. Furthermore, we discovered 163 unique genes (38 genes at a 2-fold cutoff), whose expressions are regulated by the cAMP pathway.

The present study further characterized one of the cAMP-dependent genes, whose serotype A ID is CNAG_06576.2, since it appeared dramatically downregulated in all of the cAMP mutants but its function had not been hitherto characterized. Presently, Northern blot analysis was used to confirm that the gene was indeed positively regulated by the cAMP-pathway. Appropriately, it was designated cAMP-regulated gene 1 (CAR1). The results indicate that Car1 does not play any significant roles in growth, differentiation, and virulence factor production of C. neoformans.

Materials and Methods

Strains and media

Strains used in this study are listed in Table 1. Yeast extract-peptone-dextrose (YPD) medium was used for culturing C. neoformans strains. L-DOPA or Niger seed medium for melanin synthesis and agarbased Dulbecco modified Eagle (DME) medium for capsule production were prepared as previously described [18, 25].

Table 1

Cryptococcus neoformans strains used in this study

Each NAT-STM# indicates the Natr marker with a unique signature tag.

MAT, mating type.

Gene disruption of the CAR1 gene

For CAR1 gene disruption, information about the CAR1 genomic structure was obtained from the C. neoformans genomic database. (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html). The CAR1 gene-disruption cassette was constructed by double joint polymerase chain reaction (DJ-PCR) with NAT-split markers as previously described [26]. The CAR1Δ mutants were generated by introduction of the CAR1-deletion cassette into the C. neoformans serotype A H99 strain by biolistic transformation [26]. Primers for amplification of the 5' and 3' flanking regions of the CAR1 gene (Fig. 2A) are described in Table 2. The primers were used to amplify dominant selectable NAT (nourseothricin acetyltransferase) marker. Stable transformants selected on YPD medium containing nourseothricin were screened by diagnostic PCR and their correct genotypes were verified by Southern blot analysis.

Fig. 2

Construction of the car1Δ mutant. A, Diagram for disruption of the cAMP-regulated gene 1 (CAR1) gene. Primers for the first-round PCR and double joint (DJ)-PCR are indicated as bent arrows. Through triple recombination between 5'- and 3' flanking regions of CAR1 and split NAT selectable marker, the CAR1 gene was specifically replaced with the intact NAT selectable marker. B, Nourseothricin-resistant transformants were selected and screened by diagnostic PCR with the CAR1-specific screening primer as listed in Table 2.

Table 2

Primers used in this study

Northern blot analysis

Total RNAs that have been prepared for the previous DNA microarray analysis [21] were used for Northern blot analysis. Electrophoresis, washing, membrane transfer, and hybridization were carried out by following standard protocols [27]. To construct the specific probe for the CAR1 gene, specific primers summarized in Table 2 were used.

Melanin and capsule assay

Each mutant and wild type strain were grown in YPD liquid medium overnight at 30℃. Three microliters of cells were spotted on agar-based DME medium for capsule production and on agar-based L-DOPA or Niger seed medium for melanin production, which all contained the indicated concentrations of glucose. The plates were incubated at 30℃ or 37℃, daily monitored for up to 4 days, and photographed using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with camera (Diagnostic Instrument Inc., Sterling Heights, MI, USA). For visual inspection of capsule production, cells scraped from DME medium were resuspended in distilled water and stained with India ink. Relative capsule size was quantitatively measured as previously described [18, 25].

Mating assay

Mating assay was performed as previously described [18, 25]. Briefly, all C. neoformans strains for mating were grown overnight at 30℃ in liquid YPD medium. Next, the same concentration of mating type (MAT)α and MATa cells (107 cells/mL) were mixed, spotted (5 µL of the mixture) onto the V8 medium adjusted to pH 5, and then incubated in the dark at room temperature for 2 weeks. All colonial and cellular images were monitored and photographed using Olympus BX51 microscope (Olympus).

Stress sensitivity test

Each mutant and wild type strain were cultured overnight at 30℃ in YPD liquid medium. These strains were washed, serially diluted (1~104), and spotted (4 mL) onto solid YP or YPD medium containing diamide (diazenedicarboxylic acid bis (N,N-dimethylamide)), hydrogen peroxide (H2O2), or menadione for oxidative stress test, CdSO4 for heavy metal stress test, Congo-red for cell wall integrity test, and NaCl or KCl for osmosensitivity. To test antifungal drug sensitivity, cells were spotted onto YPD medium containing indicated concentrations of fluconazole, itraconazole, ketoconazole, amphotericin B, or fludioxonil. The plates were incubated at 30℃ for 2~5 days and photographed.

Results

Identification of the CAR1 gene in C. neoformans

Among the cAMP-dependent genes previously identified through transcriptome analysis [21], the expression patterns of CNAG_06576.2 was confirmed by Northern blot analysis. Expression levels of the CNAG_06576.2 gene significantly decreased in the cac1Δ, gpa1Δ, and pka1Δ pka2Δ mutants compared to the wild type strain, echoing DNA microarray data previously reported [21]. Appropriately, the gene was designated CAR1 (Fig. 1).

Fig. 1

Transcript levels of the cAMP-regulated gene 1 (CAR1) gene in cAMP-signaling mutants. A, Each bar indicates reduction of the CAR1 gene in microarray data in aca1Δ (YSB6), cac1Δ (YSB42), gpa1Δ (YSB83), and pka1Δ pka2Δ (YSB200) mutants compared to the wild-type strain (H99) of our previous report [16]. B, Transcript levels of the CAR1 gene in the wild-type strain (H99) and gpa1Δ, aca1Δ, cac1Δ, and pka1Δ pka2Δ mutants were verified by Northern blot analysis.

To further characterize the role of Car1, mutant analysis was carried out by constructing the car1Δ mutant in the background of the serotype A H99 strain. For amplifying each 5'- and 3' flanking region of the CAR1 gene and truncated NAT selectable marker, the specific primers summarized in Table 2 were used. Through triple homologous recombination between 5'- and 3' flanking regions of the CAR1 gene and NAT-split markers that were produced by double joint PCR as previously described [26], the CAR1 gene was specifically disrupted (Fig. 2A). Nine transformants were selected by diagnostic PCR with the CAR1-specific screening primer (Fig. 2B). Among these, six transformants were verified by Southern blot analysis (data not shown).

Role of Car1 in capsule and melanin production and mating

The cAMP pathway plays a major role in production of virulence factors such as capsule and melanin in C. neoformans. The cac1Δ mutant is defective in capsule and melanin production as well as in mating [17]. Similarly, deletion of the ACA1 gene results in reduction of capsule and melanin production, and causes decreased levels of sexual differentiation [18]. To test the hypothesis that Car1 may play a role in regulation of virulence factors, a capsule production assay was carried out. Interestingly, deletion of the CAR1 gene did not lead to any capsule defects, unlike that of CAC1 gene (Fig. 3A). The possible involvement of Car1 in production of antioxidant melanin was assessed using L-DOPA and Niger seed media (Fig. 3B). Unlike the cac1Δ mutant, melanin production levels in the car1Δ mutant were similar to those in the wild-type strain (Fig. 3B). Finally to address the role of Car1 in mating response, a mating assay was performed. Contrary to the CAC1 gene, the CAR1 gene was not required for sexual differentiation (Fig. 3C). The observations were consistent with the suggestion that Car1 is not directly involved in capsule and melanin production, as well as in mating.

Fig. 3

Car1 is not involved in capsule and melanin production and mating response. A, Capsule production in the wild-type strain (H99) and cac1Δ and car1Δ mutants was visualized by staining with india ink and cellular images were monitored and recorded. B, For melanin assay, the wild-type strain (H99) and cac1Δ, and car1Δ mutants were spotted onto agar-based L-DOPA and Niger seed media and grown at either 30℃ or 37℃ for 5 days. Colony images of melanin production in the wild-type strain and cac1Δ, and car1Δ mutants were acquired. C, The following mating type (MAT)α and MATa strains were co-cultured on V8 medium at dark room temperature up to 2 weeks: H99 × KN99 (α × a), YSB639 × KN99 (car1Δ × a), and YSB42 × KN99 (cac1Δ × a) and photographed after 9 days.

Role of Car1 in stress response of C. neoformans

We recently reported that cac1Δ, aca1Δ, gpa1Δ, and pka1Δ cAMP-dependent mutants display hypersensitivity to a polyene drug, amphotericin B, and an azole drug, itraconazole [21]. Furthermore, these cAMP mutants were more susceptible to methylglyoxal (MG), a toxic metabolite, and CdSO4, a heavy metal stress inducer, than the wild-type strain. However, these mutants exhibited more resistance to diamide than the wild-type strain. Taken together, the results are consistent with the suggestion that cAMP-pathway contributes to diverse stress responses in C. neoformans. To address the role of Car1 in stress response in C. neoformans, we performed diverse stress response and antifungal drug sensitivity test. The car1Δ mutants exhibited similar resistance to osmotic shock as the wild-type strain (Fig. 4). Furthermore, the CAR1Δ mutants exhibited wild-type levels of resistance to antifungal drugs, unlike the other cAMP dependent mutants (Fig. 4) [18]. In response to oxidative stress-inducing agents including menadione, diamide, and hydrogen peroxide, the car1Δ mutants showed wild type levels of sensitivity (Fig. 4). Furthermore, Car1 was not involved in maintenance of cell wall integrity (Congo-red), genotoxic stress (methyl methanesulfonate, MMS), toxic metabolite stress (MG), and heavy metal stress (CdSO4). Taken together, these data support the suggestion that Car1 is not involved in diverse stress responses of C. neoformans.

Fig. 4

Car1 is not involved in a variety of stress response against oxidative, osmotic, and genotoxic agents and treatment of cell wall destabilizer, antifungal drug, and toxic metabolites. For stress-sensitivity test, each C. neoformans strain (wild-type strain [H99] and cac1Δ [YSB42], hog1Δ [YSB64], car1Δ [YSB639 and YSB640] mutants) was cultured overnight at 30℃ in liquid yeast extract-peptone-dextrose (YPD) medium and 4 µL of cells were spotted with 10-fold serial dilution onto YPD agar containing indicated concentrations of NaCl, KCl, H2O2, diamide, menadione, CdSO4, methyl methanesulfonate, Congo-red, methylglyoxal, and antifungal drugs (amphotericin B, fluconazole, itraconazole, ketoconazole, and fludioxonil).

Discussion

The present study aimed to functionally characterize the role of one of cAMP-regulated genes, CNAG_06576.2, that we previously discovered in the human fungal pathogen C. neoformans using comparative transcriptome analysis [21]. Here, the expression patterns were confirmed by Northern blot analysis and the gene was named CAR1. Unlike other cAMP dependent mutants, however, the deletion of the CAR1 gene did not affect the production levels of two major Cryptococcus virulence factors, including capsule and melanin. Furthermore, the car1Δ mutant was as normal in mating as the wild-type strain. In addition, the car1Δ mutant exhibited wild type-like stress-response phenotypes against diverse environmental stresses, such as osmotic and oxidative shock, treatment of antifungal drugs, cell wall integrity destabilizers, and toxic metabolite. Taken together, the data support the view that Car1 is regulated by the cAMP-pathway, but appears not to play any significant role in C. neoformans.

However, it is still possible that Car1 may have some functional roles in the cAMP-pathway. In S. cerevisiae, the transcriptional levels of three genes named GRE1 (Genes de Respuesta a Estres [stress responsive genes]), GRE2, and GRE3 increase in response to hyperosmotic, oxidative, ionic, and heat shock stress [28]. These three genes are controlled by the HOG pathway, and GRE1 and GRE3 are regulated negatively by the cAMP-PKA pathway [28]. However, the functional roles of GRE genes have not been characterized during diverse stress responses. Furthermore, a similar case is found in other genes of C. neoformans. Expression of the SMG1 gene is repressed in the gpa1Δ mutant and SMG1 overexpression can restore melanin deficiency of the gpa1D mutant. However, the smg1Δ mutant exhibits wild type-levels of melanin production, unlike the gpa1Δ mutant [29, 30]. Therefore, it is possible that the function of CAR1 gene may overlap with that of other genes regulated by the cAMP pathway, which explains why disruption of the CAR1 gene does not result in any phenotypes in C. neoformans. Our previous transcriptome analysis identified many other candidate cAMP-regulated genes, which do not have any known function or orthologs in other fungi. This indicates that some of the cAMP-dependent phenotypes could be controlled by multiple target genes. Therefore, to further address the function of Car1, it may be necessary to overexpress the CAR1 gene in some of the cAMP mutants and observe whether any of the cAMP-related phenotypes could be restored (at least partly) by CAR1 overexpression.

Another possibility is that Car1 may play a role in some of cAMP-signaling related phenotypes that have not yet been elucidated. It is unlikely that all of the cAMP-signaling dependent phenotypes have been discovered in C. neoformans. Therefore, it is too early to conclude that Car1 does not play any role in the cAMP-signaling pathway of C. neoformans, since only a limited number of phenotypic analyses has been performed. Particularly, Car1 may be required for conferring full virulence during host infection and disease progress. To address this question, animal studies should be done.