Ubiquitin Degradation of the AICAR Transformylase/IMP Cyclohydrolase Ade16 Regulates the Sexual Reproduction of Cryptococcus neoformans.

F-box protein is a key protein of the SCF E3 ubiquitin ligase complex, responsible for substrate recognition and degradation through specific interactions. Previous studies have shown that F-box proteins play crucial roles in Cryptococcus sexual reproduction. However, the molecular mechanism by which F-box proteins regulate sexual reproduction in C. neoformans is unclear. In the study, we discovered the AICAR transformylase/IMP cyclohydrolase Ade16 as a substrate of Fbp1. Through protein interaction and stability experiments, we demonstrated that Ade16 is a substrate for Fbp1. To examine the role of ADE16 in C. neoformans, we constructed the iADE16 strains and ADE16OE strains to analyze the function of Ade16. Our results revealed that the iADE16 strains had a smaller capsule and showed growth defects under NaCl, while the ADE16OE strains were sensitive to SDS but not to Congo red, which is consistent with the stress phenotype of the fbp1Δ strains, indicating that the intracellular protein expression level after ADE16 overexpression was similar to that after FBP1 deletion. Interestingly, although iADE16 strains can produce basidiospores normally, ADE16OE strains can produce mating mycelia but not basidiospores after mating, which is consistent with the fbp1Δmutant strains, suggesting that Fbp1 is likely to regulate the sexual reproduction of C. neoformans through the modulation of Ade16. A fungal nuclei development assay showed that the nuclei of the ADE16OE strains failed to fuse in the bilateral mating, indicating that Ade16 plays a crucial role in the regulation of meiosis during mating. In summary, our findings have revealed a new determinant factor involved in fungal development related to the post-translational regulation of AICAR transformylase/IMP cyclohydrolase.

The F-Box Protein Fbp1 Regulates Virulence of Cryptococcus neoformans Through the Putative Zinc-Binding Protein Zbp1.

The ubiquitin-proteasome system (UPS) is the major protein turnover mechanism that plays an important role in regulating various cellular functions. F-box proteins are the key proteins of the UPS, responsible for the specific recognition and ubiquitination of downstream targets. Our previous studies showed that the F-box protein Fbp1 plays an essential role in the virulence of C. neoformans. However, the molecular mechanism of Fbp1 regulating the virulence of C. neoformans is still unclear. In this study, we analyzed the potential Fbp1 substrates using an iTRAQ-based proteomic approach and identified the zinc-binding protein Zbp1 as a substrate of Fbp1. Protein interaction and stability assays showed that Zbp1 interacts with Fbp1 and is a downstream target of Fbp1. Ubiquitination analysis in vivo showed that the ubiquitination of Zbp1 is dependent on Fbp1 in C. neoformans. Subcellular localization analysis revealed that the Zbp1 protein was localized in the nucleus of C. neoformans cells. In addition, both deletion and overexpression of the ZBP1 gene led to the reduced capsule size, while overexpression has a more significant impact on capsule size reduction. Fungal virulence assays showed that although the zbp1Δ mutants are virulent, virulence was significantly attenuated in the ZBP1 overexpression strains. Fungal load assay showed that the fungal burdens recovered from the mouse lungs decreased gradually after infection, while no yeast cells were recovered from the brains and spleens of the mice infected by ZBP1 overexpression strains. Thus, our results revealed a new determinant of fungal virulence involving the post-translational regulation of a zinc-binding protein.

Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores' production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

The Role of Oxidoreductase-Like Protein Olp1 in Sexual Reproduction and Virulence of Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycete human fungal pathogen causing lethal meningoencephalitis, mainly in immunocompromised patients. Oxidoreductases are a class of enzymes that catalyze redox, playing a crucial role in biochemical reactions. In this study, we identified one Cryptococcus oxidoreductase-like protein-encoding gene OLP1 and investigated its role in the sexual reproduction and virulence of C. neoformans. Gene expression patterns analysis showed that the OLP1 gene was expressed in each developmental stage of Cryptococcus, and the Olp1 protein was located in the cytoplasm of Cryptococcus cells. Although it produced normal major virulence factors such as melanin and capsule, the olp1Δ mutants showed growth defects on the yeast extract peptone dextrose (YPD) medium supplemented with lithium chloride (LiCl) and 5-fluorocytosine (5-FC). The fungal mating analysis showed that Olp1 is also essential for fungal sexual reproduction, as olp1Δ mutants show significant defects in hyphae growth and basidiospores production during bisexual reproduction. The fungal nuclei imaging showed that during the bilateral mating of olp1Δ mutants, the nuclei failed to undergo meiosis after fusion in the basidia, indicating that Olp1 is crucial for regulating meiosis during mating. Moreover, Olp1 was also found to be required for fungal virulence in C. neoformans, as the olp1Δ mutants showed significant virulence attenuation in a murine inhalation model. In conclusion, our results showed that the oxidoreductase-like protein Olp1 is required for both fungal sexual reproduction and virulence in C. neoformans.

A Predicted Mannoprotein Cmp1 Regulates Fungal Virulence in Cryptococcus neoformans.

The capsule of the fungal pathogen Cryptococcus neoformans consists of glucuronoxylomannan (GXM), glucuronoxylomannogalactan (GXMGal), and mannoproteins (MPs). MPs are a kind of glycoproteins with low content but high immunogenicity, which can stimulate the immune protection of the host. However, there is not much information about the role of mannoproteins in virulence of the human fungal pathogen C. neoformans. In this study, we reported the identification and functional analysis of a predicted mannoprotein Cmp1 that regulates fungal virulence in C. neoformans. Gene expression pattern analysis indicates that the CMP1 gene was ubiquitously expressed at all stages of cryptococcal development. Subcellular localization analysis indicated that Cmp1 was localized in the cytoplasm of cryptococcal cells. Disruption or overexpression of CMP1 results in impairing capsule formation in Cryptococcus, but it does not affect the melanin production and sensitivity under various stress conditions, nor does it affect the sexual reproduction process of Cryptococcus. Survival assay showed that the pathogenicity of the cmp1Δ mutant or the CMP1 overexpression strain was significantly attenuated in a murine inhalation model of cryptococcosis. In conclusion, our findings implied that the mannoprotein Cmp1 is required for the virulence of C. neoformans.

Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans.

Autophagy (macroautophagy) is an evolutionarily conserved degradation pathway involved in bulk degradation of cytoplasmic organelles, old protein, and other macromolecules and nutrient recycling during starvation. Extensive studies on functions of autophagy-related genes have revealed that autophagy plays a role in cell differentiation and pathogenesis of pathogenic fungi. In this study, we identified and characterized 14 core autophagy machinery genes (ATGs) in C. neoformans. To understand the function of autophagy in virulence and fungal development in C. neoformans, we knocked out the 14 ATGs in both α and a mating type strain backgrounds in C. neoformans, respectively, by using biolistic transformation and in vivo homologous recombination. Fungal virulence assay showed that virulence of each atgΔ mutants was attenuated in a murine inhalation systemic-infection model, although virulence factor production was not dramatically impaired in vitro. Fungal mating assays showed that all the 14 ATGs are essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between each atgΔ mutants. Fungal nuclei development assay showed that nuclei in the bilateral mating of each atgΔ mutants failed to undergo meiosis after fusion, indicating autophagy is essential for regulating meiosis during mating. Overall, our study showed that autophagy is essential for fungal virulence and sexual reproduction in C. neoformans, which likely represents a conserved novel virulence and sexual reproduction control mechanism that involves the autophagy-mediated proteolysis pathway.

The Cys2His2 zinc finger protein Zfp1 regulates sexual reproduction and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous yeast pathogen that often infects the human central nervous system (CNS) to cause meningitis in immunocompromised individuals. Although numerous signaling pathways and factors important for fungal sexual reproduction and virulence have been investigated, their precise mechanism of action remains to be further elucidated. In this study, we identified and characterized a novel zinc finger protein Zfp1 that regulates fungal sexual reproduction and virulence in C. neoformans. qRT-PCR and ZFP1 promoter regulatory activity assays revealed a ubiquitous expression pattern of ZFP1 in all stages during mating. Subcellular localization analysis indicates that Zfp1 is targeted to the cytoplasm of C. neoformans. In vitro assays of stress responses showed that zfp1Δ mutants and the ZFP1 overexpressed strains ZFP1OE are hypersensitive to SDS, but not Congo red, indicating that Zfp1 may regulate cell membrane integrity. Zfp1 is also essential for fungal sexual reproduction because basidiospore production was blocked in bilateral mating between zfp1Δ mutants or ZFP1 overexpressed strains. Fungal nuclei development assay showed that nuclei in the bilateral mating of zfp1Δ mutants or ZFP1 overexpressed strains failed to undergo meiosis after fusion, indicating Zfp1 is important for regulating meiosis during mating. Although zfp1Δ mutants showed normal growth and produced normal major virulence factors, virulence was attenuated in a murine model. Interestingly, we found that the ZFP1 overexpressed strains were avirulent in a murine systemic-infection model. Overall, our study showed that the zinc finger protein Zfp1 is essential for fungal sporulation and virulence in C. neoformans.