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

Abstract

Opportunistic 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°C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1Δ 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.

Effects of 5′-3′ Exonuclease Xrn1 on Cell Size, Proliferation and Division, and mRNA Levels of Periodic Genes in Cryptococcus neoformans

“…In previous reports, Xrn1 affects growth ability at 37 • C, capsule synthesis, laccase activity and mating [22]. In addition, the xrn1∆ mutant exhibits reduced virulence in invertebrate models [22]. Our results further confirmed growth defects of xrn1∆ mutant at 37 • C and found that the cell became larger and the cell growth rate slowed.…”

“…The slow-growth phenomenon was not caused by a decrease in the budding rate of the xrn1∆ mutant, because we found that the budding rate of the xrn1∆ mutant was not significantly different from that of the wild type ( Figure 3b). We also analyzed the growth of the xrn1∆ mutant on YPD medium at 37 °C and found that the growth was defective in the xrn1∆ strain at 37 °C , which is consistent with previous research [22]. The addition of osmotic stabilizers such as NaCl, KCl and sorbitol did not fully restore the growth defect ( Figure 3c).…”

“…Uncontrolled exchange between the nuclear interior and cytoplasm leads to DNA damage [30]. In previous reports, Xrn1 affects growth ability at 37 • C, capsule synthesis, laccase activity and mating [22]. In addition, the xrn1∆ mutant exhibits reduced virulence in invertebrate models [22].…”

“…Three major virulence-associated factors have been established, including the ability to grow at 37 • C, the formation of polysaccharide capsule and the production of pigment melanin [19][20][21]. Deletion of cytoplasmic exonuclease XRN1 in C. neoformans has been reported to lead to growth defects at 37 • C, smaller capsule, reduced melanin formation and impaired uni-and bisexual mating [22]. Here, we further find that the deletion of XRN1 led to an increase in cell size, which was lethal when the temperature was set at 37 • C. The KEGG analysis of the transcriptome showed a significant difference in the cell cycle pathway between WT and mutant cells, and the expression of many genes involved in cell cycle progression was up-regulated in mutant cells.…”

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3′UTR Introns

“…This second branch would have a role in quality control in destroying certain incorrectly or alternatively spliced mRNAs as well as governing levels of mRNAs that normally contain spliced 39-UTR introns. Determining where there are differences between fungi and higher eukaryotes with respect to specific elements that comprise these mechanisms could provide targets for the development of new therapeutics; for example, the fungal pathogen C. neoformans is avirulent when it lacks XRN1, an exonuclease that has a central role in mRNA degradation (Wollschlaeger et al 2014). The similarities between fungal systems and higher eukaryotes, and the experimental advantages that fungi offer, will enable experimental approaches for understanding the different branches of NMD and provide new opportunities for insights into fundamental mechanisms governing RNA stability.…”