The Mechanism Study Of Macrophages Uptake Saccharomyces Cerevisiae Spores

Saccharomyces cerevisiae has been used to produce beneficial products to us and in recent years has been found that its derived glucan can enhance animal immunity function.Saccharomyces cerevisiae has two forms: vegetative yeast and four-spores packaged inside ascus.Yeast vegetative and spore wall structures are different: The outermost layer of spores is the dityrosine layer;the outer layer is the chitosan;the inner layer is glucan,and mannose being the most inner layer.The yeast vegetative cell wall constitutes the mannose glycoprotein outermost layer,the glucan inner layer,and chitin a the innermost layer.Saccharomyces cerevisiae derived glucan,zymosan,is a kind of alkali insoluble glucan that consists of beta-1,3 and beta-1,6 key linked glucan polymer,which can be recognized and internalized by the macrophage.Zymosan enhances the immune function,stimulate immune cells to produce cytokines,and also has an antiviral and an antitumor effect,as well as an antimicrobial role.Yeast chs3? mutants spore has chitosan layer defect and thus exposes the glucan layer,which can be used as glucan microparticles,of which its immunity function was tested in this study.And the dit1? mutant spore wall exposes chitosan layer which is also useful.Chitosan is composed of amino polysaccharide which can be used as an immune adjuvant,has good biocompatibility,has an antibacterial and anti-inflammatory effect and at the same time,it can also stimulate immune response.By using chs3? mutant spores as glucan and dit1? mutants as chitosan microparticles,compared with the wild type yeast spores or vegetative yeast effect on macrophages,we found that wild type spores,dit1?,chs3?,and vegetative yeast can stimulate macrophages to produce cytokine TNF-a,IL-12 without a significant difference.However,in this study,we found that yeast wild type spore phagocytosis efficiency is much higher than that of the dit1?and chs3? mutant spore or vegetative yeast.For this special phenomenon,we conducted intensive research.Macrophages recognize and uptake microbes depending on the the pattern recognition receptors(PRPs)recognized the molecules that the microbial related molecular pattern(MRMP).Thus,the efficiently uptake of yeast spores by macrophage is based on its unique spore wall molecular structure,as wild types yeast spores have a dityrosine layer,which does not exist on dit1?,chs3? spore wall and vegetative yeast wall.This study explores the ligands and identifies receptors and signaling pathways that are involved in phagocytosis of wild type yeast spores,based on the aspect of efficient internalization of wild-type spores.Once macrophage receptor recognizes ligands activation of the receptor and recruits tyrosine kinase,Syk to the receptors activated ITAM motif,it sequentially activates the downstream signal.But macrophages can also uptake exotic material by macropinocytosis pathway,and the difference between macropinocytosis and phagocytosis is that macropinocytosis is independent of PI3 K,and receptor-mediated phagocytosis relies on PI3 K kinase to reorganize actin to form the phagosome.The main contents of this study are as follows:(1)Actin polymerization is formed around yeast spores phagosome.Spores phagosome formation can be suppressed by Syk and PI3 K inhibitors,which suggests that yeast spores engulfed by macrophages are receptor mediated.(2)To explore the molecules on wild type spores that are recognized by macrophages though inhibited by sugars and polysaccharides,it was found that yeast-derived glucan only inhibits vegetative yeast phagocytosis without affecting the yeast spores internalization.This indicates that the macrophages recognition molecules of yeast spores and vegetative yeast are different.In this study,we determined that yeast spores containing dityrosine layer have much more higher phagocytosis efficiency compared with dityrosine defective spores.chs3? and osw2? ascue lysate,which both contain dityrosine compared with dit1? ascue lysate,can inhibit yeast spores phagocytosis,which showed that the dityrosine is required for yeast spores internalization.(3)Through purification of yeast spores and vegetative yeast phagosome,the spore phagosome enriched proteins was analyzed.Through a series of knockout of possible receptors,it was found that knockout macrophages MAC-1 receptor alpha chain(ITGAM)or beta chain(ITGB2)can lead to the spore internalization defect.(4)Through the mutation of small GTPase,which is enriched in spores phagosome,it was found that overexpressing RAP1 A continuous active mutation can increase phagocytosis efficiency of spores,not vegetative yeast,and overexpressing the RAP1 A dominant negative mutations in RAW264.7 can specifically inhibit yeast spores internalization.This is consistent with the previously reported results that RAP1 A can regulate MAC-1 induced phagocytosis pathway.All in all,this is the first detailed study of the phagocytosis mechanism of yeast spores in mouse macrophage which found that MAC-1 mediates yeast spores internalization,and RAP1 A regulate yeast spores phagocytosis.