Characterization of antagonistic interbacterial pathways at the single-cell level

Thesis (Ph.D.)--University of Washington, 2015Antagonistic pathways are nearly universal among free-living bacteria, and may determine which organisms co-exist in polymicrobial communities. Bacteria have also evolved pathways that can sense and respond to assaults from competitors. In this work, I characterize interbacterial interactions mediated by the type VI secretion system (T6SS), and describe the integration of this antagonistic factor into a global regulatory pathway that senses and responds to danger from bacterial competitors. I developed tools to investigate interbacterial antagonism at the single-cell level, and applied these tools to the characterization of interbacterial interactions mediated by the Hcp-secretion Island I T6SS (H1-T6SS) of Pseudomonas aeruginosa. This approach revealed that T6S-dependent interactions require direct cell–cell contact and that T6SS effectors induce recipient cell lysis. Furthermore, I discovered that H1-T6S-mediated intoxication is more likely to occur when the recipient has an active T6SS itself. The mechanistic underpinnings of this observation are two-fold. First, preferential localization of the apparatus towards cells with an active T6SS suggests that assembly of the apparatus is spatiotemporally coordinated between neighboring cells. Second, I found that H1-T6SS expression is induced when cells are in direct contact with a competitor – a form of innate immunity that we term danger sensing. P. aeruginosa senses antagonism by detecting lysed kin cells via the Gac/Rsm regulatory pathway. This global regulatory pathway stimulates increased expression of the T6SS, as well as other offensive and defensive factors that collectively promote fitness in co-culture with a competitor. Finally, I utilized the single-cell analysis tools to characterize the outcomes and mechanisms of a potential programmed cell-death pathway of P. aeruginosa. This pathway responds to DNA damage, and provides an advantage in an in vivo infection model. Utilizing a single-cell approach to study bacteria within communities has uncovered the environmental significance and ramifications of interbacterial pathways