Western blots were developed according to the manufacturer’s instructions using the Hybond ECL kit (AmershamPharmacia Biotech)
Western blots were developed according to the manufacturer’s instructions using the Hybond ECL kit (AmershamPharmacia Biotech). C. although did not account for the reduced virulence of mutants in a mouse model of systemic infection. All these data support a role for the Cek1?mediated pathway in fungal cell wall maintenance, virulence and antifungal discovery. is an opportunistic fungus that forms part of the human microbiota. Upon alteration of the host defenses, this microbe may disseminate within the human body and gain access to internal organs causing severe infections. Although several virulence factors have been identified in the last years,1 cell wall components are considered to be most relevant: their external location makes them essential in processes such as adhesion, colonization and immune recognition, playing therefore a major role during infection.2 The cell wall is a complex KRAS G12C inhibitor 13 dynamic structure based on a core assembly of ?1,3?glucan (covalently linked to ?1,6?glucan) and chitin and an outer layer of mannose?glycosylated proteins.3,4 Chitin, a polymer of ?1,4?linked N?acetylglucosamine, is bound to a network of glucan which is usually masked by the cell wall outer layer and is only exposed due to certain cell wall alterations.5 The outer layer KRAS G12C inhibitor 13 of the fungal cell wall is mainly composed of mannoproteins and phosphopeptidomannan (PPM), which is a polymer of O?linked mannoses (?1,2 type or ?1,2/?1,3 type depending on the fungus) and N?linked mannoses (an inner core elongated by ?1,6 linear chain with branches of ?1,2 and ?1,3 mannose). PPM and mannoproteins carry ? and ?1,2?mannosides; however, while and ?linked mannosides are rather similar, the ?1,2?type of linkage is specific to the pathogenic yeast, contributing to its virulence and immunomodulatory responses.6-8 ?1,2?mannosides (?M) are also a main component of Mouse monoclonal to CD45 the glycosphingolipid phospholipomannan (PLM) which is thought to be distributed through the cell wall, both in the inner and outer layer.9 Since the fungal cell wall is the most external structure of the cell, it is at the interface between the host and the infective microbe, constituting the main source of KRAS G12C inhibitor 13 pathogen-associated molecular patterns (PAMPs). These structures are recognized by the pattern recognition receptors (PRRs) from immune cells, mediating microbial uptake and killing, and modulating the immune response.10 Toll?like receptors (TLRs) have been identified as a major class of PRRs involved in the recognition of microbial structures, being TLR2 and TLR4, which recognize PLMs and O?linked mannans respectively, the main TLRs involved in the signaling induced by opsonisation and uptake.17 Finally, galectin?3, a member of a ?galactoside?binding protein family, contributes to the recognition of by macrophages in cooperation with TLR2 and Dectin?1,7,18 a C?type lectin that binds exposed ?1,3?glucan.19 Galectin?3 is highly produced and secreted by macrophages and is also expressed in dendritic cells (DCs), activated lymphocytes and epithelial cells.6,20 It participates in a variety of cellular processes through either intracellular or extracellular mechanisms.21 Intracellular galectin?3 regulates cell survival, pre?mRNA splicing and phagocytosis22 while extracellular galectin?3 modulates cell adhesion, activation and migration23 and it has been shown to possess a direct fungicidal activity against through recognition of ?M.24 Adaptation to a changing environment and coping with host defenses is vital for pathogen survival. MAPK?mediated signal transduction pathways are mechanisms that organisms employ for this purpose. In mutant is also associated to a reduced virulence in the mouse model for systemic candidiasis although KRAS G12C inhibitor 13 it doesnt show hypersensitivity to macrophages or neutrophil-mediated killing.44,45 In this study we have characterized the cell wall of mutants and their response to drugs that interfere with cell wall assembly. We demonstrate here that cell wall ?1,2 and ?1,2?mannosides are differentially exposed in the surface of mutants (as well as in mutants) and that this altered cell wall structure influences recognition by host immune cells. We also address the role of galectin?3 in mediating the recognition of and its possible implication during the infection by cells. Results KRAS G12C inhibitor 13 Transcriptional analysis reveals differentially expressed cell wall-related genes in cek1 mutants Previous.