| dc.description.abstract | The type III secretion system (T3SS) is a bacterial injection system expressed by many Gram-negative bacteria. During the last two decades, the repertoire of T3SS effectors has been greatly explored, and several mechanisms of these effectors have been discovered. The identified host targets of T3SS effectors are involved in different biological events including cytoskeleton rearrangement, cellular signaling, transcription and protein degradation. A/E (attaching and effacing) pathogens including EHEC (Enterohaemorrhaigic E. coli), EPEC (Enteropathogenic E. coli) and C. rodentium (Citrobacter rodentium), a pathogen of mice, inhibit NF-κB transcriptional activity by employing unidentified T3SS effectors. However, the identity of these effector(s) was unknown. In this thesis, my goals were to identify T3SS effectors from attaching and effacing (A/E) pathogens responsible for modulating NF-κB activation and reveal the working mechanism of these identified effectors. In the first project, NleH1 and NleH2, which share C-termini similarity with the S. flexneri T3SS effector OspG, were studied. OspG targets ubiquitin-conjugating enzyme UbcH5 to prevent IκBα degredation, which results in the inhibition of NF-κB activation. We discovered that both NleH1 and NleH2 interact with the N-terminus of ribosomal protein S3 (RPS3) after their translocation into host cells. RPS3 is a non-Rel NF-κB subunit which promotes the DNA binding affinity of NF-κB. We found that NleH1, but not NleH2, blocks the nuclear translocation of RPS3 stimulated by TNF and by bacterial infection. By this process, NleH1 selectively attenuates RPS3-mediated NF-κB dependent gene transcription. In addition, we discovered NleH proteins as Ser/Thr kinases and that kinase activity is critical for the effect of NleH1 on RPS3. By collaborating with Dr. Lenardo's group in National Institute of Allergy and Infectious Diseases (NIAID), we discovered that RPS3 is inducibly associated with and phosphorylated by IKKβ at serine 209 (S209) and that NleH1 efficiently blocks this phosphorylation. Moreover, by using a gnotobiotic pig model, we found piglets infected with wild-type E. coli O157:H7 exhibits diffuse and low intensity phospho-RPS3 staining. Surprisingly, although infection by ΔnleH1 EHEC causes mild diarrhea and displays significantly reduced bacterial colonization in piglets, this mutant becomes hypervirulent to the host, as infected piglets die more rapidly and develop systemic intoxication compared to infection with the wild-type strain. Therefore, our data suggests a complex role for NleH1 in mediating bacterial virulence in the host to maximize bacterial survival and growth. In the second project, we identified NleB, another T3SS effector known to target NF-κB activation, as an O-GlcNAc (O-linked N-acetylglucosamine) transferase. We found NleB directly interacts with host glycolytic protein GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and O-GlcNAcylates GAPDH during infection. GAPDH has many nonglycolytic roles and is involved in a broad range of biological events, such as transcription, cell signaling, membrane integrity and cell survivial. In our study, we demonstrated an essential role for GAPDH in NF-κB activation. We found that GAPDH serves as a co-activator of TRAF2 (TNF receptor-associated factor 2) and promotes TRAF2 polyubiquitination under stress conditions. Targeting the catalytic site C150 of GAPDH by a chemical inhibitor or by site-directed mutagenesis specifically impairs TNF-induced TRAF2 polyubiquitination and NF-κB activation. This function is unrelated to glycolysis, as targeting the rate-limiting glycolytic enzymes does not imipair TRAF2 activation and only leads to a moderate inhibition on NF-κB activation, which is likely due to an unrelated mechanism. Moreover, O-GlcNAcylated GAPDH fails to interact with TRAF2 resulting in the attenuation of TRAF2 polyubiquitination. Eliminating the O-GlcNAc transferase activity of NleB by mutating its catalytic sites or by deleting the nucleotide sugar-binding domain abolishes the effect of NleB on NF-κB activation and reduces bacteria colonization of mice. Taken together, our studies suggest an integral role of the metabolic protein GAPDH in the NF-κB signaling pathway and that the T3SS effector NleB O-GlcNAcylates GAPDH to prevent the participation of GAPDH in NF-κB signaling. | |
