Quantitative Proteomic Analysis Of A Bacterial Pathogenic Protein MgtC Functions And Host Responses To SeV Infection

Studies on infection and immunity reveal mechanisms of pathogenesis and host immunity, which promotes the development of new therapeutics for infectious disease and improve the quality of life. Quantitative proteomics, through unbiased large-scale data and robust bioinformatics analysis, is a new approach for infection and immunity related study. Using proteomic tool to establish new theories is an important direction for future infection and immunity research.In the first section of this dissertation, we used the quantitative proteomics and biochemistry approaches to investigate the function of a pathogenic protein MgtC from Salmonella enterica serovar Typhimurium (S. Typhimurium). The importance of this study lies not only for that the S. Typhimurium infected mice is the animal model of human typhoid, but also for the fact the salmonella is a common human pathogen infecting human intestinal tract, and often serves as an experimental model for the studies of bacteria-host interactions. It has been known that Salmonella reduces its growth rate once entering macrophage cells in order to adapt to restricted nutrition and anoxic host cell environment. Yet it remains unclear how Salmonella regulates its energy metabolism rate. Our findings demonstrate that MgtC protein plays a key role in the adjustment of Salmonella energy metabolism.By quantitative proteomics analysis, we found that in the low Mg2+ culture condition, a condition commonly used to mimic the host cell invasion process, compare the wild type to MgtC knock-out Salmonella strain, the aerobic energy metabolism related proteins were down-regulated, while anaerobic respiration related proteins were up-regulated. Additionally, protein synthesis and lipid metabolism related proteins were all down-regulated while stress resistance related proteins were up-regulated. Further BN-PAGE based protein complex analysis found that MgtC may interact with components of two other protein complexes, the ATP synthase F1 subunit and the bacterioferritin protein complex. MgtC is involved in the stabilization of bacterioferritin protein complex. Further biochemistry test revealed that under the low Mg2+ culture condition, ATP concentration in wild type is significantly lower than that of the MgtC mutant strain. Overexpress of MgtC in wild type further reduced ATP concentration, and this even was true in E.Coli which did not have MgtC-like gene. Membrane potential test indicated that wild type Salmonella membrane potential was significantly lower than that of the MgtC mutant. When low concentration of an oxidative phosphorylation uncoupler, CCCP, were added into the culture medium, MgtC protein expression level was increased. Further study showed that MgtC mutant was more sensitive to CCCP toxicity than wild type strain, indicating MgtC is important for anaerobic survival. Based on our data, we conclude that MgtC plays important roles in modulating Salmonella energy metabolism by at least two different means:one by interacting with F1-ATPase to inhibit ATP production, and the other is by stabilizing the bacterioferritin protein complex to reduce bacterial inner iron ion concentration which will lead to the inhibition of aerobic respiration and the enhancement of anaerobic respiration. MgtC's roles in modulating energy metabolism is crucial for Salmonella to adapt to host cell environment.In the second section of this dissertation, we used the quantitative proteomics and biochemistry approaches to investigate the roles of peroxisome-related proteins in host response to viral infection. Previous study has indicated that an important adaptor of innate immune responses, VISA, initially reported as a mitochondrial protein, is also located in peroxisomes, and the peroxisomal and mitochondrial located VISA proteins may mediate different antiviral effects. We hypothesize that peroxisomes may serve as an important platform in antiviral innate immune signal transduction pathways, and performed quantitative proteomic analysis.Peroxisome-enriched fractions (PEFs) from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and their protein profiles were compared. We identified 311 proteins that are affected by SeV infection. Among these altered proteins, 25 are immune response-related proteins, which validates our approach. Further bioinformatics analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex (MAC)-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 proteins and identified CALU and LGALS3BP as negative regulators of the virus-induced activation of the type I interferon.