Abstract
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.
Highlights
Cryptococcus neoformans is an encapsulated yeast pathogen that can cause fatal fungal meningitis in immunocompromised individuals (Chayakulkeeree and Perfect, 2006; Pukkila-Worley and Mylonakis, 2008)
We identified an F-box protein Fbp1 and revealed that it controls the virulence of C. neoformans without affecting classical virulence factors (Liu et al, 2011b)
In the process of exploring the mechanism of Fbp1 regulating the virulence of C. neoformans, we identified a downstream substrate protein, inositol phosphosphingolipid-phospholipase C1 (Isc1), and found that both the isc1D mutant and the ISC1 overexpression strain significantly reduced fungal virulence (Liu and Xue, 2014)
Summary
Cryptococcus neoformans is an encapsulated yeast pathogen that can cause fatal fungal meningitis in immunocompromised individuals (Chayakulkeeree and Perfect, 2006; Pukkila-Worley and Mylonakis, 2008). With the increasing number of immunocompromised people such as AIDS patients or those who have received organ transplants and immunosuppressive therapy (Brown et al, 2012), UPS Regulation of Cryptococcus Virulence the morbidity and mortality of cryptococcal infection has increased significantly (Kronstad et al, 2011; Erwig and Gow, 2016). Despite the high morbidity and mortality from cryptococcosis, treatment options for cryptococcosis are limited, and only three major groups of drugs are currently approved for clinical use: polyenes (e.g., Amphotericin B), azoles (e.g., Fluconazole), and the pyrimidine analogue flucytosine (5-FC) (Day et al, 2013; May et al, 2016). Given the shortage of anti-fungal drugs and the objective facts of fungal drug resistance, the pathogenesis of C. neoformans and the mechanism of drug resistance have become a hot and challenging spot that remains to be explored
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