Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

Abstract

Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1⌬ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1⌬ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1⌬ mutant is due to its inability to leave the lung. The fbp1⌬ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.C ryptococcus neoformans is a major human fungal pathogen and the causative agent of fatal cryptococcal meningoencephalitis, which is considered an AIDS-defined condition (1-5). Despite its medical importance, the treatment for cryptococcosis is limited. With increasing concerns of drug resistance and evolution of new virulent strains (6-9), there is an urgent need to understand the molecular basis of cryptococcal infection in order to discover and develop safer and more effective antifungal drugs. Against this background of clinical need are the heroic research efforts toward understanding the mechanism of Cryptococcus development and virulence that have significantly advanced the field in the past several decades. Several virulence factors, including production of polysaccharide capsule and melanin and the ability to grow at body temperature (37°C), have been well characterized (10, 11). A number of signal pathways regulating these virulence factors and important for Cryptococcus pathogenesis have also been identified (5, 12-16). Systematic genetic analysis of genes involved in fungal virulence revealed a capsule-independent antiphagocytic mechanism, as well as a number of proteins regulating fungal virulence independent of the classical virulence factors (17, 18). These studies revealed tha...

Cryptococcus inositol utilization modulates the host protective immune response during brain infection

“…virulent strain [35]. Moreover, mice vaccinated with either heat-killed Cryptococcus cells or culture filtrate antigens showed significant T-cell-dependent delayed-type hypersensitivity reaction and were better protected against subsequent challenge with a virulent strain [36,37]. Similar to these studies on animal models, elevated IFN-γ levels in the CSF, as well as administration of recombinant IFN-γ were directly correlated with better protective response against cryptococcal infection in humans [38].…”

Intracellular Action of a Secreted Peptide Required for Fungal Virulence

“…As macrophages are key determinants of the outcome of C. neoformans infection, we compared the interactions of wild-type and qsp1 Δ cells with primary macrophages and observed a defect in intracellular accumulation of qsp1Δ cells compared to wild-type. The intracellular accumulation defect of qsp1 Δ cells likely contributes to its virulence defect as other cryptococcal mutants with decreased accumulation inside macrophages are attenuated in an intranasal model of infection (Evans et al, 2015; Liu and Xue, 2014; Ma et al, 2009). An intriguing possibility is that confinement in the phagolysosome leads to the accumulation of high local concentrations of Qsp1 (Carnes et al, 2010), thereby triggering an adaptive program.…”

New technology and resources for cryptococcal research

“…Moreover, Vu and his colleagues identified a secreted metalloprotease (Mpr1) required for central nervous system infection of C. neoformans through a proteomic analysis of the extracellular proteome (Vu et al, 2014). This approach was also used for identifying the substrate of a virulence associated enzyme, components important for the interactions of extracellular vesicles with the cell wall, and metabolic changes in cryptococcal biofilms (Liu and Xue, 2014; Santi et al, 2014; Wolf et al, 2014). An important outcome of proteome analysis has been the identificaiton of potential new drug targets for the treatment of fungal infection.…”