Laure Journet

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

VgrG, Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors

The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into both prokaryotic and eukaryotic cells, therefore participating in interbacterial competition and virulence. The T6SS is functionally and structurally similar to the contractile bacteriophage cell puncturing device: the contraction of a sheath-like structure is believed to propel an inner tube terminated by a spike towards target cells, allowing the delivery of effectors. In this review, we summarize recent advances in the identification and characterization of T6SS effector proteins, highlighting the broad repertoire of enzymatic activities, and discuss recent findings relating to the secretion mechanisms.

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C‐terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original α/β fold, a P‐x‐L‐G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.

Type III secretion: a secretory pathway serving both motility and virulence (Review)

‘Type III secretion’ (T3S) refers to a secretion pathway that is common to the flagellae of eubacteria and the injectisomes of some Gram-negative bacteria. Flagellae are rotary nanomachines allowing motility but they contain a built-in secretion apparatus that exports their own distal components to the distal end of the growing structure where they polymerize. In some cases they have been shown to export non-flagellar proteins. Injectisomes are transkingdom communication apparatuses allowing bacteria docked at the surface of a eukaryotic cell membrane to inject effector proteins across the two bacterial membranes and the eukaryotic cell membrane. Both nanomachines share a similar basal body embedded in the two bacterial membranes, topped either by a hook and a filament or by a stiff short needle. Both appear to be assembled in the same fashion. They recognize their substrate by a loose N-terminal peptide signal and the help of individual chaperones of a new type.

Real Time Fluorescent Resonance Energy Transfer Visualization of Ferric Pyoverdine Uptake in Pseudomonas aeruginosa A ROLE FOR FERROUS IRON

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.

TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System

Background: The T6SS assembles from 13 proteins that form two sub-assemblies. Results: TssK is a cytoplasmic protein that interacts with Hcp, TssC, TssL, and TssA. Conclusion: The TssK complex is three-arm shaped and links the membrane and phage-like complexes in T6SS. Significance: The structural and functional characterization of TssK leads to a better understanding of T6SS assembly. 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.

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

Significance Gram-negative bacteria use the type VI secretion system (T6SS) to deliver effectors into adjacent cells. Salmonella Typhimurium is an enteric pathogen that causes disease in millions of individuals each year. Its ability to infect the mammalian gut is a key factor that contributes to its virulence and transmission to new hosts. However, many of the details on how Salmonella successfully colonizes the gut and persists among members of the gut microbiota remain to be deciphered. In this work, we provide evidence that Salmonella uses an antibacterial weapon, the type VI secretion system, to establish infection in the gut. In addition, our results suggest that S. Typhimurium selectively targets specific members of the microbiota to invade the gastrointestinal tract. 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.

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.

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems

Background: TssL is a core component of the T6SS and has homologue in the T4bSS. Results: The TssL cytoplasmic domain adopts a globular α-helical domain and forms dimers. Conclusion: Dimer formation involves the trans-membrane segment, but contacts mediated by the cytoplasmic domain are important for TssL function. Significance: The structural and functional characterization of TssL leads to a better understanding of T6SS and T4bSS assembly. 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 C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC

Type VI secretion systems (T6SS) are macromolecular complexes present in Gram‐negative bacteria. T6SS are structurally similar to the bacteriophage cell‐puncturing device and have been shown to mediate bacteria–host or bacteria–bacteria interactions. T6SS assemble from 13 to 20 proteins. In enteroaggregative Escherichia coli (EAEC), one of the subassemblies is composed of four proteins that form a trans‐envelope complex: the TssJ outer membrane lipoprotein, the peptidoglycan‐anchored inner membrane TagL protein, and two putative inner membrane proteins, TssL and TssM. In this study, we characterized the TssL protein of the EAEC Sci‐1 T6SS in terms of localization, topology, and function. TssL is a critical component of the T6SS, anchored to the inner membrane through a single transmembrane segment located at the extreme C‐terminus of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK.