Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans

Abstract

Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect of the ubi1 strain could be closely associated with its morphological abnormalities, such as its reduced ribosome particles. In addition, the ubi1 mutant also displayed increased cell ploidy, cell cycle arrest, and decreased intracellular survival inside macrophages. All these phenotypes were reversed by the reconstitution of the full-length UBI1 gene or RPL40a domain. Mouse survival and fungal burden assays further revealed a severely attenuated pathogenicity for the ubi1 mutant, which is probably associated with its reduced stress tolerance and the induction of T-helper 1-type immune response. Taken together, Ubi1 is required for maintaining the vegetative growth, morphological homeostasis, cell cycle progression, and pathogenicity in vivo of C. neoformans. The pleiotropic roles of Ubi1 are dependent on the presence of Rpl40e and associated with its regulation of cryptococcal ribosome biogenesis.

Cryptococcus neoformans Csn1201 Is Associated With Pulmonary Immune Responses and Disseminated Infection

“…Stress media were prepared by adding different stress inducing agents to YNB agar medium (0.67% yeast nitrogen base without amino acids, 2% glucose) or YPD agar medium before autoclaving. Capsule- and melanin-inducing media were prepared as previously reported ( 16 ). DMEM with 10% FBS (Transgen Biotech, Beijing, China) was used for cell culture in the murine-derived J774 macrophage killing assay.…”

“…For gene disruption, overlap PCR was used to generate the knock-out cassette of CSN1201 , including the flanking fragments and NAT resistance gene ( 17 ). The purified PCR products were precipitated onto gold microparticles and introduced into H99 cells by biolistic transformation ( 16 ). Stable transformants were screened using selective medium containing nourseothricin and confirmed by diagnostic PCR, DNA sequencing, and Southern blotting.…”

More Than Just Cleaning: Ubiquitin-Mediated Proteolysis in Fungal Pathogenesis

“…Reconstitution of the full length of UBI1 or the C-terminal Rpl40a could both reverse the phenotypes of ubi1D mutation, indicating a role for Ubi1 in cryptococcal ribosome biogenesis (Zhao et al, 2020). Deletion of UBI1 also led to the differential expression of ubiquitin-conjugating enzymes (Zhao et al, 2020), suggesting a regulatory function for Ubi1 in the UPS, but the role of ubiquitin moiety in the pathogenicity of C. neoformans requires further investigation (Zhao et al, 2020). Deletion of the polyubiquitin gene MGG_01282 resulted in abnormal morphology and virulence defects in the filamentous ascomycete fungus Magnaporthe oryzae, the leading cause of fungal diseases in rice globally (Oh et al, 2012).…”

“…UBI4 encodes a polyubiquitin precursor containing five ubiquitin repeats ( Spitzer and Spitzer, 1995 ). Deletion of UBI1 results in a vegetative growth defect, morphological changes, a melanin production defect, decreased intracellular survival inside macrophages and virulence attenuation during infection ( Zhao et al., 2020 ). Reconstitution of the full length of UBI1 or the C-terminal Rpl40a could both reverse the phenotypes of ubi1 Δ mutation, indicating a role for Ubi1 in cryptococcal ribosome biogenesis ( Zhao et al., 2020 ).…”

“…Deletion of UBI1 results in a vegetative growth defect, morphological changes, a melanin production defect, decreased intracellular survival inside macrophages and virulence attenuation during infection ( Zhao et al., 2020 ). Reconstitution of the full length of UBI1 or the C-terminal Rpl40a could both reverse the phenotypes of ubi1 Δ mutation, indicating a role for Ubi1 in cryptococcal ribosome biogenesis ( Zhao et al., 2020 ). Deletion of UBI1 also led to the differential expression of ubiquitin-conjugating enzymes ( Zhao et al., 2020 ), suggesting a regulatory function for Ubi1 in the UPS, but the role of ubiquitin moiety in the pathogenicity of C. neoformans requires further investigation ( Zhao et al., 2020 ).…”