Browsing by Subject "Phospholipids"
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Item The Assembly of Pathogenic Signaling Circuits by a Family of Bacterial Secreted Effector Proteins(2013-04-02) Orchard, Robert Charles 1987-; Hendrixson, David R.; Sperandio, Vanessa; Sternweis, Paul C.; Alto, NealBacterial type III secreted effector proteins facilitate Gram-negative bacterial replication, dissemination, and immune evasion in the infected host organism. While much attention has been focused on the cell inhibitory mechanisms of these virulence factors, there is emerging evidence that bacterial effectors exert direct control over host cellular behavior by assembling new signaling circuits from pre-existing regulatory modules. However, these mechanisms are poorly understood. In this work, we utilize the WxxxE family of effector proteins as a model system to understand how pathogens rewire host signaling cascades. These effectors share a core catalytic domain that functions as a guanine-nucleotide exchange factor (GEF) for Rho family GTPases. Using a structure to function approach, we uncover a GEF-GTPase pairing mechanism important for signaling fidelity and pathogenic diversity. Guided by these structural insights, we next wanted to know how E. coli, an extracellular pathogen, induces the polarization of host actin molecules. By using synthetic derivatives of the enteropathogenic E. coli GEF Map, we discover that Cdc42 GTPase activity cycles are controlled in space and time by Map’s interaction with F-actin. Mathematical modeling reveals how actin dynamics coupled to a Map-dependent positive feedback loop spontaneously polarizes Cdc42. By reconstituting the system, we further show how cells polarize in response to an extracellular spatial cue. These results demonstrate how pathogens gain systems level control over host signaling networks and suggest a new view of cellular polarity centered on the interaction between GEFs and F-actin. To explore alternative mechanisms that bacteria utilize to assemble circuits, we utilize yeast genetics to identify novel membrane-interactions. We identify for the first time the direct association of the Shigella GEF IpgB1 with acidic phospholipids. Surprisingly, we find that these protein-lipid interactions are not required for IpgB1’s known role in Shigella invasion. However, we do find that IpgB1’s interactions with eukaryotic membranes are essential for bacterial replication and persistence within host cells. Furthermore, we identify a pathogenic circuit that connects GTPase activity with phospholipid metabolism. In summation, our findings illustrate the complex evolutionary relationship between pathogen and host, and how investigating these interactions provide insight into endogenous signaling systems.Item Phospholipid fatty liver: a tale of four drugs(1985-02-21) Combes, BurtonItem Studies on Plasma Membrane Proteins Involved in Membrane Traffic: Syntaxins and E-Syts(2007-05-22) Min, Sang-Won; Südhof, Thomas C.Fusion of synaptic vesicles is catalyzed by SNARE complex assembly which requires that the SNARE proteins syntaxin-1A and -1B, two isoforms of syntaxin-1, switch from a 'closed' to an 'open' conformation. To test the physiological significance of this switch, I analyzed mutant mice with a point mutation in syntaxin-1B which renders it predominantly 'open' in the syntaxin-1A null background. Whereas deletion of syntaxin-1A caused no detectable phenotype, opening of syntaxin-1B produced lethal epilepsy, independent of the presence of syntaxin-1A. Morphological and electrophysiological analyses revealed that opening of syntaxin-1B impaired steps in exocytosis upstream of vesicle priming, but enhanced Ca2+Item A Systems Biology Approach to Study Type III and Type IV Bacterial Effector Properties(2014-07-23) Weigele, Bethany Auten; Shiloh, Michael; Alto, Neal; Yan, Nan; Goodman, Joel M.The interaction between Type III and Type IV bacterial effector proteins and host signal transduction enzymes is a critical interface that, in many cases, determines the outcome of infectious disease. While many pathogenic strategies, such as evasion of phagolysosomal fusion, have been identified as necessary for microbial survival within the host, the effectors responsible are still largely unknown. Since most Gram-negative bacterial pathogens secrete between 5 to 300 effector proteins, a "systems biology" approach offers an enormous discovery potential. To approach the problem of effector protein biology from a global perspective, I first developed a comprehensive library of Type III and Type IV effector proteins (from six diverse pathogens) and assayed this library of effectors for their ability to associate with eukaryotic membranes. Unexpectedly, 30% of the virulence factor repertoire exhibited transmembrane-spanning domains, fatty-acid acceptor sites, peripheral membrane-binding properties, and/or cryptic phospholipid-targeting motifs. From a global analysis of phospholipid-binding mechanisms and from specific studies on the Shigella flexneri invasion program, a membrane-dependent autocatalytic feedback loop that regulates bacterial effector protein functions in space and time was identified. Additionally, new tools to further understand the potential role(s) of bacteria effector molecules in usurping the tightly regulated endocytic trafficking pathway were developed. These tools were then used to identify and characterize the location at which three bacterial effectors EspG, VirA, and IpaJ disrupt the global secretory pathway. Lastly, the effector library was utilized in a bioinformatics approach to identify bacterial effectors from a newly sequenced pathogen found to encode a Type III secretion system. Exploiting previous knowledge of homologous characterized effectors within the library, the first bacterial effectors from the pathogen, Providencia alcalifaciens were identified. Taken together, these findings suggest that the evolution of bacterial membrane binding motifs promote higher-order signaling functions in host cells and provide a resource for further interrogation of these virulence properties across a broad range of bacterial pathogens.