Cell width dictates Type VI secretion tail length

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Antibacterial Weapons: Targeted Destruction in the Microbiota

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Structure and Activity of the Type VI Secretion System

International audienceThe type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells. The injection apparatus is composed of a baseplate on which is built a contractile tail tube/sheath complex. The inner tube, topped by the spike complex, is propelled outside of the cell by the contraction of the sheath. The injection system is anchored to the cell envelope and oriented towards the cell exterior by a trans-envelope complex. Effectors delivered by the T6SS are loaded within the inner tube or on the spike complex and can target prokaryotic and/or eukaryotic cells. Here we summarize the structure, assembly, and mechanism of action of the T6SS. We also review the function of effectors and their mode of recruitment and delivery

Cell width dictates Type VI secretion tail length

International audienceThe type VI secretion system (T6SS) is a multiprotein apparatus that injects protein effectors into target cells, hence playing a critical role in pathogenesis and in microbial communities [1, 2, 3, 4]. The T6SS belongs to the broad family of ontractile injection systems (CISs), such as Myoviridae bacteriophages and R-pyocins, that use a spring-like tail to propel a needle loaded with effectors [5, 6]. The T6SS tail comprises an assembly baseplate on which polymerizes a needle, made of stacked Hcp hexamers, tipped by the VgrG-PAAR spike complex and wrapped by the contractile sheath made of TssB and TssC [7, 8, 9, 10, 11, 12, 13]. The T6SS tail is anchored to the cell envelope by a membrane complex that also serves as channel for the passage of the needle upon sheath contraction [14, 15, 16]. In most CISs, the length of the tail sheath is invariable and is usually ensured by a dedicated protein called tape measure protein (TMP) [17, 18, 19, 20, 21, 22]. Here, we show that the length of the T6SS tail is constant in enteroaggregative Escherichia coli cells, suggesting that it is strictly controlled. By overproducing T6SS tail subunits, we demonstrate that component stoichiometry does not participate to the regulation of tail length. The observation of longer T6SS tails when the apparatus is relocalized at the cell pole further shows that tail length is not controlled by a TMP. Finally, we show that tail stops its elongation when in contact with the opposite membrane and thus that T6SS tail length is determined by the cell width

Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system

International audiencehe Type VI secretion system (T6SS) is an anti-bacterial weapon comprising a contractile tail anchored to the cell envelope by a membrane complex. The TssJ, TssL and TssM proteins assemble a 1.7-MDa channel complex that spans the cell envelope, including the peptidoglycan layer. The electron microscopy structure of the TssJLM complex revealed that it has a diameter of ∼ 18 nm in the periplasm, which is larger that the size of peptidoglycan pores (∼ 2 nm), hence questioning how the T6SS membrane complex crosses the peptidoglycan layer. Here, we report that the MltE housekeeping lytic transglycosylase (LTG) is required for T6SS assembly in enteroaggregative E. coli. Protein-protein interactions studies further demonstrated that MltE is recruited to the periplasmic domain of TssM. In addition, we show that TssM significantly stimulates MltE activity in vitro and that MltE is required for the late stages of T6SS membrane complex assembly. Collectively, our data provide the first example of domestication and activation of a LTG encoded within the core genome for the assembly of a secretion system

Transcriptional frameshifting rescues Citrobacter rodentium Type VI secretion by the production of two length variants from the prematurely interrupted tssM gene

The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting

Chapter 12. Measure of peptidoglycan hydrolase activity Running head: peptidoglycan remodelling enzymes

International audienceMost of the gene clusters encoding multiprotein complexes of the bacterial cell envelope, such as conjugation and secretion systems, Type IV pili and flagella, bear a gene encoding an enzyme with peptidoglycan hydrolase activity. These enzymes are usually glycoside hydrolases that cleave the glycan chains of the peptidoglycan. Their activities are spatially controlled to avoid cell lysis and to create localized rearrangement of the cell wall. This is assured by interaction with structural subunits of the apparatus. Here, we describe protocols to test the peptidoglycan hydrolase activity of these proteins in vitro and in solution

In vivo TssA proximity labeling reveals temporal interactions during Type VI secretion 1 biogenesis and TagA, a protein that stops and holds the sheath.

International audienceThe Type VI secretion system (T6SS) is a multiprotein weapon used by bacteria to destroy competitor cells. The T6SS contractile sheath wraps an effector-loaded syringe that is injected into the target cell. This tail structure assembles onto the baseplate that is docked to the membrane complex. In entero-aggregative Escherichia coli TssA plays a central role at each stage of the T6SS assembly pathway by stabilizing the baseplate and coordinating the polymerization of the tail. Here we adapted an assay based on APEX2-dependent biotinylation to identify the proximity partners of TssA in vivo. By using stage-blocking mutations, we define the temporal contacts of TssA during T6SS biogenesis. This proteomic mapping approach also revealed an additional partner of TssA, TagA. We show that TagA is a cytosolic protein tightly associated with the membrane. Analyses of sheath dynamics further demonstrate that TagA captures the distal end of the sheath to stop its polymerization and to maintain it under the extended conformation

Role and recruitment of the TagL peptidoglycan-binding protein during Type VI secretion system biogenesis

International audienceThe type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking

Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS