Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade

Abstract

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Galpha protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Galpha protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

FIG. 1.

Northern analysis confirms the gpa1 mutant microarray results. The wild-type (GPA1) and gpa1 mutant strains were incubated to mid-logarithmic phase in YPD and exposed for 1 h to glucose-rich (+) or glucose-poor (−) conditions. Total RNA was extracted from these strains and used for Northern analysis with the CAS1, CAS8, SMG1, and GPA1 genes as probes, with the ACT1 (actin) gene as a loading control.

FIG. 2.

LAC1 and LAC2 transcript levels are regulated by Gpa1. (A) Gene microarray analysis identified six genomic sequences of the serotype A strain H99 that exhibit an increased ratio of cDNA hybridization in the wild-type strain compared to the gpa1 mutant (GPA1/gpa1), suggesting that a gene present in these sequences is differentially expressed in these two strains. (B) One of these sequences (s0011P0069Z_A11) localized to the region of the LAC1 gene, and two sequences (s0011P0048Z_A9 and s0011P0037Z_E9) localized 4 to 5 kb upstream of LAC1, in a region containing a second putative laccase gene, LAC2. (C) To confirm the microarray results, Northern analysis using the LAC1 gene as a probe was performed with total RNA from the wild type (H99), the gpa1 mutant (AAC1), and the gpa1+GPA1 reconstituted strain (AAC3) after 1 h of incubation under glucose-rich (+) or glucose-poor (−) conditions. The rRNA signal in the ethidium bromide-stained RNA gel is shown to demonstrate equal RNA loading.

FIG. 3.

Measuring LAC2 transcript using real-time PCR and Northern analysis. (A) Real-time PCR was used to determine the relative transcript levels of LAC2 in each of the following strains after 1 h of glucose starvation: (A) wild-type (H99), (B) lac2 mutant (RPC27), (C) lac1 mutant (MDC16), (D) lac2+pGPD-LAC2 mutant (QGC1), (E) lac1 lac2 mutant (QGC9), (F) gpa1 mutant (AAC1), (G) gpa1+pGPD-LAC2 mutant (RPC18), and (H) wild type + pGPD-LAC2 (RPC21). The results are demonstrated as expression relative to the wild-type strain H99. Each data point represents the average for triplicate samples with error bars indicated. (B) The wild-type strain H99 was incubated to mid-logarithmic phase in YPD medium and subsequently exposed to several different growth conditions for 1 h: (1) YNB (pH 7), (2) YNB (pH 4), (3) YNB (pH 7) plus 10 mM Na hydro-gen peroxide, (6) YPD plus 10 mM paraquat, (7) YNB plus 0.01% glucose, and (8) YPD plus 100 μM copper sulfate. Total mRNA was isolated from these strains, treated with DNase-free RNase, and converted to cDNA. Relative LAC2 transcript levels, indicated as n-fold induction compared to the baseline condition 1, were determined for each of the samples with real-time PCR. Each data point represents the average for triplicate samples with error bars indicated. (C) Northern analysis documenting LAC2 overexpression. Total RNA was isolated from the wild-type (H99), gpa1+pGPD-LAC2 (RPC18), and wild type + pGPD-LAC2 (RPC21) strains after 1 h of incubation in a glucose-rich (+) or glucose-poor (−) medium and subjected to Northern analysis with the LAC2 gene as a probe. The rRNA signal in the ethidium bromide-stained RNA gel is shown to demonstrate equal RNA loading.

FIG. 4.

Southern blot of lac2 and lac1 lac2 mutants. (A) Genomic DNA from the wild type (H99), lac2 mutants (RPC26, RPC27, and RPC28), and lac1 lac2 double mutants (RPC30 and QGC9) was digested with PstI and XhoI and examined by Southern analysis using the indicated region of the LAC2 locus as a probe. (B) Restriction maps of the LAC2 and LAC1 loci.

FIG. 5.

Laccase activity. (A) The wild-type (H99), lac2 mutant (RPC27), lac1 mutant (MDC16), and lac1 lac2 double mutant (QGC9) were incubated in YNB plus 0.1% glucose with 10 mM epinephrine. Laccase activity was quantified at 48 and 72 h by the appearance of pigment in the supernatant as assessed by measuring the absorbance at 475 nm. (B) The wild-type (H99), wild-type + pGPD-LAC2 (RPC 21), gpa1 (AAC1), gpa1+pGPD-LAC2 (RPC18), lac1 (MDC16), lac1+pGPD-LAC2 (QGC1), lac2 (RPC27), and lac2+pGPD-LAC2 (QGC4) strains were incubated on niger seed medium with either 2 or 0.1% glucose for 72 h at 30°C. Melanin-producing strains make brown pigments on this medium.

FIG. 6.

Substrate utilization for melanin. The wild-type, lac2 mutant (RPC27), lac1 mutant (MDC16), lac1 lac2 mutant (QGC9), and lac1+pGPD-LAC2 strains (QGC1) were incubated for 72 h on YNB medium with 0.1% glucose containing one of the following substrates for melanin production: epinephrine, dopamine, ABTS, or niger seed extract.

FIG. 7.

LAC2 and the murine model of inhalational cryptococcosis. Female A/Jcr mice were intranasally inoculated with 10⁵ cells of the following strains: wild type (H99), lac2 mutant (RPC27), lac1 mutant (MDC16), lac1+pGPD-LAC2 strain (QGC1), gpa1 mutant (AAC1), or gpa1+pGPD-LAC2 strain (RPC18). The mice were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical end points that predict imminent mortality.