Bacterial Avoidance of Innate Immune Detection

Pathogenic bacteria, such as enterohemorrhagic E. coli (EHEC), subvert their human hosts through the Type III secretion of ~40-60 effector proteins. We recently identified the Type III secreted effector protein E. coli secreted protein G (EspG) as a potent disruptor of the Golgi. Biochemical and crystallographic evidence revealed that EspG interacted specifically with GTP-bound ADP Ribosylation Factor 1 and 6 (Arf1 and Arf6). We hypothesize that EspG has evolved to bind ArfGTP and lock it in the constitutively active conformation which leads to Golgi fragmentation and disruption of vesicular secretion, and in turn, results in inhibition of innate immune signaling. Arf1 and Arf6 are signaling GTPases that recruit vesicular coat proteins, termed coatomer, to regulate endomembrane traffic. Therefore, it reasons that pathogenic bacteria evolved an effector like EspG to inhibit the general secretory pathway, which is responsible for cytokine signaling and innate immune responses. Our preliminary data show that EspG induces Golgi disruption and can bind to Arf1GTP proteoliposomes and protect Arf1 from GAP activity. These data strongly suggest that EspG targets a conserved eukaryotic regulatory and signaling pathway to disrupt normal cellular function during a bacterial infection. <p/>Therefore, in Aim 1, we intend to functionally reconstitute the virulence mechanism of EspG in vitro using a Golgi-mimetic liposome system. Vesicle coat assays will then be performed on synthetic Golgi membranes to determine if EspG biases coatomer recruitment or assembly. Extending this further, we will test if EspG alters vesicular budding and/or scission by performing Golgi vesiculation assays using isolated rabbit liver Golgi membranes and mouse brain cytosol. Because the EspG- and VirA-induced Golgi disruption pattern is strikingly similar to Golgi fragmentation during mitosis, we propose that pathogenic bacteria induce a mitotic-like Golgi phenotype in order to evade the innate immune response during infection. We will therefore perform Golgi stacking assays to differentiate mitotic Golgi fragmentation from EspG-induced Golgi disruption. <p/> In Aim 2, we propose to define innate immune signaling disruption caused by EspG and its homologue from Shigella, VirA. To test this, cytokine release assays will be carried out using epithelial cells either transfected with EspG or VirA as well as during infections with EHEC and Shigella. Although homologous to EspG, VirA from Shigella does not target the same mammalian host substrates, namely Arf1 and Arf6. Therefore, we will determine the host substrates of VirA. Elucidation of the molecular mechanism of the EspG/VirA effector protein family will represent a large step forward in understanding bacterial immune evasion and could have significant applications in development of therapeutic agents important for combating bacterial infectious diseases.