Antibacterial effectors of the type VI secretion system

Abstract

With the advent of high-throughput culture-independent sequencing it has become increasingly apparent that bacteria often live in complex communities, both in the environment and in association with the human body. Moreover, in polymicrobial settings there is often fierce competition for both space and resources, the results of which can have drastic effects on community composition. The evolutionary pressure exerted by competition is reflected by the significant portion of the coding capacity of many bacterial genomes dedicated to the production and regulation of antagonistic pathways. One such pathway, the type VI secretion system (T6SS), has emerged as a mechanism mediating the delivery of potent antibacterial effectors between contacting Gram-negative bacteria, granting the attacking organism a fitness benefit over sensitive neighbors. Initial studies of interbacterial T6S provided evidence that this system plays an important role in antagonism, however its mechanism of action on recipient cells remained elusive. This thesis describes the first biochemical characterization of antibacterial T6SS effector proteins, finding that they compromise basal features of bacterial physiology such as cell envelope integrity. By targeting highly conserved processes the T6SS has the capacity to mediate antagonism between highly disparate organisms. The cost of such versatility is that bacteria are susceptible to their own T6SS effectors. In order to overcome this hurdle antibacterial effectors are invariably associated with cognate immunity proteins that prevent their toxicity, protecting the producing cell. Beyond initial biochemical characterization of antibacterial effector proteins, this work describes informatic efforts to identify substrates secreted by the interbacterial T6SS throughout sequenced bacterial genomes. This strategy uncovered highly divergent effector sequences comprising distinct families within superfamilies defined by a common enzymatic target. These divergent effectors not only exhibit distinct substrate specificities, but also vary in their capacity to be neutralized by different sets of immunity proteins. Lastly, our informatic efforts led to the discovery of antibacterial effector proteins in a phylum of Gram-negative bacteria not predicted to encode a T6SS, the Bacteroidetes. My work on this abundant environmental and human-associated phylum of bacteria has found that they possess a highly divergent T6S-like pathway, one that, like its Proteobacterial homolog, takes part in interbacterial antagonism. Together, these findings represent a significant advancement in the field of interbacterial T6S, and serve as a platform for further work defining the in situ benefit of this antagonistic pathway.