ABSTRACT
The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.
Subject(s)
Bacterial Proteins/chemistry , Oxygenases/chemistry , Pseudomonas putida/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , FMN Reductase/metabolism , Flavin Mononucleotide/metabolism , Models, Molecular , Molecular Sequence Data , Oxygenases/genetics , Oxygenases/metabolism , Plasmids/genetics , Protein Conformation , Protein Folding , Pseudomonas putida/genetics , Pseudomonas putida/metabolism , Sequence AlignmentABSTRACT
Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.
Subject(s)
Bacterial Secretion Systems/physiology , Cytoskeleton/metabolism , Salmonella Infections/metabolism , Salmonella/metabolism , Salmonella/pathogenicity , Virulence Factors/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytoskeleton/genetics , Female , Flagella/genetics , Flagella/metabolism , Gene Deletion , Genomic Islands/physiology , Mice , Salmonella/genetics , Salmonella Infections/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Virulence Factors/geneticsABSTRACT
The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant WRC and activate it, but when both GTPases were bound at the membrane, recruitment and concomitant activation of WRC were dramatically enhanced. This cooperativity between the two GTPases was sufficient to induce WAVE-dependent bead motility in cell extracts. Our findings suggest that Arf GTPases may be central components in WAVE signalling, acting directly, alongside Rac1.
Subject(s)
ADP-Ribosylation Factor 1/physiology , Actins/chemistry , Wiskott-Aldrich Syndrome Protein Family/physiology , rac1 GTP-Binding Protein/physiology , Animals , Humans , Liposomes/chemistry , Signal TransductionABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) generate F-actin-rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspF(U), a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspF(U) repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspF(U) are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspF(U) fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspF(U). Whereas clustering of a single EspF(U) repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspF(U) derivatives promote actin assembly more efficiently. Moreover, the EspF(U) repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3-mediated signaling pathways.
Subject(s)
Actin Cytoskeleton/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Enterohemorrhagic Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Repetitive Sequences, Amino Acid , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Carrier Proteins/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Enterohemorrhagic Escherichia coli/genetics , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , SwineABSTRACT
A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].
ABSTRACT
Salmonella pathogenesis relies upon the delivery of over thirty specialised effector proteins into the host cell via two distinct type III secretion systems. These effectors act in concert to subvert the host cell cytoskeleton, signal transduction pathways, membrane trafficking and pro-inflammatory responses. This allows Salmonella to invade non-phagocytic epithelial cells, establish and maintain an intracellular replicative niche and, in some cases, disseminate to cause systemic disease. This review focuses on the actions of the effectors on their host cell targets during each stage of Salmonella infection.
Subject(s)
Bacterial Proteins/metabolism , Epithelial Cells/microbiology , Salmonella/pathogenicity , Virulence Factors/metabolism , Animals , HumansABSTRACT
A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.
Subject(s)
Bacterial Proteins/metabolism , Cholesterol/metabolism , Membrane Proteins/metabolism , 3T3 Cells/microbiology , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Membrane Microdomains , Mice , Microscopy, Electron, Transmission/methods , Protein Transport/physiology , Salmonella typhimurium/metabolism , Salmonella typhimurium/pathogenicity , Shigella flexneri/metabolism , Shigella flexneri/pathogenicity , Virulence/physiologyABSTRACT
Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.
Subject(s)
Actins/metabolism , Bacterial Proteins/metabolism , Salmonella/pathogenicity , Actin Depolymerizing Factors , Actins/chemistry , Actins/ultrastructure , Animals , Binding Sites , Cell Extracts , Cytoskeleton/metabolism , Destrin , Gelsolin/metabolism , Host-Parasite Interactions , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/metabolism , Models, Molecular , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , XenopusABSTRACT
Entry into non-phagocytic mammalian cells by the invasive pathogens Salmonella and Shigella is triggered by the delivery of bacterial virulence effector proteins into the host cell. This is dependent upon Salmonella SipB or its Shigella homologue IpaB, which insert into the eukaryotic cell plasma membrane. Here we show that a SipB-derived 166 residue alpha-helical polypeptide is a potent inhibitor of SipB-directed liposome fusion in vitro, preventing the membrane-associated form of SipB from inserting deeply into the bilayer. This polypeptide blocks Salmonella entry into cultured mammalian cells at 10(-10) M, and is a heterologous inhibitor of analogous IpaB activity and Shigella cell entry. These findings reveal a potential strategy to identify inhibitors of the 'trigger' mechanism underlying cell entry by these major invasive pathogens.
Subject(s)
Bacterial Proteins/metabolism , Eukaryotic Cells/microbiology , Membrane Proteins/metabolism , Peptides/metabolism , Salmonella/pathogenicity , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Membrane/metabolism , HeLa Cells , Humans , Liposomes/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Peptides/chemistry , Peptides/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Shigella/metabolism , Shigella/pathogenicity , Virulence/drug effectsABSTRACT
A critical early event in Salmonella infection is entry into intestinal epithelial cells. The Salmonella invasion protein SipB is required for the delivery of bacterial effector proteins into target eukaryotic cells, which subvert signal transduction pathways and cytoskeletal dynamics. SipB inserts into the host plasma membrane during infection, and the purified protein has membrane affinity and heterotypic membrane fusion activity in vitro. We used complementary biochemical and biophysical techniques to investigate the topology of purified SipB in a model membrane. We show that the 593 residue SipB is predominantly alpha-helical in aqueous solution, and that no significant change in secondary structural content accompanies lipid interaction. SipB contains two -helical transmembrane domains (residues 320-353 and 409-427), which insert deeply into the bilayer. Their integration allowed the hydrophilic region between the hydrophobic domains (354-408) to cross the bilayer. SipB membrane integration required both the hydrophobic domains and an additional helical C-terminal region (428-593). Further spectroscopic analysis of these domains in isolation showed that the hydrophobic regions insert obliquely into the bilayer, whereas the C-terminal domain associates with the bilayer surface, tilted parallel to the membrane. The combined data suggest a topological model for membrane-inserted SipB.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Lipid Bilayers/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Structure, Secondary , Salmonella/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Circular Dichroism , Humans , Light , Liposomes/chemistry , Liposomes/metabolism , Membrane Proteins/genetics , Molecular Probes/chemistry , Molecular Probes/metabolism , Molecular Sequence Data , Molecular Structure , Protein Structure, Tertiary , Spectroscopy, Fourier Transform InfraredABSTRACT
An essential early event in Shigella and Salmonella pathogenesis is invasion of non-phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full-length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N-terminal domain predicted to form a trimeric coiled-coil, and is predominantly alpha-helical. Upon lipid interaction, two transmembrane domains (residues 313-333 and 399-419) penetrate the bilayer, allowing the intervening hydrophilic region (334-398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB-derived 162 residue alpha-helical polypeptide (IpaB(418-580)) is a potent inhibitor of IpaB-directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10(-8) M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB(418-580) failed to prevent the contact-dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact-dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.