Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike.

The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG)3 -(Tle1)3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.

Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3/Tli3 T6SS Effector/Immunity Complex Determined Using an AlphaFold2 Predicted Model.

The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3-an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3AIEC has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3AIEC. Tli3AIEC binds Tle3 in a 1:1 stoichiometric ratio. Tle3AIEC, Tli3AIEC and the Tle3AIEC-Tli3AIEC complex were purified and subjected to crystallization. The Tle3AIEC-Tli3AIEC complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3AIEC exhibits an α/ß-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3AIEC displays a new fold of three stacked ß-sheets and a protruding loop that inserts in Tle3AIECcatalytic crevice. We showed, experimentally, that Tle3AIEC interacts with the VgrG AIEC cargo protein and AlphaFold2 prediction of the VgrGAIEC-Tle3AIEC complex reveals a strong interaction between the VgrGAIEC C-terminus adaptor and Tle3AIEC N-terminal loop.

Activity, delivery, and diversity of Type VI secretion effectors.

The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery.

Priming and polymerization of a bacterial contractile tail structure.

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

TssA: The cap protein of the Type VI secretion system tail.

The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.

The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.

Architecture and assembly of the Type VI secretion system.

The Type VI secretion system (T6SS) delivers protein effectors to diverse cell types including prokaryotic and eukaryotic cells, therefore it participates in inter-bacterial competition and pathogenesis. The T6SS is constituted of an envelope-spanning complex anchoring a cytoplasmic tubular edifice. This tubular structure is evolutionarily, functionally and structurally related to the tail of contractile phages. It is composed of an inner tube tipped by a spike complex, and engulfed within a sheath-like structure. This structure assembles onto a platform called "baseplate" that is connected to the membrane sub-complex. The T6SS functions as a nano-crossbow: upon contraction of the sheath, the inner tube is propelled towards the target cell, allowing effector delivery. This review focuses on the architecture and biogenesis of this fascinating secretion machine, highlighting recent advances regarding the assembly of the membrane or tail complexes. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

TssK is a trimeric cytoplasmic protein interacting with components of both phage-like and membrane anchoring complexes of the type VI secretion system.

The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.

The type VI secretion system of Stenotrophomonas rhizophila CFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings.

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild-type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS-deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris.

Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems.

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families.

The tip protein PAAR is required for the function of the type VI secretion system.

IMPORTANCE: The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

Mechanism of ferripyoverdine uptake by Pseudomonas aeruginosa outer membrane transporter FpvA: no diffusion channel formed at any time during ferrisiderophore uptake.

To get access to iron, Pseudomonas aeruginosa produces the siderophore pyoverdine (PVD), composed of a fluorescent chromophore linked to an octapeptide, and its corresponding outer membrane transporter FpvA. This transporter is composed of three domains: a ß-barrel inserted into the membrane, a plug that closes the channel formed by the barrel, and a signaling domain in the periplasm. The plug and the signaling domain are separated by a sequence of five residues called the TonB box, which is necessary for the interaction of FpvA with the inner membrane TonB protein. Genetic deletion of the plug domain resulted in the presence of a ß-barrel in the outer membrane unable to bind and transport PVD-Fe. Expression of the soluble plug domain with the TonB box inhibited PVD-(55)Fe uptake most likely through interaction with TonB in the periplasm. A reconstituted FpvA in the bacterial outer membrane was obtained by the coexpression of separately encoded plug and ß-barrel domains, each endowed with a signal sequence and a signaling domain. This resulted in polypeptide complementation after secretion across the cytoplasmic membrane. The reconstituted FpvA bound PVD-Fe with the same affinity as wild-type FpvA, indicating that the resulting transporter is correctly folded and reconstituted in the outer membrane. PVD-Fe uptake was TonB-dependent but 75% less efficient compared to wild-type FpvA. These data are consistent with a gated mechanism in which no open channel with a complete removal of the plug domain for PVD-Fe diffusion is formed in FpvA at any point during the uptake cycle.

The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome.

The length of the Yersinia injectisome needle is determined by the protein YscP, which could act as a molecular ruler. The analysis of the correlation between the size of YscP and the needle length in seven wild-type strains of Yersinia enterocolitica reinforced this hypothesis but hinted that the secondary structure of YscP might influence needle length. Hence, 11 variants of YscP(515) were generated by multiple Pro or Gly substitutions. The needle length changed in inverse function of the helical content, indicating that not only the number of residues but also their structure controls length. Taking the secondary motifs into account, Pro/Gly-variants were subjected to in silico modelling to simulate the extension of YscP upon needle growth. The calculated lengths when the helical content is preserved correlated strikingly with the measured needle length, with a constant difference of approximately 29 nm, which corresponds approximately to the size of the basal body. These data support the ruler model and show that the functional ruler has a helical structure.

Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation.

Pyoverdines, the main siderophores produced by fluorescent Pseudomonads, comprise a fluorescent dihydroxyquinoline chromophore attached to a strain-specific peptide. These molecules are thought to be synthesized as non-fluorescent precursor peptides that are then modified to give functional pyoverdines. Using the fluorescent properties of PVDI, the pyoverdine produced by Pseudomonas aeruginosa PAO1, we were able to show that PVDI was not present in the cytoplasm of the bacteria, but large amounts of a fluorescent PVDI precursor PVDIp were stored in the periplasm. Like PVDI, PVDIp is able to transport iron into P. aeruginosa cells. Mutation of genes encoding the periplasmic PvdN, PvdO and PvdP proteins prevented accumulation of PVDIp in the periplasm and secretion of PVDI into the growth medium, indicating that these three enzymes are involved in PVDI synthesis. Mutation of the gene encoding PvdQ resulted in the presence of fluorescent PVDI precursor in the periplasm and secretion of a functional fluorescent siderophore that had different isoelectric properties to PVDI, suggesting a role for PvdQ in the periplasmic maturation of PVDI. Mutation of the gene encoding the export ABC transporter PvdE prevented PVDI production and accumulation of PVDIp in the periplasm. These data are consistent with a model in which a PVDI precursor peptide is synthesized in the cytoplasm and exported to the periplasm by PvdE where siderophore maturation, including formation of the chromophore moiety, occurs in a process involving the PvdN, PvdO, PvdP and PvdQ proteins.

A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif.

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.

Cell Width Dictates Type VI Secretion Tail Length.

The 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 [1-4]. The T6SS belongs to the broad family of contractile 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 [7-13]. 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 [14-16]. In most CISs, the length of the tail sheath is invariable and is usually ensured by a dedicated protein called tape measure protein (TMP) [17-22]. 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.

Structure and Activity of the Type VI Secretion System.

The 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.

In vivo TssA proximity labelling during type VI secretion biogenesis reveals TagA as a protein that stops and holds the sheath.

The 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 enteroaggregative 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.

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity.

The type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer-membrane lipoprotein and the TssL and TssM inner-membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH). Here, we show that TssL TMH dimerization is required for the stability of the protein and for T6SS function. Using the TOXCAT assay and point mutations of the 23 residues of the TssL TMH, we identified Thr194 and Trp199 as necessary for TssL TMH dimerization. NMR hydrogen-deuterium exchange experiments demonstrated the existence of a dimer with the presence of Trp185 and Trp199 at the interface. A structural model based on molecular dynamic simulations shows that TssL TMH dimer formation involves π-π interactions resulting from the packing of the two Trp199 rings at the C-terminus and of the six aromatic rings of Tyr184, Trp185 and Trp188 at the N-terminus of the TMH.