Mechanisms of type VI secretion system effector transport and toxicity

Abstract

The type VI secretion system (T6SS) is a protein export pathway that mediates competition between Gram-negative bacteria by facilitating the injection of toxic effector proteins from attacking cells into target cells. To function properly, many T6SSs require at least one protein that possesses a proline-alanine-alanine-arginine (PAAR) domain. These PAAR domains are often found within large, multi-domain effectors that possess additional N- and C-terminal extension domains whose function in type VI secretion is not well understood. The work described herein uncovers the function of these accessory domains across multiple PAAR-containing effectors. First, I demonstrated that thousands of PAAR effectors possess N-terminal transmembrane domains (TMDs) and that these effectors require a family of molecular chaperones for stability in the cell prior to their export by the T6SS. Our findings are corroborated by co-crystal structures of chaperones in complex with the TMDs of their cognate effectors, capturing the first high-resolution structural snapshots of T6SS chaperone-effector interactions. Second, I characterize a previously undescribed prePAAR effector named Tas1. My work shows that the C-terminus of Tas1 possesses a toxin domain that pyrophosphorylates ADP and ATP to synthesize the nucleotides adenosine penta- and tetraphosphate (hereafter referred to as (p)ppApp). Delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ADP and ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. These findings reveal a new mechanism of interbacterial antagonism, the first characterization of a (p)ppApp synthetase and the first demonstration of a role for (p)ppApp in bacterial physiology. TMD- and toxin-containing PAAR proteins constitute a large family of over 6,000 T6SS effectors found in Gram-negative bacteria. My work on these proteins has uncovered that different regions found within effectors have distinct roles in trafficking between bacterial cells and in the growth inhibition of the target cell.