Solving the Puzzle: Connecting a Heterologous Agrobacterium tumefaciens T6SS Effector to a Pseudomonas aeruginosa Spike Complex

Abstract

The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic and prokaryotic target cells. Such T6SS spike consists of a needle-shaped trimer of VgrG proteins topped by a conical and sharp PAAR protein that facilitates puncturing of the target membrane. T6SS-delivered effector proteins can be either fused to one of the two spike proteins or interact with either in a highly specific manner. In Agrobacterium tumefaciens the T6SS effector Tde1 is targeted to its cognate VgrG1 protein. Here, we attempted to use a VgrG shuttle to deliver a heterologous T6SS effector by directing Tde1 onto a T6SS spike in Pseudomonas aeruginosa. For this, we designed chimeras between VgrG1 from A. tumefaciens and VgrG1a from P. aeruginosa and showed that modification of the spike protein hampered T6SS functionality in the presence of the Tde1 effector complex. We provide evidence suggesting that Tde1 specifically binds to the VgrG spike in the heterologous environment and propose that there are additional requirements to allow proper effector delivery and translocation. Our work sheds light on complex aspects of the molecular mechanisms of T6SS delivery and highlights some limitations on how effectors can be translocated using this nanomachine.

Bioinformatic Analysis of the Campylobacter jejuni Type VI Secretion System and Effector Prediction

“…The Gp5 C-terminal domain is commonly found in the bacteriophage T4 tail lysozyme protein Gp5 and VgrG proteins of bacteria, forming the membrane-puncturing β-helix structure of the spike proteins (Kanamaru et al, 2002;Pukatzki et al, 2007;El-Gebali et al, 2019). The C-termini Gp5 regions of some VgrG proteins may be also extended with catalytic domains (Hachani et al, 2011;Wettstadt et al, 2020). Furthermore, these extensions may also contribute to the recruitment of additional effectors (Flaugnatti et al, 2020).…”

“…The T6SS puncturing structure is composed by a VgrG trimer further sharpened with a capping PAAR domaincontaining protein tip (Wettstadt et al, 2020). To assess the prevalence of these "effector markers, " the amino acid sequences of representative proteins belonging to all classes of PAAR subgroups found in the superfamily CL21497 were aligned against a local protein database of C. jejuni genomes (Supplementary Data 1).…”

Activity, delivery, and diversity of Type VI secretion effectors

“…This information is crucial in order to design inhibitors that could block the T6SS‐based translocation of clinically important effectors. Moreover, first attempts to use the T6SS for delivery of heterologous proteins were successful but revealed some important limitations likely related to steric hindrances (Ho et al, 2017; Ma et al, 2009; Wettstadt et al, 2019; Wettstadt et al, 2020). Therefore, a better understanding of effector translocation at the molecular level could help harness T6SS for therapeutic interests, such as the injection of antibodies or antibiotics directly into the target cells.…”

Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase

“…Notably, effectors can be noncovalently attached for delivery (cargo effectors) or included as extended domains of the structural proteins, which are termed evolved or specialized effectors. Past attempts at delivering recombinant proteins using T6SSs have revealed key challenges ( 18 ) as well as some successes, which include delivery of β-lactamase into eukaryotic cells ( 19 – 21 ) and fusion of two exogenous effectors for secretion ( 21 , 22 ). Vibrio cholerae encodes one of the best-characterized T6SS, and the T6SS has been particularly well characterized in strain V52, which is a nonpandemic strain with a constitutively active T6SS ( 9 ).…”