scholarly journals UGE1 and UGE2 Regulate the UDP-Glucose/UDP-Galactose Equilibrium in Cryptococcus neoformans

2008 ◽  
Vol 7 (12) ◽  
pp. 2069-2077 ◽  
Author(s):  
Frédérique Moyrand ◽  
Ingrid Lafontaine ◽  
Thierry Fontaine ◽  
Guilhem Janbon
Keyword(s):  
Plasma Membrane ◽  
Carbon Source ◽  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Dependent Manner ◽  
Pathogenic Yeast ◽  
Intracellular Vesicles ◽  
Yeast Genomes ◽  
Fungal Kingdom ◽  
Affect Cell Growth

ABSTRACT The genome of the basidiomycete pathogenic yeast Cryptococcus neoformans carries two UDP-glucose epimerase genes (UGE1 and UGE2). UGE2 maps within a galactose cluster composed of a galactokinase homologue gene and a galactose-1-phosphate uridylyltransferase. This clustered organization of the GAL genes is similar to that in most of the hemiascomycete yeast genomes and in Schizosaccharomyces pombe but is otherwise not generally conserved in the fungal kingdom. UGE1 has been identified as necessary for galactoxylomannan biosynthesis and virulence. Here, we show that UGE2 is necessary for C. neoformans cells to utilize galactose as a carbon source at 30°C but is not required for virulence. In contrast, deletion of UGE1 does not affect cell growth on galactose at this temperature. At 37°C, a uge2Δ mutant grows on galactose in a UGE1-dependent manner. This compensation by UGE1 of UGE2 mutation for growth on galactose at 37°C was not associated with upregulation of UGE1 transcription or with an increase of the affinity of the enzyme for UDP-galactose at this temperature. We studied the subcellular localization of the two enzymes. Whereas at 30°C, Uge1p is at least partially associated with intracellular vesicles and Uge2p is on the plasma membrane, in cells growing on galactose at 37°C, Uge1p colocalizes with Uge2p to the plasma membrane, suggesting that its activity is regulated through subcellular localization.

Prostaglandin E2 interaction with AVP: effects on AQP2 phosphorylation and distribution

2000 ◽  
Vol 278 (3) ◽  
pp. F388-F394 ◽  
Author(s):  
Marina Zelenina ◽  
Birgitte Mønster Christensen ◽  
Johan Palmér ◽  
Angus C. Nairn ◽  
Søren Nielsen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Collecting Duct ◽  
Water Channel ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Centrifugation Technique ◽  
Renal Inner Medulla ◽  
Intracellular Vesicles ◽  
Dose Dependent

Prostaglandin E2 (PGE2) antagonizes the action of arginine vasopressin (AVP) on collecting duct water permeability. To investigate the mechanism of this antagonism, rat renal inner medulla (IM) was incubated with the two hormones, and the phosphorylation and subcellular distribution of the water channel, aquaporin-2 (AQP2) were studied. Using a phosphorylation state-specific AQP2 antibody, we demonstrated that AVP stimulates AQP2 phosphorylation at the Ser256 protein kinase A consensus site in a time- and dose-dependent manner. In parallel studies using a differential centrifugation technique, we demonstrated that AVP induced translocation of AQP2 from an intracellular vesicle-enriched fraction to a plasma membrane-enriched fraction. PGE2(10− 7 M) added after AVP (10− 8 M) did not decrease AQP2 phosphorylation but reversed AVP-induced translocation of AQP2 to the plasma membrane. Preincubation of IM with PGE2 did not prevent the effects of AVP on AQP2 phosphorylation and trafficking. PGE2 alone did not influence AQP2 phosphorylation and subcellular distribution. Our data indicate that 1) recruitment of AQP2 to the plasma membrane and its retrieval to a pool of intracellular vesicles may be regulated independently, 2) PGE2 may counteract AVP action by activation of AQP2 retrieval, 3) dephosphorylation of AQP2 is not a prerequisite for its internalization.

scholarly journals Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

2008 ◽  
Vol 8 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Connie B. Nichols ◽  
Jessica Ferreyra ◽  
Elizabeth R. Ballou ◽  
J. Andrew Alspaugh
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Sexual Differentiation ◽  
Signaling Cascades ◽  
Ras Signaling ◽  
Signaling Specificity ◽  
Downstream Signaling ◽  
Functional Flexibility ◽  
Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.

scholarly journals The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers

2000 ◽  
Vol 151 (4) ◽  
pp. 919-930 ◽  
Author(s):  
E.J. Kamsteeg ◽  
I. Heijnen ◽  
C.H. van Os ◽  
P.M.T. Deen
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Subcellular Localization ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Principal Cells ◽  
Intracellular Vesicles ◽  
And Function ◽  
Fine Tune

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non–p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.

scholarly journals Internalization of the host alkaline pH signal in a fungal pathogen

2020 ◽  
Author(s):  
Hannah E. Brown ◽  
Kaila M. Pianalto ◽  
Caroline M. Fernandes ◽  
Katherine D. Mueller ◽  
Maurizio Del Poeta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Fungal Pathogen ◽  
Cellular Response ◽  
Microbial Pathogens ◽  
Dependent Manner ◽  
Human Host ◽  
Human Fungal Pathogen ◽  
Stress Signals

AbstractThe ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. This adaptability is especially important for microbial pathogens that must sense and respond to drastic changes when encountering the human host. Cryptococcus neoformans is an environmental fungus and opportunistic pathogen that naturally lives in slightly acidic reservoirs, but must adapt to the relative increase in alkalinity in the human host in order to effectively cause disease. The fungal-specific Rim alkaline response signaling pathway effectively converts this extracellular signal into an adaptive cellular response allowing the pathogen to survive in its new environment. The newly identified Rra1 protein, the most upstream component of the C. neoformans Rim pathway, is an essential component of this alkaline response. Previous work connected Rra1-mediated signaling to the dynamics of the plasma membrane. Here we identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner, and that this dynamic pattern of localization further depends on specific residues in its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. The data presented here continue to unravel the complex and intricate processes of pH-sensing in a relevant human fungal pathogen. These studies will further elucidate general mechanisms by which cells respond to and internalize extracellular stress signals.Author SummaryThe work described here explores the genetics and mechanics of a cellular signaling pathway in a relevant human fungal pathogen, Cryptococcus neoformans. The findings presented in this manuscript untangle the complex interactions involved in the activation of a fungal-specific alkaline response pathway, the Rim pathway. Specifically, we find that C. neoformans is able to sense an increase in pH within the human host, internalize a membrane-bound pH-sensor, and activate a downstream signaling pathway enabling this pathogen to adapt to a novel host environment and effectively cause disease. Revealing the mechanisms of Rim pathway activation within the larger context of the fungal cell allows us to understand how and when this microorganism interprets relevant host signals. Furthermore, understanding how this pathogenic organism converts extracellular stress signals into an adaptive cellular response will elucidate more general mechanisms of microbial environmental sensing and stress response.

scholarly journals Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells

1994 ◽  
Vol 303 (2) ◽  
pp. 481-487 ◽  
Author(s):  
V Le Cabec ◽  
I Maridonneau-Parini
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Membrane Fraction ◽  
Immunofluorescence Microscopy ◽  
Resting Cells ◽  
Dependent Manner ◽  
Intact Cells ◽  
Cytoplasmic Granules ◽  
Calcium Dependent ◽  
Specific Granules

Annexins are soluble proteins capable of binding to phospholipid membranes in a calcium-dependent manner. Annexin 3, a 33 kDa protein mainly expressed in neutrophils, aggregates granules in cell-free assays, and a 36 kDa variant of this protein, specifically expressed in monocytes, has recently been identified. To obtain further information on these proteins, we defined their subcellular localization in resting and activated cells by immunofluorescence microscopy. Both proteins were associated with cytoplasmic granules in resting cells. We obtained evidence to indicate that, in neutrophils which possess a heterogenous granule population, annexin 3 was more likely to be associated with the specific granules. In cells activated with phorbol 12-myristate 13-acetate or opsonized zymosan, the 33 kDa and 36 kDa proteins translocated to the plasma or the phagosome membrane. Upon stimulation with A23187, annexin 3 translocated to the plasma membrane only in neutrophils. We also report that while annexin 3 was associated with restricted membranes in intact cells, it binds indiscriminately to every membrane fraction in cell-free assay. In conclusion, association of both forms of annexin 3 with granules suggests that these proteins could be implicated in processes of granule fusion.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen, Cryptococcus neoformans

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3476
Author(s):  
Kwang-Woo Jung ◽  
Moon-Soo Chung ◽  
Hyoung-Woo Bai ◽  
Byung Yeoup Chung ◽  
Sungbeom Lee
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Reverse Genetics ◽  
Mapk Pathway ◽  
Global Climate ◽  
Mitogen Activated Protein Kinase ◽  
Ergosterol Biosynthesis ◽  
Thymus Vulgaris ◽  
Dependent Manner ◽  
Expression Levels

Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.

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