Structural analysis of a specialized type III secretion system peptidoglycan-cleaving enzyme.

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 ß-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.

The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes.

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.

Assembly and structure of the T3SS.

The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding of T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Structure of Salmonella effector protein SopB N-terminal domain in complex with host Rho GTPase Cdc42.

SopB is a type III secreted Salmonella effector protein with phosphoinositide phosphatase activity and a distinct GTPase binding domain. The latter interacts with host Cdc42, an essential Rho GTPase that regulates critical events in eukaryotic cytoskeleton organization and membrane trafficking. Structural and biochemical analysis of the SopB GTPase binding domain in complex with Cdc42 shows for the first time that SopB structurally and functionally mimics a host guanine nucleotide dissociation inhibitor (GDI) by contacting key residues in the regulatory switch regions of Cdc42 and slowing Cdc42 nucleotide exchange.

Identification of Small Molecule Inhibitors of the Pathogen Box against Vibrio cholerae.

Antimicrobial resistance (AMR) has become a serious public and economic threat. The rate of bacteria acquiring AMR surpasses the rate of new antibiotics discovery, projecting more deadly AMR infections in the future. The Pathogen Box is an open-source library of drug-like compounds that can be screened for antibiotic activity. We have screened molecules of the Pathogen Box against Vibrio cholerae, the cholera-causing pathogen, and successfully identified two compounds, MMV687807 and MMV675968, that inhibit growth. RNA-seq analyses of V. cholerae after incubation with each compound revealed that both compounds affect cellular functions on multiple levels including carbon metabolism, iron homeostasis, and biofilm formation. In addition, whole-genome sequencing analysis of spontaneous resistance mutants identified an efflux system that confers resistance to MMV687807. We also identified that the dihydrofolate reductase is the likely target of MMV675968 suggesting it acts as an analog of trimethoprim but with a MIC 14-fold lower than trimethoprim in molar concentration. In summary, these two compounds that effectively inhibit V. cholerae and other bacteria may lead to the development of new antibiotics for better treatment of the cholera disease. IMPORTANCE Cholera is a serious infectious disease in tropical regions causing millions of infections annually. Vibrio cholerae, the causative agent of cholera, has gained multi-antibiotic resistance over the years, posing greater threat to public health and current treatment strategies. Here we report two compounds that effectively target the growth of V. cholerae and have the potential to control cholera infection.

A type VI secretion system effector delivery mechanism dependent on PAAR and a chaperone-co-chaperone complex.

The type VI secretion system (T6SS) is used by many Gram-negative bacteria as a molecular weapon to modulate neighbouring bacterial and eukaryotic cells, thereby affecting the dynamics of community structure in multiple species environments. The T6SS injects its inner-needle Hcp tube, the sharpening tip complex consisting of VgrG and PAAR, and toxic effectors into neighbouring cells. Its functions are largely determined by the activities of its delivered effectors. Six mechanisms of effector delivery have been described: two mediated by the inner tube and the others mediated by the VgrG and PAAR tip complex. Here, we report an additional effector delivery mechanism that relies on interaction with a chaperone complex and a PAAR protein as a carrier. The Pseudomonas aeruginosa PAO1 TOX-REase-5 domain-containing effector TseT directly interacts with PAAR4 and the chaperone TecT for delivery, and an immunity protein, TsiT, for protection from its toxicity. TecT forms a complex with its co-chaperone, co-TecT, which is disrupted by the carboxy-terminal tail of PAAR4. In addition, we delineate a complex, multilayered competitive process that dictates effector trafficking. PAAR delivery provides an additional tool for engineering cargo protein translocation.

Structural analysis of SepL, an enteropathogenic Escherichia coli type III secretion-system gatekeeper protein.

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.