This dissertation summarizes advances made toward understanding of the composition, structure, mechanism, and regulation of the bacterial type VI secretion system (T6SS). The T6SS is a widely conserved bacterial nanomachine used by Gram-negative bacteria to deliver toxic effector proteins into the extracellular environment or into adjacent target cells. Systematic deletion of open reading frames present in the Vibrio cholerae T6SS gene cluster revealed the genes essential for T6SS activity and provided key insights into understanding the mechanism by which this organelle is assembled and its components are recycled. Characterization of one phage-related T6SS component yielded insight into the mechanism by which many effectors associate with the T6SS organelle and are delivered into target cells. This T6SS component serves both to sharpen the tip of the membrane-puncturing T6SS spike complex and as a vehicle for attaching a diverse set of effector proteins. Time-lapse fluorescence microscopy of GFP-labeled T6SS components revealed key insights into the behavior and regulation of the T6SS in Pseudomonas aeruginosa. The T6SS in this organism assembled in response to exogenous T6SS attack by adjacent sister cells as well as heterologous T6SS+ species V. cholerae and Acinetobacter baylyi. This retaliatory T6SS counterattack was precisely aimed and caused no collateral damage to neighboring, non-aggressive bacteria. These counterattacks are mediated by phosphorylation cascade that recognizes exogenous attacks and post-translationally activates the T6SS in P. aeruginosa. Deletion of genes in this pathway eliminated the retaliatory response while retaining T6SS functionality. This pathway also induced T6SS counterattacks in response to mating pair formation associated with type IV secretion system (T4SS)-mediated DNA conjugation as well as treatment with membrane-disrupting natural product polymyxin B, suggesting that the signal needed to induce T6SS activity was mechanical perturbation of the P. aeruginosa cell membrane. Interestingly, these T4SS-induced counterattacks were able to confer resistance to the acquisition of horizontally transferred foreign DNA by selectively killing conjugative donor cells. As such, the T6SS of P. aeruginosa may represent a type of general bacterial innate immune system capable of responding to a wide range of exogenous threats.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11745715 |
| Date | 25 February 2014 |
| Creators | Ho, Brian Thomas |
| Contributors | Mekalanos, John J. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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