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VopE of Vibrio cholerae mediates mitochondrial dynamics by the Miro GTPase


Success of a bacterial pathogen depends on its ability to avoid recognition by the host innate immune system. A plethora of conserved pathogen-associated molecular patterns are recognized by host receptors including nucleic acids, lipoproteins, flagellin, lipopolysaccharides, and peptidoglycans. To counteract recognition of structures conserved in bacteria that, in theory, should be sufficient for the induction of antibacterial mechanisms and clearance of any pathogen, Gram-negative bacteria evolved strategies that rely on a use of species-specific effectors that target induction of the innate immune response. Delivery of such effectors by bacteria requires complex nanomachineries such as Type III Secretion System (T3SS). Our group showed that virulence of non-O1, non-O139 Vibrio cholerae strain AM-19226 that lacks cholera toxin and toxin-coregulated pilus relies on the activity of T3SS. We showed that at least 4 different effectors, including VopE, are required for Epithelial surface disruption and diarrheal response. However, targets and mechanisms of these effectors are not fully characterized. In this proposal I aim to identify molecular mechanisms that govern avoidance of innate immune responses by V. cholerae AM-19226. Our preliminary data showed that the effector VopE is targeted to Mitochondria during infection. Furthermore, mitochondrial morphology was drastically changed in cells infected with the VopE mutant in a T3SS-dependent manner, which was apparent by a significant increase in perinuclear mitochondrial clustering compared to the wild-type-infected cells. Modulation of mitochondrial dynamics was due to a specific interaction between VopE and mitochondrial Rho GTPases Miro-1 and Miro-2, and was required for clustering of the mitochondrial outer membrane protein MAVS and inhibition of type I interferon signaling. These data indicate that VopE activity is required for inhibition of a conserved pathogen-recognition pathway that is essential for recognition of V. cholerae AM-19226. In Aim 1, I will identify bacterial effectors tht induce clustering of Mitochondria in the absence VopE and will determine mechanism(s) of their activity utilizing microscopy, biochemistry, and cell biology approaches. In Aim 2, I will determine the mechanism by which Miro-1/2 induces clustering of MAVS using a combination of live-cell microscopy and knockdown experiments. My hypothesis is that Miro-1/2 induces clustering of MAVS through interaction with MAVS-regulating proteins such as Mfn-2, a protein essential for mitochondrial fusion that is known to interact both with Miro and MAVS. In Aim 3, I will determine the cytokines that are released in response to V. cholerae, and will define the importance of the innate immune response during infection. These findings will define key signaling events that V. cholerae subvert to avoid host recognition and induction of the innate immune response, and can have a large impact on subsequent development of novel antibacterial and anti-inflammatory agents.

Danilchanka, Olga V
Harvard University Medical School
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