Abstract
Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
Highlights
Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells
(b) LITESEC-act, a system that confers activation of T3SSdependent protein translocation by blue light illumination. Both systems rely on two interaction partners which we have engineered: (i) A membrane-bound anchor protein, which is a fusion between the N-terminal transmembrane helix (TMH) of a well-characterized transmembrane protein, Escherichia coli TatA, extended by two amino acids (Val-Leu) for more stable insertion in the inner membrane (IM), a Flag peptide for detection and spacing, and the larger domain of the respective optogenetic interaction switches, iLID or LOV2
SctQ, an essential and dynamic cytosolic component of the T3SS32, was genetically fused to one interaction domain of two optogenetic sequestration systems, the iLID and LOVTRAP systems[36,37,45], while the membrane-bound interaction domain was coexpressed in trans
Summary
Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. The core components of the injectisome, or type III secretion system (T3SS) are shared with the bacterial flagellum[3,4]. In this manuscript, T3SS refers to the virulence-associated T3SS. The injectisome is an essential virulence factor for many pathogenic Gram-negative bacteria, including Salmonella, Shigella, pathogenic Escherichia coli, and Yersinia[7]. It is usually assembled upon entry into a host organism, but remains inactive until contact to a host cell has been established. Secretion of effector proteins can be triggered in vivo by host cell contact or in vitro by low Ca2+ levels in the medium[10]
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