Control of actin turnover by a salmonella invasion protein.

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

Crystallization and preliminary X-ray diffraction studies of the oxygenating subunit of 3,6-diketocamphane monooxygenase from Pseudomonas putida.

Journal ArticleResearch Support, Non-U.S. Gov'tThe oxygenating constituent of the 3,6-diketocamphane monooxygenase isozyme from Pseudomonas putida NCIMB 10007 has been crystallized under two different conditions. Crystals were initially grown from polyethylene glycol (PEG) 8000 and sodium acetate using the vapour-phase diffusion method. The crystals were of orthorhombic P212121 space group, with cell dimensions a = 55.8, b = 94.5 and c = 163.7 A and diffracted to 2.8 A resolution. More recently, improved crystals, which diffracted beyond 2 A, have been grown from ammonium sulfate. These crystals also belong to the orthorhombic P212121 space group, with cell dimensions of a = 54.6, b = 93.2 and c = 154. 1 A. A full native data set to 2.5 A resolution has been collected from the ammonium sulfate grown crystals.BBSR

Deciphering interplay between Salmonella invasion effectors

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction

Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

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 EspFU, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspFU 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 EspFU are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspFU fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspFU. Whereas clustering of a single EspFU repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspFU derivatives promote actin assembly more efficiently. Moreover, the EspFU 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

Conservation of Salmonella Infection Mechanisms in Plants and Animals

Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection

Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

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 EspFU, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspFU 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 EspFU are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspFU fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspFU. Whereas clustering of a single EspFU repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspFU derivatives promote actin assembly more efficiently. Moreover, the EspFU 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

A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly

P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function

Characterization of Salmonella Type III Secretion Hyper-Activity Which Results in Biofilm-Like Cell Aggregation

We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Analysis of the Expression, Secretion and Translocation of the Salmonella enterica Type III Secretion System Effector SteA

Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1–10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella