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Background: Bacterial virulence-associated type III secretion system (T3SS) assembly requires a dedicated enzyme to penetrate peptidoglycan (PG).Results: We structurally characterized a T3SS PG-lytic enzyme, identified catalytically important residues, and characterized its activity.Conclusion: The active site is similar to lysozymes and lytic transglycosylases and interaction with the T3SS enhances activity.Significance: Structural information is critical for development of drugs targeting T3SS PG-lytic enzymes.The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or “effector” proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the β-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.

Equipped with a plethora of secreted toxic effectors, protein secretion systems are essential for bacteria to interact with and manipulate their neighboring environment to survive in host microbiota and other highly competitive communities. While effectors have received spotlight attention in secretion system studies, many require accessory chaperone and adaptor proteins for proper folding/unfolding and stability throughout the secretion process. Here, we review the functions of chaperones and adaptors of three protein secretions systems, type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS), which are employed by many Gram‐negative bacterial pathogens to deliver toxins to bacterial, plant, and mammalian host cells through direct contact. Since chaperone and adaptor functions of the T3SS and the T4SS are relatively well studied, we discuss in detail the methods of chaperone‐facilitated effector secretion by the T6SS and highlight commonalities between the effector chaperone/adaptor proteins of these diverse secretion systems. While the chaperones and adaptors are generally referred to as accessory proteins as they are not directly involved in toxicities to target cells, they are nonetheless vital for the biological functions of the secretion systems. Future research on biochemical and structural properties of these chaperones will not only elucidate the mechanisms of chaperone‐effector binding and release process but also facilitate custom design of cargo effectors to be translocated by these widespread secretion systems for biotechnological applications. [ABSTRACT FROM AUTHOR]

Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe-host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn2+) under oxidative stress. Next, we identified a T6SS-4- dependent Mn2+-binding effector TseM, and its interacting partner MnoT, a Mn2+-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn2+ conditions. Both TseM and MnoT are required for efficient uptake of Mn2+ across the outermembrane underMn2+-limited and -oxidative stress conditions. The TseM-MnoT-mediated active Mn2+ transport system is also involved in contact-independent bacteria-bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria-bacteria competition. [ABSTRACT FROM AUTHOR]

During infection, enteropathogenic Escherichia coli assembles a complex multi-protein type III secretion system that traverses the bacterial membranes and targets the host cell membrane to directly deliver virulence or effector proteins to the host cytoplasm. As this secretion system is composed of more than 20 proteins, many of which form oligomeric associations, its assembly must be tightly regulated. A protein called the gatekeeper, or SepL, ensures that the secretion of the translocon component, which inserts into the host membrane, occurs before the secretion of effectors. The crystal structure of the gatekeeper SepL was determined and compared with the structures of SepL homologues from other bacterial pathogens in order to identify SepL residues that may be critical for its role in type III secretion-system assembly. [ABSTRACT FROM AUTHOR]

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688-698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase. [ABSTRACT FROM AUTHOR]

Type VI secretion systems (T6SSs) translocate effector proteins, such as Rhs toxins, to eukaryotic cells or prokaryotic competitors. All T6SS Rhs-type effectors characterized thus far contain a PAAR motif or a similar structure. Here, we describe a T6SS-dependent delivery mechanism for a subset of Rhs proteins that lack a PAAR motif. We show that the N-terminal Rhs domain of protein RhsP (or VP1517) from Vibrio parahaemolyticus inhibits the activity of the C-terminal DNase domain. Upon auto-proteolysis, the Rhs fragment remains inside the cells, and the C-terminal region interacts with PAAR2 and is secreted by T6SS2; therefore, RhsP acts as a pro-effector. Furthermore, we show that RhsP contributes to the control of certain “social cheaters” (opaR mutants). Genes encoding proteins with similar Rhs and PAAR-interacting domains, but diverse C-terminal regions, are widely distributed among Vibrio species. It is unclear how Rhs toxins lacking a PAAR motif are secreted by Type VI secretion systems. Here, the authors show for one of these proteins that the mechanism requires removal of an N-terminal fragment by auto-proteolysis, followed by interaction with a PAAR protein and then secretion. [ABSTRACT FROM AUTHOR]