Infections still account for over 25% of deaths worldwide despite the development and widespread use of antimicrobials. A common theme for viral, bacterial, parasitic and fungal infections is their need to gain entry into cells to establish infection. Diverse pathogens including HIV-1, E. coli, Plasmodium falciparum and Candida albicans all manipulate mammalian tetraspanins for cell invasion or intracellular trafficking. As the name suggests, tetraspanins span lipid bilayers four times and are thought to play a structural role, interacting laterally with other proteins and forming membrane microdomains. Characterization of precise function of tetraspanins has been notoriously difficult as a result of molecular redundancy and the lack of intrinsic catalytic activity-- their function is linked to other proteins in their role as lateral organizers of membrane proteins. Adding to the complexity is the fact that the same tetraspanin expressed in different cells provide unique location-dependent functions by associating with distinct partners. The tetraspanin CD82 has been best described in the context of cancer where levels of surface expression are inversely correlated with tumor metastasis. In dendritic cells, CD82 associates with Class II MHC and other components of the endocytic pathway. In order to understand better the role of CD82 in the immune system, we have made the following key observations that are the rationale for our proposed work: 1) Proteomic data indicates that CD82 may be associated with a number of TLRs 2) CpG DNA failed to induce TLR9-dependent, TNF-αsecretion from CD82-/- DCs but other TLR signaling pathways remained intact 3) CD82 is recruited to phagosomes containing fungal pathogens including Aspergillus fumigatus as determined by time-lapse imaging of a fluorescentlytagged version of CD82 expressed in primary DCs using a spinning-disk confocal microscope 4) CD82 is recruited to pathogen-containing phagosomes prior to acidification with its recruitment unaffected by inhibitors of lysosomal acidification. CD82 recruitment is coincident with the arrival of Class II MHC and occurs before CD63 5) Biochemical evidence indicates that class II MHC and CD82 are associated 6) CD82-/- DCs loaded with ovalbumin failed to stimulate antigen-specific T cells as well as their wild-type counterparts. We hypothesize that CD82 directly participates in forming the TLR9 signaling complex and directly organizes peptide-loaded class II MHC on the surface of DCs. We propose to: 1) Determine the role of CD82 in TLR9- mediated signaling in DCs 2) Define the role of CD82 in the immune response to fungal pathogens using CD82-/- mice 3) Investigate the contribution of CD82 to antigen processing and presentation. Knowledge gained regarding the mechanism of action of CD82 in DCs will be important in furthering our understanding of TLR9 signaling and of antigen processing and presentation, and could lead to novel treatments of invasive fungal infections caused by A. fumigatus.
One common feature in infections is the universal need of pathogens to enter cells to cause disease. Tetraspanins are a family of proteins that are used by microbes to gain entry into cells. This application seeks to understand the function of one such tetraspanin, CD82 in its direct role in the immune response to viral and fungal infections.
|Mansour, Michael K; Reedy, Jennifer L; Tam, Jenny M et al. (2014) Macrophage Cryptococcus interactions: an update. Curr Fungal Infect Rep 8:109-115|
|Kasper, Lydia; Seider, Katja; Gerwien, Franziska et al. (2014) Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages. PLoS One 9:e96015|
|Mansour, Michael K; Tam, Jenny M; Khan, Nida S et al. (2013) Dectin-1 activation controls maturation of *-1,3-glucan-containing phagosomes. J Biol Chem 288:16043-54|
|Grimm, Melissa J; Vethanayagam, R Robert; Almyroudis, Nikolaos G et al. (2013) Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice. J Immunol 190:4175-84|
|Tam, Jenny M; Mansour, Michael K; Khan, Nida S et al. (2012) Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity. Integr Biol (Camb) 4:220-7|
|Mansour, Michael K; Vyas, Jatin M; Levitz, Stuart M (2011) Dynamic virulence: real-time assessment of intracellular pathogenesis links Cryptococcus neoformans phenotype with clinical outcome. MBio 2:|
|Kasperkovitz, Pia V; Khan, Nida S; Tam, Jenny M et al. (2011) Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae. Infect Immun 79:4858-67|