Mechanisms Of Effector Loading Of The Type ? Secretion System In Vibrio Cholerae

The type VI secretion system(T6SS)is a lethal yet energetically costly weapon in Gramnegative bacteria.Through contraction of a long sheath,the T6 SS ejects a few copies of effectors accompanied by hundreds of structural carrier proteins per delivery.The few ejected effectors,however,dictate T6 SS functions.It remains elusive how t he T6 SS ensures effector loading and avoids futile ejection.Here,by systemically mutating the active sites of three V.cholerae effectors,Tse L,Vas X,and Vgr G3,this thesis shows that the physical presence but not their activities is crucial for T6 SS assembly.Here catalytic mutants of Tse L and Vgr G3 and truncated Vas X mutants were constructed.These mutations abolished the killing of the effector-cognate immunity mutants.The Vas X-mediated antimicrobial activity was solely dependent on the C-terminal colicin-domain.Removal of the colicin domain abolished Vas X secretion and reduced T6 SS assembly,while deletion of the colicin internal loop abolished its toxicity but had little effect on secretion and assembly.The triple effector-inactive mutant maintains an active T6 SS that is capable of delivering chimeric Vgr G,PAAR,and Tse L proteins fused with a cargo nuclease,indicating effector-activities are not required for T6 SS assembly nor penetration into the cytosol of recipient cells.Therefore,by recruiting effectors as critical components for T6 SS assembly,it represents an effective on-board checking mechanism that ensures effectors are loaded in place to prevent futile secretion.This thesis also demonstrates a detoxified secretion platform by inactivating native effector activities that could translocate engineered cargo proteins via multiple routes.