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MAPK, MKP1 and c-Fos Discriminate Candida albicans Yeast from Hyphae in Epithelial Cells
Moyes, David L.
Runglall, Manohursingh
Murciano, Celia
Shen, Chengguo
Nayar, Deepa
Thavaraj, Selvam
Kohli, Arinder
Islam, Ayesha
Mora-Montes, Hector
Challacombe, Stephen J
Naglik, Julian R
Cell host & microbe
Host mechanisms enabling discrimination between the commensal and pathogenic states of opportunistic pathogens are critical in mucosal defense and homeostasis. Here, we demonstrate that oral epithelial cells orchestrate an innate response to the human fungal pathogen Candida albicans via NF-κB and a bi-phasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to the recognition of fungal cell wall structures. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha-formation and fungal burdens, and correlates with proinflammatory responses. This MAPK-based discriminatory pathway may provide a mechanism for epithelial tissues to remain quiescent in the presence of low fungal burdens whilst responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may thus comprise a `danger response' pathway in vivo and may be critical in identifying when this normally commensal fungus has become pathogenic.
Related Articles
Candida albicans Yeast and Hyphae are Discriminated by MAPK Signaling in Vaginal Epithelial Cells
Moyes, David L.
Murciano, Celia
Runglall, Manohursingh
Islam, Ayesha
Thavaraj, Selvam
Naglik, Julian R.
PLoS ONE
We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The 'second' or 'late' MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
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Activation of MAPK/c-Fos induced responses in oral epithelial cells is specific to Candida albicans and Candida dubliniensis hyphae
Moyes, David L.
Murciano, Celia
Runglall, Manohursingh
Kohli, Arinder
Islam, Ayesha
Naglik, Julian R.
Medical Microbiology and Immunology
Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.
Candida albicans; Candida dubliniensis; Hypha formation; MAPK; MKP1; c-Fos; NF-κB; Oral epithelium; Innate immunity
Related Articles
Candida albicans Cell Wall Glycosylation May Be Indirectly Required for Activation of Epithelial Cell Proinflammatory Responses ▿
Murciano, Celia
Moyes, David L.
Runglall, Manohursingh
Islam, Ayesha
Mille, Celine
Fradin, Chantal
Poulain, Daniel
Gow, Neil A. R.
Naglik, Julian R.
Infection and Immunity
Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or β-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
Related Articles
Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions
Murciano, Celia
Moyes, David L.
Runglall, Manohursingh
Tobouti, Priscila
Islam, Ayesha
Hoyer, Lois L.
Naglik, Julian R.
PLoS ONE
The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
Related Articles
Protection Against Epithelial Damage During Candida albicans Infection Is Mediated by PI3K/Akt and Mammalian Target of Rapamycin Signaling
Moyes, David L.
Shen, Chengguo
Murciano, Celia
Runglall, Manohursingh
Richardson, Jonathan P.
Arno, Matthew
Aldecoa-Otalora, Estibaliz
Naglik, Julian R.
The Journal of Infectious Diseases
Background. The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.
Methods. Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans–induced damage protection was determined using chemical inhibitors.
Results. Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation.
Conclusions. PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling.
Akt; Candida albicans; epithelial; inflammation; fungal; PI3 kinase; damage; MAPK; c-Fos; microarray; mTOR
Related Articles
Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages
Strijbis, Karin
Tafesse, Fikadu G.
Fairn, Gregory D.
Witte, Martin D.
Dougan, Stephanie K.
Watson, Nicki
Spooner, Eric
Esteban, Alexandre
Vyas, Valmik K.
Fink, Gerald R.
Grinstein, Sergio
Ploegh, Hidde L.
PLoS Pathogens
Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Author Summary
The opportunistic yeast Candida albicans is a commensal organism of the human digestive tract, but also the most common cause of human fungal infections. Phagocytosis, the process by which innate immune cells engulf pathogens, is vital to prevent C. albicans infections. The major phagocytic receptor involved in anti-fungal immunity is Dectin-1. We identify two new interacting proteins of Dectin-1 in macrophages: Bruton's Tyrosine Kinase (BTK) and Vav1. In the course of phagocytosis, different phosphoinositides (PIs) are formed in the phagosomal membrane to allow the recruitment of proteins equipped with specialized lipid-interaction domains. We show that BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 at the phagocytic cup, but not with diacylglycerol (DAG), which marks more mature phagosomal membranes. Inhibition of BTK affects the production of DAG and the recruitment of DAG-interacting proteins. BTK and Vav1 are essential for C. albicans immune responses, as BTK- or Vav1-deficient macrophages show reduced uptake of C. albicans and BTK- or Vav1-deficient deficient mice are more susceptible to systemic C. albicans infection. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Related Articles
Therapeutic Potential of Thiazolidinedione-8 as an Antibiofilm Agent against Candida albicans
Feldman, Mark
Al-Quntar, Abed
Polacheck, Itzhak
Friedman, Michael
Steinberg, Doron
PLoS ONE
Candida albicans is known as a commensal microorganism but it is also the most common fungal pathogen in humans, causing both mucosal and systemic infections. Biofilm-associated C. albicans infections present clinically important features due to their high levels of resistance to traditional antifungal agents. Quorum sensing is closely associated with biofilm formation and increasing fungal pathogenicity. We investigated the ability of the novel bacterial quorum sensing quencher thiazolidinedione-8 (S-8) to inhibit the formation of, and eradication of mature C. albicans biofilms. In addition, the capability of S-8 to alter fungal adhesion to mammalian cells was checked. S-8 exhibited specific antibiofilm and antiadhesion activities against C. albicans, at four- to eightfold lower concentrations than the minimum inhibitory concentration (MIC). Using fluorescence microscopy, we observed that S-8 dose-dependently reduces C. albicans–GFP binding to RAW macrophages. S-8 at sub-MICs also interfered with fungal morphogenesis by inhibiting the yeast-to-hyphal form transition. In addition, the tested agent strongly affected fungal cell wall characteristics by modulating its hydrophobicity. We evaluated the molecular mode of S-8 antibiofilm and antiadhesion activities using real-time RT-PCR. The expression levels of genes associated with biofilm formation, adhesion and filamentation, HWP1, ALS3 and EAP1, respectively, were dose-dependently downregulated by S-8. Transcript levels of UME6, responsible for long-term hyphal maintenance, were also significantly decreased by the tested agent. Both signaling pathways of hyphal formation-cAMP-PKA and MAPK-were interrupted by S-8. Their upstream general regulator RAS1 was markedly suppressed by S-8. In addition, the expression levels of MAPK cascade components CST20, HST7 and CPH1 were downregulated by S-8. Finally, transcriptional repressors of filament formation, TUP1 and NRG1, were dramatically upregulated by our compound. Our results indicate that S-8 holds a novel antibiofilm therapeutic mean in the treatment and prevention of biofilm-associated C. albicans infections.
Related Articles
PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90
LaFayette, Shantelle L.
Collins, Cathy
Zaas, Aimee K.
Schell, Wiley A.
Betancourt-Quiroz, Marisol
Gunatilaka, A. A. Leslie
Perfect, John R.
Cowen, Leah E.
PLoS Pathogens
Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.
Author Summary
Treating fungal infections is challenging due to the emergence of drug resistance and the limited number of clinically useful antifungal drugs. We screened a library of 1,280 pharmacologically active compounds to identify those that reverse resistance of the leading human fungal pathogen, Candida albicans, to the most widely used antifungals, the azoles. This revealed a new role for protein kinase C (PKC) signaling in resistance to drugs targeting the cell membrane, including azoles, allylamines, and morpholines. We dissected mechanisms through which PKC regulates resistance in C. albicans and the model yeast Saccharomyces cerevisiae. PKC enabled survival of cell membrane stress at least in part through the mitogen-activated protein kinase (MAPK) cascade in both species. In S. cerevisiae, inhibition of PKC signaling blocked activation of a key regulator of membrane stress responses, calcineurin. In C. albicans, Pkc1 and calcineurin independently regulate resistance via a common target. Deletion of C. albicans PKC1 rendered fungistatic drugs fungicidal and reduced virulence in a mouse model. The molecular chaperone Hsp90, which stabilizes client proteins including calcineurin, also stabilized the terminal C. albicans MAPK, Mkc1. We establish new circuitry connecting PKC with Hsp90 and calcineurin and suggest a promising strategy for treating life-threatening fungal infections.
Related Articles
A GATA Transcription Factor Recruits Hda1 in Response to Reduced Tor1 Signaling to Establish a Hyphal Chromatin State in Candida albicans
Lu, Yang
Su, Chang
Liu, Haoping
PLoS Pathogens
Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. One critical virulence attribute is its morphogenetic plasticity. Hyphal development requires two temporally linked changes in promoter chromatin, which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of the cAMP/PKA pathway and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling. Molecular mechanisms for the temporal connection and the link to Tor1 signaling are not clear. Here, through a forward genetic screen, we report the identification of the GATA family transcription factor Brg1 as the factor that recruits Hda1 to promoters of hypha-specific genes during hyphal elongation. BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin; therefore, it is a sensitive readout of Tor1 signaling. Interestingly, promoters of hypha-specific genes are not accessible to Brg1 in yeast cells. Furthermore, ectopic expression of Brg1 cannot induce hyphae, but can sustain hyphal development. Nucleosome mapping of a hypha-specific promoter shows that Nrg1 binding sites are in nucleosome free regions in yeast cells, whereas Brg1 binding sites are occupied by nucleosomes. Nucleosome disassembly during hyphal initiation exposes the binding sites for both regulators. During hyphal elongation, Brg1-mediated Hda1 recruitment causes nucleosome repositioning and occlusion of Nrg1 binding sites. We suggest that nucleosome repositioning is the underlying mechanism for the yeast-hyphal transition. The hypha-specific regulator Ume6 is a key downstream target of Brg1 and functions after Brg1 as a built-in positive feedback regulator of the hyphal transcriptional program to sustain hyphal development. With the levels of Nrg1 and Brg1 dynamically and sensitively controlled by the two major cellular growth pathways, temporal changes in nucleosome positioning during the yeast-to-hypha transition provide a mechanism for signal integration and cell fate specification. This mechanism is likely used broadly in development.
Author Summary
Candida is part of the gut microflora in healthy individuals, but can disseminate and cause systemic disease when the host's immune system is suppressed. Its ability to grow as yeast and hyphae in response to environmental cues is a major virulence attribute. Hyphal development requires temporary clearing of the transcription inhibitor Nrg1 upon activation of cAMP/PKA for initiation and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling for maintenance. Here, we show that, during hyphal initiation when Nrg1 is gone, expression of the GATA family transcription factor Brg1 is activated under reduced Tor1 signaling. Accumulated Brg1 recruits Hda1 to hyphal promoters to reposition nucleosomes, leading to obstruction of Nrg1 binding sites and sustained hyphal development. The nucleosome repositioning during the yeast-hyphal transition provides a mechanism for temporal integration of extracellular signals and cell-fate specification. The hypha-specific transcription factor Ume6 functions after Brg1 in this succession of feed-forward regulation of hyphal development. Since misregulation of either Nrg1 or Ume6 causes altered virulence, and Brg1 regulates both Nrg1 accessibility and Ume6 transcription, our findings should provide a better understanding of how Candida controls its morphological program in different host niches to exist as a commensal and a pathogen.
Related Articles
The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages
Uwamahoro, Nathalie
Verma-Gaur, Jiyoti
Shen, Hsin-Hui
Qu, Yue
Lewis, Rowena
Lu, Jingxiong
Bambery, Keith
Masters, Seth L.
Vince, James E.
Naderer, Thomas
Traven, Ana
The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.
Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae.
Related Articles
A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod
Neumann, Aaron K.
Jacobson, Ken
PLoS Pathogens
Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the "fungipod", formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion.
Author Summary
Yeasts are normal microbial commensals of humans and a significant source of opportunistic infections, especially in immunocompromised individuals. We report a novel cellular protrusive structure, the fungipod, which participates in the host-microbe interaction between human immature dendritic cells (DC) and yeasts. The fungipod's structure is based on and propelled by a robust process of local actin cytoskeleton growth at the DC-yeast contact site, and this cytoskeletal remodeling results in a durable tubular structure over 10 µm long connecting the dorsal DC membrane and yeast. The fungal cell wall polysaccharides mannan and chitin trigger fungipod formation by stimulating the carbohydrate pattern recognition receptor CD206. Fungipods are part of a specific response to large particulate objects (i.e., yeast), and they may promote the human immature DC's relatively poor phagocytosis of yeast. The human fungal pathogen, Candida parapsilosis, induces a strong fungipod response from DC, and this response is highly species specific since the related pathogens Candida albicans and Candida tropicalis induce fungipods rarely. Our work highlights a novel cell biological element of fungal recognition by the innate immune system.
Related Articles
Cell Cycle-Independent Phospho-Regulation of Fkh2 during Hyphal Growth Regulates Candida albicans Pathogenesis
Greig, Jamie A.
Sudbery, Ian M.
Richardson, Jonathan P.
Naglik, Julian R.
Wang, Yue
Sudbery, Peter E.
PLoS Pathogens
The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.
Author Summary
The fungus Candida albicans is a commensal in the human microbiota, responsible for superficial infections such as oral and vaginal thrush. However, it can become highly virulent, causing life-threatening systemic candidemia in severely immunocompromised patients, including those taking immunosuppressive drugs for transplantation, sufferers of AIDS and neutropenia, and individuals undergoing chemotherapy or at extremes of age. With a rapidly increasing ageing population worldwide, C. albicans and other fungal pathogens will become more prevalent, demanding a greater understanding of their pathogenesis for the development of effective therapeutics. Fungal pathogenicity requires a coordinated change in the pattern of gene expression orchestrated by a set of transcription factors. Here we have discovered that a transcription factor, Fkh2, is modified by phosphorylation under the control of the kinases Cdc28 and Cbk1 in response to conditions that activate virulence factor expression. Fkh2 is involved in a wide variety of cellular processes including cell proliferation, but this phosphorylation endows it with a specialized function in promoting the expression of genes required for tissue invasion, biofilm formation, and pathogenesis in the host. This study highlights the role of protein phosphorylation in regulating pathogenesis and furthers our understanding of the pathogenic switch in this important opportunistic fungal pathogen.
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Dynamic, Morphotype-Specific Candida albicans β-Glucan Exposure during Infection and Drug Treatment
Wheeler, Robert T.
Kombe, Diana
Agarwala, Sudeep D.
Fink, Gerald R.
PLoS Pathogens
Candida albicans, a clinically important dimorphic fungal pathogen that can evade immune attack by masking its cell wall β-glucan from immune recognition, mutes protective host responses mediated by the Dectin-1 β-glucan receptor on innate immune cells. Although the ability of C. albicans to switch between a yeast- or hyphal-form is a key virulence determinant, the role of each morphotype in β-glucan masking during infection and treatment has not been addressed. Here, we show that during infection of mice, the C. albicans β-glucan is masked initially but becomes exposed later in several organs. At all measured stages of infection, there is no difference in β-glucan exposure between yeast-form and hyphal cells. We have previously shown that sub-inhibitory doses of the anti-fungal drug caspofungin can expose β-glucan in vitro, suggesting that the drug may enhance immune activity during therapy. This report shows that caspofungin also mediates β-glucan unmasking in vivo. Surprisingly, caspofungin preferentially unmasks filamentous cells, as opposed to yeast form cells, both in vivo and in vitro. The fungicidal activity of caspofungin in vitro is also filament-biased, as corroborated using yeast-locked and hyphal-locked mutants. The uncloaking of filaments is not a general effect of anti-fungal drugs, as another anti-fungal agent does not have this effect. These results highlight the advantage of studying host–pathogen interaction in vivo and suggest new avenues for drug development.
Author Summary
Candida is a common human commensal but disseminated candidiasis is a serious clinical problem, especially among immunocompromised patients. The innate immune system controls Candida infection, in part through the germline-encoded β-glucan receptor Dectin-1. However, during in vitro growth, Candida albicans mutes Dectin-1 recognition by cloaking its β-glucan underneath a layer of mannan. Bridging these two seemingly contradictory observations, we demonstrate that C. albicans masks β-glucan early during infection, but it becomes exposed later, allowing Dectin-1 to recognize the fungi and mediate immunity. Remarkably, treatment of mice with sub-therapeutic doses of the antifungal drug caspofungin causes exposure of β-glucan on C. albicans even when it would not be exposed naturally. We introduce a new technique for monitoring of epitope exposure during infection, which can be used to monitor the availability of any epitope for immune recognition. This technique allowed us to show that natural unmasking of β-glucan is not morphotype-specific, but drug-mediated unmasking is biased towards the invasive filamentous form of C. albicans. These results highlight the unexplored area of dynamic epitope exposure during infection and therapy, which might be targetable to enhance immune recognition and fungal clearance.
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Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants
Lewis, Leanne E.
Bain, Judith M.
Lowes, Christina
Gillespie, Collette
Rudkin, Fiona M.
Gow, Neil A. R.
Erwig, Lars-Peter
PLoS Pathogens
Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.
Author Summary
Host defence against systemic candidiasis relies mainly on the ingestion and elimination of fungal cells by cells of the innate immune system, especially neutrophils and macrophages. Here we have used live cell video microscopy coupled with sophisticated image analysis to generate a temporal and spatial analysis in unprecedented detail of the specific effects of C. albicans viability, cell wall composition, morphogenesis and spatial orientation on two distinct stages (macrophage migration and engulfment of bound C. albicans) of the phagocytosis process. The novel methods employed here to study phagocytosis of C. albicans could be applied to study other pathogens and uptake of dying host cells. Thus, our studies have direct implications for a much broader community and provide a blueprint for future studies with other phagocytes/microorganisms that would significantly enhance our understanding of the mechanisms that govern effective phagocytosis and ultimately the innate immune response to infection.
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Dendritic Cells Discriminate between Yeasts and Hyphae of the Fungus Candida albicans
d'Ostiani, Cristiana Fè
Del Sero, Giuseppe
Bacci, Angela
Montagnoli, Claudia
Spreca, Antonio
Mencacci, Antonella
Ricciardi-Castagnoli, Paola
Romani, Luigina
The Journal of Experimental Medicine
The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces. The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of debate. Here we show the interaction, and consequences, of different forms of C. albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C. albicans saprophytism and infections.
Candida albicans; yeast; hyphae; dendritic cells; cytokines
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Integrin αXβ2 Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections
Jawhara, Samir
Pluskota, Elzbieta
Verbovetskiy, Dmitriy
Skomorovska-Prokvolit, Olena
Plow, Edward F.
Soloviev, Dmitry A.
The Journal of Immunology Author Choice
The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in αXβ2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of αXβ2-deficient mice within 9 d. Organs taken from αXβ2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use αXβ2 to control fungal invasion.
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Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p
Peters, Brian M.
Ovchinnikova, Ekaterina S.
Krom, Bastiaan P.
Schlecht, Lisa Marie
Zhou, Han
Hoyer, Lois L.
Busscher, Henk J.
van der Mei, Henny C.
Jabra-Rizk, Mary Ann
Shirtliff, Mark E.
2012;158(Pt 12):2975-2986.
The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive hyphae. This differed from tongues infected with St. aureus alone or in conjunction with the als3 mutant strain of C. albicans, where bacterial presence was limited to the outer layers of the oral tissue. Collectively, the findings generated from this study identified a key role for C. albicans Als3p in mediating this clinically relevant fungal–bacterial interaction.
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Mucins Suppress Virulence Traits of Candida albicans
Kavanaugh, Nicole L.
Zhang, Angela Q.
Nobile, Clarissa J.
Johnson, Alexander D.
Ribbeck, Katharina
Candida albicans is the most prevalent fungal pathogen of humans, causing a variety of diseases ranging from superficial mucosal infections to deep-seated systemic invasions. Mucus, the gel that coats all wet epithelial surfaces, accommodates C. albicans as part of the normal microbiota, where C. albicans resides asymptomatically in healthy humans. Through a series of in vitro experiments combined with gene expression analysis, we show that mucin biopolymers, the main gel-forming constituents of mucus, induce a new oval-shaped morphology in C. albicans in which a range of genes related to adhesion, filamentation, and biofilm formation are downregulated. We also show that corresponding traits are suppressed, rendering C. albicans impaired in forming biofilms on a range of different synthetic surfaces and human epithelial cells. Our data suggest that mucins can manipulate C. albicans physiology, and we hypothesize that they are key environmental signals for retaining C. albicans in the host-compatible, commensal state.
The yeast Candida albicans causes both superficial infections of the mucosa and life-threatening infections upon entering the bloodstream. However, C. albicans is not always harmful and can exist as part of the normal microbiota without causing disease. Internal body surfaces that are susceptible to infection by C. albicans are coated with mucus, which we hypothesize plays an important role in preventing infections. Here, we show that the main components of mucus, mucin glycoproteins, suppress virulence attributes of C. albicans at the levels of gene expression and the corresponding morphological traits. Specifically, mucins suppress attachment to plastic surfaces and human cells, the transition to cell-penetrating hyphae, and the formation of biofilms (drug-resistant microbial communities). Additionally, exposure to mucins induces an elongated morphology that physically resembles the mating-competent opaque state but is phenotypically distinct. We suggest that mucins are potent antivirulence molecules that have therapeutic potential for suppressing C. albicans infections.
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Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance
Lu, Yang
Su, Chang
Wang, Allen
Liu, Haoping
PLoS Biology
Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.
Author Summary
Many organisms are able to change their phenotype in response to changes in the environment, a phenomenon referred to as plasticity. Candida albicans, a major opportunistic fungal pathogen of humans, can undergo reversible morphological changes between yeast (spherical) and hyphal (filamentous) forms of growth in response to environmental cues. This morphological plasticity is essential for its pathogenesis and survival in its hosts. In this study, we show that hyphal development is initiated and maintained by two major nutrient-sensing cellular growth pathways that act by removing the inhibition provided by the transcriptional repressor Nrg1. While initiation requires a rapid but temporary disappearance of Nrg1 via activation of the cAMP-dependent protein kinase A pathway, maintenance requires the recruitment to promoters of the Hda1 histone deacetylase under conditions of reduced signaling by the target of rapamycin (TOR) kinase, leading to chromatin remodeling that blocks Nrg1 access to the promoters of hypha-specific genes. We observed that recruitment of Hda1 to promoters happens only during the time window when Nrg1 is absent. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals in the regulation of gene expression and phenotypic plasticity during development and cell fate specification.
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Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis
Puri, Sumant
Kumar, Rohitashw
Chadha, Sonia
Tati, Swetha
Conti, Heather R.
Hube, Bernhard
Cullen, Paul J.
Edgerton, Mira
PLoS ONE
Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.
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A Drug-Sensitive Genetic Network Masks Fungi from the Immune System
Wheeler, Robert T
Fink, Gerald R
PLoS Pathogens
Fungal pathogens can be recognized by the immune system via their β-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of "masking" this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its β-glucan, leading to increased β-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask β-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics.
Opportunistic fungal pathogens such as Candida albicans often cause fatal infections in patients with a compromised immune system. Unfortunately, current drugs often fail to halt fungal disease, are ineffective against drug-resistant strains, and have severe side effects. Despite the clear clinical significance of fungal infections, it is still not understood how fungi are recognized by the immune system. Candida has high levels of the structural molecule β-glucan in its cell wall, but the majority of its β-glucan is masked by a mannoprotein coat and is therefore invisible to the immune system. Masking of β-glucan may be a fungal virulence factor, because exposed β-glucan provokes a proinflammatory response that is important for mounting an effective immune response against the fungus and clearing the infection. By surveying the genome of the model fungus Saccharomyces cerevisiae (bakers' yeast), the authors discovered a genetic network required for masking β-glucan from the immune system. Mutation of genes in this network in C. albicans caused unmasking of β-glucan and an increased immune response to the fungus. The authors also found that sublethal doses of the antifungal drug caspofungin cause unmasking and lead to a greater immune response. Drugs targeting this fungally conserved masking network may provide new tools to fight fungal infections.
Related Articles
Contribution of Candida albicans Cell Wall Components to Recognition by and Escape from Murine Macrophages ▿
McKenzie, C. G. J.
Koser, U.
Lewis, L. E.
Bain, J. M.
Mora-Montes, H. M.
Barker, R. N.
Gow, N. A. R.
Erwig, L. P.
Infection and Immunity
The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Δ, pmr1Δ, and mnt3 mnt5Δ), whereas O- and N-linked mannan defects (mnt1Δ mnt2Δ and mns1Δ) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Δ, hwp1Δ, and als3Δ) and yeast-locked mutants (clb2Δ, hgc1Δ, cph1Δ, efg1Δ, and efg1Δ cph1Δ), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.
Related Articles
Genome-Wide Transcriptional Profiling of the Cyclic AMP-Dependent Signaling Pathway during Morphogenic Transitions of Candida albicans▿ †
Bahn, Yong-Sun
Molenda, Matthew
Staab, Janet F.
Lyman, Courtney A.
Gordon, Laura J.
Sundstrom, Paula
Eukaryotic Cell
Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.
Related Articles
Candida albicans β-Glucan Exposure Is Controlled by the Fungal CEK1-Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1 ▿ †
Galán-Díez, Marta
Arana, David M.
Serrano-Gómez, Diego
Kremer, Leonor
Casasnovas, José M.
Ortega, Mara
Cuesta-Domínguez, Álvaro
Corbí, Angel L.
Pla, Jesús
Fernández-Ruiz, Elena
Infection and Immunity
Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as β-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall β-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IκB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in β-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.
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Tiruchendur Hotels
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Hotels in Tiruchendur : 4, Tiruchendur Hotels Booking Online | Book Budget, Luxury, Star & Cheap Hotels in Tiruchendur, India
TIRUCHENDUR Hotels Reviews
By Rathish kumar R on 10 March 2015
Hotel Chitra Park
I enjoyed the day. Hotel looks very neat and clean. Staff are kind to interact and guide. Hotel is very near to temple. No noise around the hotel. Very peaceful to stay. I will recommend to all my family and friends. Show More
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Hotel Udhayam International
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Tiruchendur Hotels Overview:
Thirucheer Alaiwai is a sobriquet to Tiruchendur. In the Thoothukudi district of Tamil Nadu, Tiruchendur is a panchayat town at the southern tip of India. By dint of its position at the southern tip of the country, it is situated at the Indian Ocean. Surrounding the town, Arid forests are located in numerous suburban hamlets. The densely grown palm trees are what the big thing about the red soil forests is. Canopy of the well forested-trees range under the spectrum: palm trees, cashew plantations and other crops, which are typical in the region. The town of Arumuganaer is at a stone's throw from Tiruchendur – at the meager distance of some 8 km. The town is very finely linked via both the modes of rail and road. The nearby airport is Tuticorin Airport; which is 40 kilometers (25 mi) from Tuticorin, 55 km (34 mi) from Tirunelveli and 171 km (106 mi) from Madurai. One inbound and the same number of outbound flight to Chennai is operated by Kingfisher Airlines presently. Also, the bus connections are provided and the places to which the services are available include Aruppukootai, Ettiyapuram, Tuticorin, kovilpatti, madurai and Tirunelveli. The town's connection with Tirunelveli is via a broad-gauge train track, with a train station at Tiruchendur.
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Abu Dhabi's TAQA says not holding merger talks
Gold languishes below $1,170 ahead of U.S. jobs data
Kuwait parliament approves budget with $27 billion deficit
Saudi's Mobily to reissue 2014, Q1 2015 results after regulator probe
Dubai Holding appoints new managing director, CEO
HSBC Bank Oman agrees to sell Pakistan business to Meezan Bank
European tour operators evacuate thousands from Tunisia after attack
Fitch Ratings has placed the individual ratings of...
December 5, 2009 7:14 by kippreport
Fitch Ratings has placed the individual ratings of four Dubai-based banks on Rating Watch Negative (RWN) because of Dubai World's recent request to postpone debt repayments, it said on Saturday. The banks include Commercial Bank of Dubai, Emirates NBD, Mashreqbank, and HSBC Bank Middle East. Fitch said its actions reflect the belief that "these banks have significant exposures to Dubai World and other Dubai government-related entities," but that it expects to resolve these RWN over the next two weeks.
"The lack of clarity about the full extent of the UAE banking sector's exposures to Dubai GREs and the knock-on effects on the wider Dubai economy, combined with higher provision requirements by the Central Bank of the UAE against two Saudi corporates (Saad and Al-Gosaibi), rising retail impairments and narrowing funding options means that 2009 and 2010 profits are likely to be adversely affected," Fitch added in the statement.
Saudi stock market falls on Dubai debt crisis
December 5 2009
Oil prices are perfect, says Saudi oil minister
December 5 2009


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Bleeding Edge TV 215: Beats by Dr. Dre Private Listening Session and Impressions
Posted by Andru Edwards Categories: Full Episodes, Gizmatic, Accessories, CES, CES 2008, Design, Features, Home Entertainment, Music, Podcasts, Portable Audio / Video, Product Reviews, Videocasts
The Gear Live crew got the only private listening session of the newly-announced Beats by Dr. Dre headphones during CES, and our thoughts are in this episode. Be sure to check out our other Beats by Dr. Dre video, and our Beats by Dr. Dre photo gallery.
I'm what you would refer to as a cost-sensitive buyer. I'm obsessive about value propositions with my purchases. And I would buy the $400 Beats by Dr. Dre headphones by Dre & Monster Cable.
We got a private demo with Monster and had the opportunity to listen to what's amounted to more than two years of obsessive research and attention to detail. All the materials, the drivers, the amplifier and more are clearly thought out and it shows. The response from these is absolutely incredible. Their intent was to allow you to listen to the music as the producer intended and dare I say it's as faithful reproduction as you'll find on a $400 set of headphones. It's enough that I would buy these before buying the nearest-priced Logitech system for my computer. The bass response was impressive to the Nth—low bass held its tonal quality through a great deal of dynamic range, something not usually seen. The midrange was rich and warm and perfectly presented and the high ends came out just as well.
I'm sad because I really want these. I mean, I was upset that I had to go home tomorrow to my sound system, and that it's nothing close to how these performed. They were comfortable, sexy, and sounded absolutely incredible.
Try these when they come out in June. But only if you're prepared to buy them.
CES 2008 Video: Sandisk SSD Drive Technology
Posted by Andru Edwards Categories: Gizmatic, Short Bytes, CES, CES 2008, Features, PC / Laptop, Podcasts, Storage, Videocasts
Many companies showed of Solid State Disks (SSD) at CES, but none so cost effectively as Sandisk. As usual Sandisk is bringing a product to market that beats the price of it's competitors while still not skimping on features. SSD drives are an effective way to increase performance and battery life on notebooks without requiring re-engineering. Available now in a variety of capacities and price points - look for them in stores or devices soon.
Bleeding Edge TV 207: Asus Eee PC RAM Upgrade
Posted by Andru Edwards Categories: Hack Attack, Short Bytes, Features, Handhelds, Mods / Hacks, PC / Laptop, Podcasts, Videocasts
The Asus Eee PC is an amazing little wonder of a device. However, unless you buy the 8G version, your little Linux mobile laptop isn't fully powered. Luckily, getting inside the Eee PC is not much of a challenge at all, and adding in some extra RAM typically works nicely for any computer user. In this episode, Nate True show us how to upgrade the RAM in the Eee PC. All you need is a tiny Philips screwdriver, a 1 or 2 GB RAM module, an Eee PC, and a few minutes of time. Check out the video for the full details, and let us know how it goes if you try it out. Remember, we will have more Eee PC tips and hacks for you soon.
MacWorld 2008: Apple MacBook Air Notebook Announced
Posted by Andru Edwards Categories: Apple, PC / Laptop, Wireless / WiFi
During this morning's MacWorld keynote, Steve Jobs announced the new MacBook Air notebook. We've all been expecting some sort of Apple sub-notebook, and this one looks amazing. Apple it touting this as the "world's thinnest notebook". The MacBook Air is 0.16-inches to 0.76-inches. The thickest part of the Air is thinner than the thinnest part of a Sony TZ series notebook. That is impressive. The device features an 80 GB 1.8-inch hard drive, with a 64 GB SSD drive as an option. It ships standard with 2 GB of RAM and a 1.6 GHz Intel Core 2 Duo chip, with the option to switch to 1.8 GHz. Other features include a 45-watt MagSafe, 1 USB 2.0 port, a Micro-DVI port, and audio out. There is no Ethernet port (otherwise they couldn't have gotten it this thin) so you will have to rely on the built-in 802.11n WiFI and Bluetooth 2.1+EDR. The MacBook Air also leaves out the optical drive, but you can purchase an external SuperDrive accessory for $99 USD. It also ships with software that allows it to "borrow" the optical drive of another Mac or PC. Expect 5 hours of battery life out of this one - or more if you opt for the SSD drive. The MacBook Air will hit retail in about two weeks, and will cost $1799. Pre-orders start today.
Read More | MacBook Air Product Page
MacWorld 2008: Apple announces TIme Capsule Airport Extreme with built-in hard drive
Posted by Andru Edwards Categories: Apple, Storage, Wireless / WiFi
During this morning's MacWorld keynote, Apple announced a brand new router specifically meant for those of us running Leopard on Apple notebooks, called Time Capsule. Basically an Airport Extreme NAS, the Time Capsule router allows those running Leopard while connected over WiFi to run Time Machine. Currently, unless you use a back-end hack to enable WiFi backups, you have to plug and unplug your laptop to an external drive to run Time Machine backups. Time Capsule removes that requirement, thus making WiFi Time Machine backups simple. Of course, it comes at a price. A 500 GB Time Capsule retails for $299 USD, which a 1 TB version costs $499.
Read More | Time Capsule Product Page
CES 2008 Video: Samsung's Q1 Ultra UMPC Line
Posted by Andru Edwards Categories: Full Episodes, Gizmatic, CES, CES 2008, Features, Handhelds, PC / Laptop, Podcasts, Portable Audio / Video, Videocasts
We visited Samsung and learned about their new Ultra Mobile Personal Computers (UMPC). They're basically palm-sized computers with the power of a laptop. Their line starts with the Q1 Ultra, which runs Windows XP or Vista has a standard 80GB hard drive, a battery life of 4 ½ hours with WiFi enabled, includes a webcam, 2 USB ports and an SD Card slot, weighing only 1.5 pounds at a price of $1300 USD. There's also the Q1 Ultra SSD, with a 32 GB solid-state drive for increased boot time and access to data. Finally, there's the just-launched, award-winning Q1 Ultra Premium, with a powerful Intel Pentium Mobile Processor, 80GB hard drive, and a battery life up to 7.5 hours with WiFi enabled. Available at the end of the month for $1500.
CES 2008 Video: Sparky gets tasered / Taser MP3 player
Posted by Andru Edwards Categories: Gizmatic, Short Bytes, CES, CES 2008, Features, Misc. Tech, Podcasts, Videocasts
When going to CES one expects a rough week involving countless interviews, hour after hour of writing, and defending oneself from PR reps from round the world. One thing you don't expect is to get the living daylights shocked out of you. First and foremost, watch the video above for a chance to see me get tasered. After all, it's hard to pass up an opportunity to see some poor sot get tasered - particularly when they signed themself up for it (full waiver and everything).
Oh yeah, a bit of background - we didn't go for the 'shock treatment' but rather went to check out Tasers new MPH or Music Playing Holster. With this faux-leather device one can carry their original, pink, or leopard skin taser in style and listen to 1GB of tunes at the same time. It doesn't fully make sense to us, but the Taser company claims this makes it an ideal exercise companion for those jogs through the seedy underbelly of your favorite big city.
CES 2008 Video: Beats by Dr. Dre First Look with Noel Lee
Posted by Andru Edwards Categories: Full Episodes, Gizmatic, Accessories, CES, CES 2008, Design, Features, Home Entertainment, Music, Podcasts, Videocasts
We got a chance to catch up with Noel Lee, Head Monster of Monster Cable to talk to him about their newly announced Beats by Dr. Dre headphones. Noel goes into the background and development of these amazing headphones, the two-year development, and the relationship with Dr. Dre. Be sure to also check out our Beats by Dr. Dre photo gallery to get a good look at these things.
CES 2008 Video: TiVo Desktop 2.6 First Look
Posted by Andru Edwards Categories: Full Episodes, Gizmatic, CES, CES 2008, Design, Features, PC / Laptop, Podcasts, Videocasts
TiVo takes some time at Digital Experience from CES to show us their latest iteration of TiVo Desktop, version 2.6. Version 2.6 includes some very cool features, including the ability for users to automatically push web-based video and podcasts (like like ours) straight down to their TiVos automatically. We talk about availability and what else to expect coming down the pipeline from TiVo in the near future in this video.
CES 2008: Pioneer AVIC f500bt GPS - voice command iPod control
Posted by Sparky Categories: CES, CES 2008, GPS, Music, Portable Audio / Video, Transportation
For the ultimate in convergence check out Pioneer's latest in-dash GPS stereo unit. Like some other units the GPS the high-resolution 5.8" screen can pop out to provide GPS and tunes on the go, but unlike the competition this new head unit keeps drivers safe with voice activated navigation and music controls. When the GPS unit is popped out you can take it on the go for directions either while walking, or when driving another car.
Outfitted with Voicebox technology the driver can give commands verbally like "Play Honeycut - Aluminum City" or "Navigate to the Space Needle in Seattle" so as to not have to take their hands or eyes off the road. The Bluetooth capabilities also allow for hands free calling to further enhance the drivers safety. Music in WMA or MP3 format can be played from SD cards, or streamed from Bluetooth phones. The AVIC f500bt also provides strong iPod integration allowing for playback and control of iPod stored music.
Available soon for a yet unknown price. For the inconvenience and high technology packed into this car entertainment don't expect it to be cheap though.
Read More | Pioneer Press
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I read many of the same Dr Zeuss books with my children that I now read with my grandchildren. They love the nonsense rhymes to learn how words are formed and they soon learn the words and will readily tell you that you have said something wrong. It makes a very bonding atmosphere for you and your little one. Horton was one of my children.s favorites and is now becoming a grandchild favorite.
I bought this for my 3 year old niece for the holidays. She hasn't opened it yet, but it seemed very cute when I looked at it. It's nice and fuzzy. I bought it to go with a Horton shirt I got her.It will make a nice gift.
No much to describe. What you see is what you get. It's a cute puppet book.


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Summer Corn And Tomatoes
18 Aug 2010
|Ears of corn
|Yogurt based spread
|1 Tablespoon (Such As Brummel & Brown)
|Diced onion
|1 Cup (16 tbs) (1 Small One)
|Cherry tomatoes
|1 Pint, cut in half (Grape Tomatoes)
|Chopped fresh parsley
|1 Tablespoon
|1⁄4 Teaspoon
|Freshly ground black pepper
|1⁄8 Teaspoon
1. Remove and discard husks and silks from corn. Cut off corn kernels into a bowl; scrape milk and remaining pulp from cob with a paring knife to measure 3 cups. Set corn aside.
2. Heat yogurt-based spread in a large nonstick skillet over low heat; add onion. Cook over medium heat 3 minutes, stirring occasionally. Add corn and cook 4 minutes, stirring frequently. Add tomatoes; cook 3 minutes. Stir in parsley, salt, and pepper.
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Summer Corn And Tomatoes Recipe


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Featuring Pumpkins Aglow in October (Fri-Sun) and the Annual Festival of Lights, (November-January) when during the Christmas season, Edaville is transformed into a winter wonderland of holiday delights! Passengers can relax comfortably in warm and dry coaches while riding through a spectacular holiday setting featuring an explosio Read more about Edaville USA
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Join us for a special tour and seminar focused on the significance of the Magna Carta.
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The Plymouth Rock Foundation's mission is to "preserve and propagate the Pilgrim story". In addition, its charter goes on to state, "we also seek to make better known the ideas and achievements of later generations of Americans who followed after the Pilgrims in both spirit and deed, forging our nation's Christian heritage." Read more about Plymouth Rock Foundation - Pilgrim Progress
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Originally written on Fox Sports | Last updated 9/14/13
Kieran McDonagh passed for a career-high 377 yards, fueling Portland State to a 43-6 pounding of Division-II Humboldt State. It makes the second straight week that McDonagh has set a career high. He passed for 308 yards in a narrow loss to Cal, a performance that earned the sophomore Big Sky Offensive Player of the Week honors. The Vikings' (2-1) potent offense did not kick in until the second half, breaking open a tight 7-6 contest with three TDs and a safety in the third quarter alone. Portland State finished with 622 total yards. The Vikings lead the FCS in total offence, averaging over 612 yards, after their first two games. Humboldt State (0-2) stayed close in the first half on Matt Bruder field goals of 32 and 28 yards less than a minute apart in the second quarter. Portland State's Kasey Closs caught 10 passes for a career-best 205 yards.
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ISON was discovered by a pair of amateur Russian astronomers and named after the telescope array (the International Scientific Optical Network) that they used. It should achieve perihelion (the point in its orbit at which it is nearest to the Sun) on the 28th of November, and may become the brightest comet of the century.
NASA, ESA, and the Hubble Heritage Team (STScI/AURA)
© independent.co.uk


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In present day Japan, the life of a school girl is never dull. The easily-bored Konata never finds time to study because of her otaku habits, which frustrates hard-working Kagami to no end. On the other hand, laid-back Tsukasa always manages to go with the flow, while Miyuki is concerned with keeping her status as resident know-it-all. Join these four girls as they muse and meander their way through everyday events such as eating chocolate cones, doing homework, gaming, and trips to the beach galore.
68 votes
29 votes
25 votes
11 votes
Okay, this is actually my first time reviewing this anime show called Lucky Star. Before I started to review it, theres actually a lot of reviews about it. Some have positive, some have negative reviews. I'm going to be one of the positive reviewers for this one. When I first saw Lucky Star, it has a fun sort of talk conversation including food, culture, video games, anime, and etc. There's also anime parodies... read more
Let me put it this way, this is anime does NOT have a storyline. It's mainly about 4 high school girls always trying to enjoy their lives and get their last two years of high school done. The anime is pretty random sometimes and it also has humor thrown in too so you might get a good laugh every now and then, it's better than having boring parts but this anime doesn't however, you might hear a lot of... read more
|Lucky Star OVA
|Same Franchise
|Miyakawa-ke no Kuufuku
|Lucky Star
|Lucky Star: Comic a la Carte
|Lucky Star: Pocket Travelers
|Miyakawa-ke no Kuufuku
Character Design
Original Manga Creator