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

2013 ◽  
Vol 82 (2) ◽  
pp. 557-568 ◽  
Author(s):  
Tong-Bao Liu ◽  
Chaoyang Xue
Keyword(s):  
Cryptococcus Neoformans ◽  
Time Course ◽  
Ubiquitin Proteasome System ◽  
Phenotypic Analysis ◽  
Fungal Virulence ◽  
Content Type ◽  
Brain Infection ◽  
Virulence Attenuation ◽  
Ubiquitin Proteasome ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis 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 forCryptococcusvirulence 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 thefbp1Δ 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. Thefbp1Δ 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 thefbp1Δ mutant is due to its inability to leave the lung. Thefbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in aCryptococcus-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 regulatingCryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.

scholarly journals The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans

2011 ◽  
Vol 10 (6) ◽  
pp. 791-802 ◽  
Author(s):  
Tong-Bao Liu ◽  
Yina Wang ◽  
Sabriya Stukes ◽  
Qing Chen ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Membrane Integrity ◽  
Infection Model ◽  
Calcofluor White ◽  
Fungal Virulence ◽  
Content Type ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Althoughfbp1mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, asfbp1mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating betweenfbp1mutants, even though normal dikaryotic hyphae were observed during mating.In vitroassays of stress responses revealed thatfbp1mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in bothSaccharomyces cerevisiaeandC. neoformansvia its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence inC. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

scholarly journals The Casein Kinase I Protein Cck1 Regulates Multiple Signaling Pathways and Is Essential for Cell Integrity and Fungal Virulence in Cryptococcus neoformans

2011 ◽  
Vol 10 (11) ◽  
pp. 1455-1464 ◽  
Author(s):  
Yina Wang ◽  
Tong-Bao Liu ◽  
Shyam Patel ◽  
Linghuo Jiang ◽  
Chaoyang Xue
Keyword(s):  
Signaling Pathways ◽  
Cryptococcus Neoformans ◽  
Casein Kinase ◽  
Cell Integrity ◽  
Casein Kinase I ◽  
Fungal Virulence ◽  
Content Type ◽  
Virulence Attenuation ◽  
Wide Range ◽  
Multiple Signaling

ABSTRACTCasein kinases regulate a wide range of cellular functions in eukaryotes, including phosphorylation of proteins that are substrates for degradation via the ubiquitin-proteasome system (UPS). Our previous study demonstrated that Fbp1, a component of the SCFFBP1E3 ligase complex, was essential forCryptococcusvirulence. Because theSaccharomyces cerevisiaehomolog of Fbp1, Grr1, requires casein kinase I (Yck1 and Yck2) to phosphorylate its substrates, we investigated the function of casein kinase I inCryptococcus neoformans. In this report, we identified aC. neoformanscasein kinase I protein homolog, Cck1. Similar to Fbp1, the expression of Cck1 is negatively regulated by glucose and during mating.cck1null mutants showed significant virulence attenuation in a murine systemic infection model, but Cck1 was dispensable for the development of classical virulence factors (capsule, melanin, and growth at 37°C).cck1mutants were hypersensitive to SDS treatment, indicating that Cck1 is required for cell integrity. The functional overlap between Cck1 and Fbp1 suggests that Cck1 may be required for the phosphorylation of Fbp1 substrates. Interestingly, thecck1mutant also showed increased sensitivity to osmotic stress and oxidative stress, suggesting that Cck1 regulates both cell integrity and the cellular stress response. Our results show that Cck1 regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases (MAPKs), demonstrating that Cck1 regulates cell integrity via the Mpk1 pathway and regulates cell adaptation to stresses via the Hog1 pathway. Overall, our study revealed that Cck1 plays important roles in regulating multiple signaling pathways and is required for fungal pathogenicity.

scholarly journals Development of Protective Inflammation and Cell-Mediated Immunity against Cryptococcus neoformans after Exposure to Hyphal Mutants

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Bing Zhai ◽  
Karen L. Wozniak ◽  
Jorge Masso-Silva ◽  
Srijana Upadhyay ◽  
Camaron Hole ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Cryptococcal Meningitis ◽  
Early Stage ◽  
Future Research ◽  
Clinical Strain ◽  
Cell Mediated Immunity ◽  
Fungal Virulence ◽  
Filamentous Form ◽  
Content Type ◽  
Virulence Attenuation

ABSTRACTMorphological switch is tightly coupled with the pathogenesis of many dimorphic fungal pathogens.Cryptococcus neoformans, the major causative agent of cryptococcal meningitis, mostly presents as the yeast form but is capable of switching to the hyphal form. The filamentous form has long been associated with attenuated virulence, yet the underlying mechanism remains elusive. We previously identified the master regulator Znf2 that controls the yeast-to-hypha transition inCryptococcus. Activation of Znf2 promotes hyphal formation and abolishes fungal virulencein vivo. Here we demonstrated that the cryptococcal strain overexpressingZNF2elicited strong and yet temporally confined proinflammatory responses in the early stage of infection. In contrast, exacerbated inflammation in mice infected with the wild-type (WT) strain showed that they were unable to control the infection. Animals inoculated with this filamentousCryptococcusstrain had fewer pulmonary eosinophils and CD11c+CD11b+cells than animals inoculated with WT yeast. Moreover, mice infected with this strain developed protective Th1- or Th17-type T cell responses. These findings suggest that the virulence attenuation of the filamentous form is likely due to its elicitation of protective host responses. The antivirulence effect of Znf2 was independent of two previously identified factors downstream of Znf2. Interestingly, mucosal immunizations with high doses ofZNF2-overexpressing cells, either in the live or heat-killed form, offered 100% protection to the host from a subsequent challenge with the otherwise lethal clinical strain H99. Our results demonstrate that heat-resistant cellular components presented in cryptococcal cells with activatedZNF2elicit protective host immune responses. These findings could facilitate future research on novel immunological therapies.IMPORTANCECryptococcal meningitis is one of the leading causes of death among AIDS patients. This disease presents a severe threat to public health. The current antifungal regimens are unsatisfactory in controlling or clearing the pathogenCryptococcus neoformans. Immunotherapies and/or vaccines could be a promising approach to prevent or manage this deadly disease. However, the lack of understanding of host-pathogen interactions during cryptococcal infection greatly hampers the development of effective immunotherapies. In this study, we discovered that inoculation of cryptococcal cells with activated Znf2, a morphogenesis regulator and an antivirulence factor, could shift the host pathological Th2 responses to the protective Th1 or Th17 responses. Importantly, we discovered that vaccination with either the viable or heat-killed form ofZNF2-overexpressing cells protected animals from the otherwise lethal infection by the highly virulent clinical strain. Our study suggests that the fungal cellular component(s) of theZNF2-overexpressing strain may provide potential vaccine candidate(s) for controlling the fatal disease.

scholarly journals The Vacuolar Morphogenesis Protein Vam6-Like Protein Vlp1 Is Required for Pathogenicity of Cryptococcus neoformans

2021 ◽  
Vol 7 (6) ◽  
pp. 418
Author(s):  
Cheng-Li Fan ◽  
Tong-Bao Liu
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Critical Role ◽  
Membrane Integrity ◽  
Infection Model ◽  
Fungal Virulence ◽  
Cell Membrane Integrity ◽  
Fungal Meningitis ◽  
Virulence Attenuation ◽  
F Box Protein

Cryptococcus neoformans is an encapsulated yeast pathogen that infects immunocompromised patients to cause fungal meningitis, resulting in hundreds of thousands of deaths each year. F-box protein Fbp1, the key component of the E3 ubiquitin ligase, plays a critical role in fungal development and virulence in fungal pathogens. In this study, we identified a potential substrate of Fbp1, the vacuolar morphogenesis protein Vam6-like protein Vlp1, and evaluated its role in virulence in C. neoformans. Deletion or overexpression of the VLP1 gene results in abnormal capsule formation and melanin production of C. neoformans. Stress tolerance assay showed that the vlp1Δ mutant was sensitive to SDS and NaCl but not to CFW or Congo red, indicating that Vlp1 might regulate the cell membrane integrity in C. neoformans. Fungal virulence assay showed that Vlp1 was essential for the pathogenicity of C. neoformans, as vlp1Δ mutants are avirulent in the mouse systematic infection model of cryptococcosis. The progression of fungal infection revealed that the vlp1Δ mutants were gradually eliminated from the lungs of the mice after infection. Moreover, the vlp1Δ mutants showed a proliferation defect inside macrophages and a viability defect in the host complement system, which likely contributes to the virulence attenuation of the vlp1Δ mutants. In summary, our results revealed that the vacuolar morphogenesis protein Vam6-like protein Vlp1 is essential for the pathogenicity of C. neoformans.

The ubiquitin–proteasome system and skeletal muscle wasting

2005 ◽  
Vol 41 ◽  
pp. 173-186 ◽  
Author(s):  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Wasting ◽  
Proteolytic Enzymes ◽  
Cathepsin L ◽  
Ubiquitin Proteasome System ◽  
Complete Breakdown ◽  
Skeletal Muscle Proteins ◽  
Ubiquitin Proteasome ◽  
Tripeptidyl Peptidase Ii ◽  
F Box Protein

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

scholarly journals An UBR-box from a non-N-recognin: Crystal Structure of the UBR domain of UBR6

2014 ◽  
Vol 70 (a1) ◽  
pp. C306-C306
Author(s):  
Juliana Muñoz-Escobar ◽  
Guennadi Kozlov ◽  
Jean-François Trempe ◽  
Kalle Gehring
Keyword(s):  
Crystal Structure ◽  
Ubiquitin Proteasome System ◽  
Zinc Binding ◽  
Binding Motifs ◽  
Clear Explanation ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Proteasome ◽  
Binding Pockets ◽  
F Box Protein

The degradation of many short-lived proteins in eukaryotic cells is carried out by the Ubiquitin Proteasome System. The N-end rule pathway links the half-life of proteins to the identity of its N-terminal residue, also called N-degron. Destabilizing N-degrons, are recognized by E3 ubiquitin ligases termed N-recognins. N-degrons are grouped into type 1, composed of basic residues, and type 2, composed of bulky hydrophobic residues. In mammals, four N-recognins mediate the N-end rule pathway: UBR1, UBR2, UBR4 and UBR5. These proteins share a ~70-residue zinc finger-like motif termed the Ubiquitin Recognin (UBR) box, responsible for their specificity. The mammalian genome encodes at least three more UBR-box proteins: UBR3, UBR6/FBXO11 and UBR7. However, these UBRs cannot recognize any type of N-degrons. Our lab reported the crystal structures of the UBR boxes from the human UBR1 and UBR2, rationalizing the empirical rules for the classification of type 1 N-degrons. Despite the valuable information obtained from those structures there is not a clear explanation for the no recognition of N-degrons by other UBR-box proteins. Here we report the crystal structure of the UBR-box domain from UBR6 also known as FBXO11. UBR6 is a F-box protein of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex and does not recognize any type of N-degrons. We crystallized a 77-residue fragment of the UBR-box of UBR6 and determined its structure at 1.7 Å resolution. Unexpectedly, this domain adopts an open conformation compared to UBR1-box, without any N-degron binding pockets. Its zinc-binding residues are conserved as in the N-recognins, but they are arranged in different zinc-binding motifs. Molecules form dimmers stabilized by zinc ions. The crystal had 4 molecules per asymmetric unit and space group P212121. For phasing we used Zn-SAD. With this structure we hope to obtain clues that explain the absence of N-degron recognition in some members of the UBR family.

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

2020 ◽  
Vol 8 (11) ◽  
pp. 1730
Author(s):  
Qi-Kun Yu ◽  
Lian-Tao Han ◽  
Yu-Juan Wu ◽  
Tong-Bao Liu
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Expression Patterns ◽  
Fungal Virulence ◽  
Growth Defects ◽  
Human Fungal Pathogen ◽  
Yeast Extract Peptone Dextrose ◽  
Virulence Attenuation ◽  
Protein Encoding ◽  
Fungal Mating

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.

scholarly journals The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1

2011 ◽  
Vol 194 (1) ◽  
pp. 17-25 ◽  
Author(s):  
Rachel Lander ◽  
Kara Nordin ◽  
Carole LaBonne
Keyword(s):  
Structural Diversity ◽  
Ubiquitin Proteasome System ◽  
Common Mechanism ◽  
Rich Domain ◽  
Transcriptional Regulatory ◽  
Snail Family ◽  
Ubiquitin Proteasome ◽  
Snail Proteins ◽  
The Stability ◽  
F Box Protein

A small group of core transcription factors, including Twist, Snail, Slug, and Sip1, control epithelial–mesenchymal transitions (EMTs) during both embryonic development and tumor metastasis. However, little is known about how these factors are coordinately regulated to mediate the requisite behavioral and fate changes. It was recently shown that a key mechanism for regulating Snail proteins is by modulating their stability. In this paper, we report that the stability of Twist is also regulated by the ubiquitin–proteasome system. We found that the same E3 ubiquitin ligase known to regulate Snail family proteins, Partner of paired (Ppa), also controlled Twist stability and did so in a manner dependent on the Twist WR-rich domain. Surprisingly, Ppa could also target the third core EMT regulatory factor Sip1 for proteasomal degradation. Together, these results indicate that despite the structural diversity of the core transcriptional regulatory factors implicated in EMT, a common mechanism has evolved for controlling their stability and therefore their function.

scholarly journals Equine Infectious Anemia Virus and the Ubiquitin-Proteasome System

2002 ◽  
Vol 76 (6) ◽  
pp. 3038-3044 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Raymond C. Sowder ◽  
Julian Adams ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Time Course ◽  
Equine Infectious Anemia Virus ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Gag Protein ◽  
Different Types ◽  
Ubiquitin Proteasome ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9Gag proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

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