Distinct and redundant roles of exonucleases in Cryptococcus neoformans: Implications for virulence and mating

International audienceOpportunistic pathogens like Cryptococcus neoformans are constantly exposed to changing environments, in their natural habitat as well as when encountering a human host. This requires a coordinated program to regulate gene expression that can act at the levels of mRNA synthesis and also mRNA degradation. Here, we find that deletion of the gene encoding the major cytoplasmic 5' -> 3' exonuclease Xrn1p in C neoformans has important consequences for virulence associated phenotypes such as growth at 37 degrees C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1 Delta mutant strains. Additionally, deletion of XRN1 impairs uni- and bisexual mating. On a molecular level, the absence of XRN1 is associated with the upregulation of other major exonuclease encoding genes (i.e. XRN2 and RRP44). Using inducible alleles of RRP44 and XRN2, we show that artificial overexpression of these genes alters LAC1 gene expression and mating. Our data thus suggest the existence of a complex interdependent regulation of exonuclease encoding genes that impact upon virulence and mating in C. neoformans. (C) 2014 Elsevier Inc. All rights reserved

Analysis of the genome and transcriptome of [i]Cryptococcus neoformans[/i] var.[i] grubii[/i] reveals complex RNA expression and microevolution leading to virulence attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

Differentially expressed gene clusters.

<p>Genes differentially expressed between the three conditions (PG, pigeon guano; SM, starvation medium; YPD, rich media) were identified from strand-specific RNA-Seq using EdgeR with two biological replicates per condition (rep1, rep2). Expression profiles are ordered based on hierarchical clustering tree; 6 clusters were defined using the kmeans algorithm (Material and Methods).</p

miscRNAs in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Two examples of a miscRNA as visualized through Artemis. The coverage of the plus stand is represented by the black curve. The coverage of the minus strand is represented by the blue curve. These results were obtained when cells grown in low glucose and nitrogen medium (starvation medium) underwent strand-specific sequencing. F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2 and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>B.</b> Schematic representation of the positions of the miscRNAs in the <i>C. neoformans</i> var. <i>grubii</i> genome as compared to coding sequences. The numbers of miscRNAs at each position is indicated. The number of miscRNAs in the antisense strand of other miscRNAs is indicated between brackets.</p

Antisense/sense transcription in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Comparison of sense/antisense transcription when an antisense transcript is present. Strand-specific data obtained from cells grown on YPD is shown. The BaseMean values represent the normalized reads count for each transcript and measure the level of sense transcription (x axis) and antisense transcription (y axis) as calculated by DESeq <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004261#pgen.1004261-Anders1" target="_blank">[126]</a>. Outliers with a BaseMean above 12,000 were not represented. <b>B.</b> Example of differential expression of miscRNA antisense of a coding gene as observed through Artemis. The red curve represents the non-strand-specific coverage observed when cells were grown in YPD to stationary phase at 30°C (condition 1); the green curve shows the non-strand-specific coverage observed when the cells were grown in YPD to log phase at 30°C (condition 2). F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2, and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>C.</b> Northern blot obtained after hybridization with strand-specific probes. RNA was extracted from cells growing in YPD (2×10⁸ cells/mL) at 30°C (condition 1), YPD (5×10⁷ cells/mL) at 30°C (condition 2), YPD with 0.01% SDS (5×10⁷ cells/mL) at 30°C (condition 3), YPD with 10 mg/mL fluconazole (5×10⁷ cells/mL) at 30°C (condition 4), YPD (5×10⁷ cells/mL) at 37°C (condition 5), and YP galactose (2×10⁸ cells/mL) at 30°C (condition 6) in duplicate. Then, 5 µg RNA were loaded on a denaturing electrophoresis agarose gel, electrophoresed, and transferred to a nylon membrane. The horizontal black line represents the position of the probes.</p

Alternative splicing in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Examples of alternative splicing. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2 and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. The numbers for each type of alternative splicing events annotated in the genome are indicated between brackets. <b>B.</b> Evaluation of intron retention level in <i>C. neoformans</i> according to the ratio of transcription intron/exon threshold used is represented.</p

Organization of the centromeres in <i>C. neoformans</i> strain H99 and a comparison with other serotypes.

<p><b>A.</b> Schematic showing the distribution of transposons, Tcn1–Tcn6, in the presumptive centromeres of all 14 chromosomes of <i>C. neoformans</i> strain H99. Each region was identified as the largest ORF-free region on its respective chromosome and contains transposons or its footprints, which are clustered in these sites. <b>B.</b> A comparative analysis of the largest ORF-free regions predicted to be centromeres between <i>C. neoformans</i> var. <i>grubii</i> (H99), <i>C. neoformans</i> var. <i>neoformans</i> (JEC21 and B3501A), and <i>C. gattii</i> (WM276 and R265) using FungiDB reveal conserved synteny of the flanking genes in chromosome 14. The grey color represents the regions that show synteny among different strains. The ORFs present in the centromeric regions are either pseudogenes or have similarity with transposons. <b>C.</b> ChIP-Seq analysis showed the enrichment of a conserved kinetochore protein, CENP-C, at the centromeric regions. Here, the enrichment on centromeric region of chromosome 14 (<i>CEN14</i>) is shown. The upper panel shows the enrichment on the whole chromosome. In the lower panel, the putative centromeric region is enlarged to show the enrichment profile of CENP-C. <b>D.</b> RNA-Seq analysis reveals the absence of poly(A) RNA from <i>CEN14</i>. <b>E.</b> Targeted truncation mutagenesis on either side of the <i>CEN14</i> centromere DNA. Four DNA fragments were produced and transformed into a diploid strain of <i>C. neoformans</i>. The stick-and-ball represents the telomeric seed sequence added to the constructs by amplification with primer GI003. No targeted recombination was observed for two constructs, whereas the other two PCR analyses indicated integration of the DNA in those regions. <b>F.</b> PCR confirmation of recombination. Lanes 1–3 contain PCRs with primers ai270-GI033, and lanes 5–7 contain PCRs with primers ai270-GI034. Lanes 1 and 5 are amplification results from the diploid strain AI187; lanes 2 and 6 are from strains with integration on the left and right sides, respectively; and lanes 3 and 7 are negative PCR controls. Lane 4 is the Invitrogen 1 kb+ size marker.</p

H99 passaged strains exhibit phenotypic variability.

<p><b>A.</b> Mating assays on V8 agar were incubated at room temperature for seven days in the dark. Each strain was mated with KN99<b>a</b> (except KN99<b>a</b>, which was mated with KN99α). Melanization assays were conducted on l–DOPA agar incubated at 30°C or 37°C for two days. <b>B.</b> H99 variants differ in virulence in the murine model of infection. A group of 10 animals was each infected with an inoculum of 5.0×10⁵ cells via intranasal instillation for each strain. The results illustrate virulence variations between these well-defined H99 lineage isolates. A PBS control in which no cells were inoculated was also included. We compared the survival data for the seven strains using the Kaplan-Meier method. The significance of the pairwise comparisons to H99O was determined by the Mantel-Cox log rank test. The average time of survival was significantly shorter for the H99S, H99F, and KN99α strains compared to H99O. The survival times of the other strains compared to those of H99O were not significantly different. <b>C.</b> H99 variants differ in virulence in the rabbit CNS model of infection. For each of the variants (H99S, H99W, and H99E), three rabbits were infected directly into the CNS. All rabbits were immunosuppressed with steroid treatment. Spinal taps were taken on days 2, 4, 7, and 10 and measured for CFU (log scale). All animals were euthanized at the conclusion of the experiment. <b>D.</b> H99 variants differ in virulence in a heterologous host model of infection. For each strain, a group of 12 <i>Galleria mellonella</i> larvae was infected with an inoculum of 1.0×10⁵ cells. Survival was monitored and plotted daily for 10 days. Isolates were significantly virulent (<i>p</i><0.005) in comparison with the mock control (sterile PBS) infection, and isolates H99C and H99E were significantly less virulent than the H99O reference strain (<i>p</i><0.05).</p