A Systems Biology Approach to Study Type III and Type IV Bacterial Effector Properties

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.