| Zinc is an essential trace clement, body need to keep cellular zinc concentration within bounds through altering the expression of ZNT and ZIP families of zinc transporters. ZIP11 is a novel gene of ZIP family, which has unknown function. Food-borne diseases in humans caused by salmonella remain a major public health concern worldwide and are commonly associated with the consumption of broiler chicken meat contaminated with salmonella. Hypozincemia is observed in E.coli infected broilers. E.coli infection caused to plasma zinc reduce and zinc is redistributed to liver. Numerous studies suggest that pathogenic microorganism infection may alter the zinc homeostasis of innate immune cells, such as neutrophile and macrophage. However, the influence and regulation mechanism of body zinc homeostasis in broiler under salmonella infection are large unknown, what's the role of zinc transporters during this process? How about the ZIP11? In order to understand and solve these questions, three experiments were conducted in this thesis.The first experiment investigates the functional characterization of ZIP 11 in zinc homeostasis by using wt mice and different cell lines. Bioinformatics analysis of the distribution and evolutionary relationships of different ZIP members in 25 rcpresentive cukarytic genomes from different cukaryotic taxa (mammals, vertebates, plants and protozoa el al) indicated that Zipl 1, the sole member of gufA subfamily, is an ancient ZIP family member that might have originated in early eukaryotic ancestors. The results of the tisssue expression of Zip11 showed that murine Zip11 mRNA is abundantly expressed in testis and the digestive system, including stomach, ileum, and cecum. To gain an appreciation of the role of Zip11 in zinc trafficking, we assessed the effect of Zip11 over expression in HEK293T cells and knockdown Zip11 in RAW264.7 cells by using siRNA technology. Analysis of cellular zinc contents and MT mRNA levels illustrated that Zip11 is a zinc importer. In mice supplemented with zinc, both mRNA and protein levels of Zip11 were slightly upregulated in certain tissues, including spleen and liver. The metal response element sequences (MREs) upstream of the first exon of Zip11 responded to elevated extracellular zinc concentrations, as assessed by luciferase reporter assays. Mutagenic analysis showed that several of MREs could regulate the Zipll promoter activity. Metal-responsive transcription factor-1 (MTF-1) was shown to be involved in this process. Collectively, these data suggest that Zip11 has unique protein sequence and structure features, its functions as a cellular zinc transporter, and its expression is at least partially regulated by zinc via hMTF-1 binding to MREs of the Zip11 promoter.The second experiment studies on the influence and regulation mechanism of body zinc homeostasis in broiler under salmonella challenge. A total 48 1-day-old Arbor Acres broilers were feeded 7 d, and then were randomly assigned to two groups:one is nonchallenged control group, the other is salmonella-challenged group. Post infection 1,3 and 7 d serum, liver, spleen, thymus, bursa of fabricius, duodenum, jejnum, ileum and cecum were collected for zinc concentration and zinc metabolism genes expression. Moreover, neutrophiles were obatained from the serum of broilers with or without slamonella challenge for calprotecin and zinc metabolism genes expression detection. In this study, Hypozincemia was observed in 7 d salmonella infected broiler. Salmonella infection resulted in plasma zinc reduction (P<0.05) and live zinc increase (P<0.05). In addition to, salmonella infection leaded to duodenum zinc decrease (P<0.01) and cecum and fabricius zinc increase (P<0.05). In order to limit salmonella to acquire zinc, host changed the systemic zinc homeostasis via limiting duodenum to absorb zinc and redistributing zinc into liver, bursa fabricius and cecum. Meanwhile, body mediated the zinc homeostasis of neutrophile and up-regulated S100A9 mRNA expression (P<0.01), which can chelate zinc in cecum lumen and limite salmonella to use zinc for surviving in host. Future study, we found that salmonella infection influences the systemic and immune cell's zinc homeostasis of broilers via altering zinc transporters mRNA expression. Zinc was redistributed into liver through up-regulation Zip14 and down-regulation ZNT1, ZNT4, ZNT5, ZNT6, ZNT8 and ZNT9 mRNA expression in liver in salmonella infected broilers (P<0.05). Meanwhile, to defense salmonella infection host limited zinc absorbtion in duodenum via reduction zinc importers such as Zip5, Zip10, Zip11, Zip12, Zip13 and Zip14(P<0.05). Moreover, Zip3, Zip8, Zip10 and Zip 14 mRNA were up-regulated in neutrophile (P<0.05). Collectively, salmonella infection greatly influences zinc homeostasis of broilers. In order to defense salmonella infection, host altered systemic and cell's zinc homeostasis through mediation zinc transporters gene expression, Zip11 was also involved.The last experiment conducts to study the role and mechanism of free zinc in the process of macrophage defenses salmonella infection. In this study, we used RAW264.7 macrophage cells as a model and altered the free zinc status of salmonella infected macrophages with zinc addition or chelation to investigate the impact of free zinc change on salmonella infetion and proliferation by using microscope, FCM, RT-PCR and WB. Meanwhile, studying the effect of free zinc altertion on NF-?B signal pathway, ROS and RNS production of host. In addition to, we verified the above results in MT1 knockout macrohages. We found free zinc to accumulate in infected macrophages and to impact on Salmonella infection. Chelation of the free zinc increased bacterial killing via enhancing reactive oxygen species (ROS) and reactive nitrogen species (RNS) production in infected macrophages. In contrast, additional free zinc decreased generation of ROS and RNS through inhibition of NF-?B p65 activation and caused better Salmonella survival in macrophages. Along with this, deletion of the key intracellular zinc-binding protein MT1 elevated free zinc concentration and reduced ROS an RNS production resulting in a better bacterial survival within the cell. Moreover, Salmonella-hosting macrophages displayed higher free zinc levels as compared with non-infected or cells which already killed Salmonella. This notion suggests that zinc accumulation may be a newly identified strategy of the pathogen to avoid NF-?B-mediated killing and foster intra-macrophage survival.In conclusion, ZIP 11 is a zinc importer, play an crucial role in zinc homeostasis. Meanwhile, zinc can regulate ZIP11 expression via MTF-1 banding to MRE sequence, which locates in Zip11 promoter. In salmonella infected broiler host denfense salmonella infection through regulation systemic and cellular zinc homeostasis. on the one hand host limits zinc duodenal absorption, on the other hand zinc is redistributed into live, bursa of fabricius and immune cells, such as neutrophile. Few zinc can be used by salmonella. In salmonella infected broiler low serum zinc may result in low free zinc in macrophage, which increases macrophage killing. However, we should note that salmonella can use free zinc to overcome host defense in macrophage. |
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