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ABSTRACT: A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C. albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C. albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C. albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C. albicans, such as secretion.
Fungal Genetics and Biology 04/2013; · 3.74 Impact Factor
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Mohlopheni J Marakalala,
Simon Vautier,
Joanna Potrykus,
Louise A Walker,
Kelly M Shepardson,
Alex Hopke, Hector M Mora-Montes,
Ann Kerrigan,
Mihai G Netea,
Graeme I Murray,
Donna M Maccallum,
Robert Wheeler,
Carol A Munro,
Neil A R Gow,
Robert A Cramer,
Alistair J P Brown,
Gordon D Brown
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ABSTRACT: The β-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.
PLoS Pathogens 04/2013; 9(4):e1003315. · 9.13 Impact Factor
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Héctor M Mora-Montes,
Steven Bates,
Mihai G Netea,
Luis Castillo,
Alexandra Brand,
Ed T Buurman,
Diana F Díaz-Jiménez,
Bart Jan Kullberg,
Alistair J P Brown,
Frank C Odds,
Neil A R Gow
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ABSTRACT: The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTDelta null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of beta-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.
Journal of Biological Chemistry 02/2010; 285(16):12087-95. · 4.77 Impact Factor
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ABSTRACT: The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, because a detailed characterization at the structural level is lacking. Antigen-presenting dendritic cells (DCs), strategically located at mucosal surfaces and in the skin, may play an important role in anti-Candida protective immunity. However, the contribution of the various Candida-associated molecular patterns and their counter-receptors to DC function remains unknown. Here, we demonstrate that two C-type lectins, DC-SIGN and the macrophage mannose receptor, specifically mediate C. albicans binding and internalization by human DCs. Moreover, by combining a range of C. albicans glycosylation mutants with receptor-specific blocking and cytokine production assays, we determined that N-linked mannan but not O-linked or phosphomannan is the fungal carbohydrate structure specifically recognized by both C-type lectins on human DCs and directly influences the production of the proinflammatory cytokine IL-6. Better insight in the carbohydrate recognition profile of C-type lectins will ultimately provide relevant information for the development of new drugs targeting specific fungal cell wall antigens.
Journal of Biological Chemistry 08/2008; 283(29):20590-9. · 4.77 Impact Factor
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Kevin M Dennehy,
Gerben Ferwerda,
Inês Faro-Trindade,
Elwira Pyz,
Janet A Willment,
Philip R Taylor,
Ann Kerrigan,
S Vicky Tsoni,
Siamon Gordon,
Friederike Meyer-Wentrup,
Gosse J Adema,
Bart-Jan Kullberg,
Edina Schweighoffer,
Victor Tybulewicz, Hector M Mora-Montes,
Neil A R Gow,
David L Williams,
Mihai G Netea,
Gordon D Brown
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ABSTRACT: Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
European Journal of Immunology 03/2008; 38(2):500-6. · 5.10 Impact Factor
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Kevin M. Dennehy,
Gerben Ferwerda,
Inês Faro-Trindade,
Elwira Pyż,
Janet A. Willment,
Philip R. Taylor,
Ann Kerrigan,
S. Vicky Tsoni,
Siamon Gordon,
Friederike Meyer-Wentrup,
Gosse J. Adema,
Bart-Jan Kullberg,
Edina Schweighoffer,
Victor Tybulewicz, Hector M. Mora-Montes,
Neil A. R. Gow,
David L. Williams,
Mihai G. Netea,
Gordon D. Brown Dr
[show abstract]
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ABSTRACT: Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1α and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
European Journal of Immunology 01/2008; 38(2):500 - 506. · 5.10 Impact Factor
