Structure-function analysis of the curli accessory protein CsgE defines surfaces essential for coordinating amyloid fiber formation

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis

Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN

The inflammatory response is one of several host alert mechanisms that recruit neutrophils from the circulation to the area of infection. We demonstrate that Bordetella, a bacterial pathogen, exploits an antiinflammatory cytokine, interleukin-10 (IL-10), to evade the host immune system. We identified a Bordetella effector, BopN, that is translocated into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Interestingly, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Using pharmacological blockade, we demonstrated that BopN-induced IL-10 production is mediated, at least in part, by its ability to block the extracellular signal-regulated kinase pathway. We also showed that BopN blocks nuclear translocation of nuclear factor κB p65 (NF-κBp65) but, in contrast, promotes nuclear translocation of NF-κBp50. A BopN-deficient strain was unable to induce IL-10 production in mice, resulting in the elimination of bacteria via neutrophil infiltration into the pulmonary alveoli. Furthermore, IL-10–deficient mice effectively eliminated wild-type as well as BopN mutant bacteria. Thus, Bordetella exploits BopN as a stealth strategy to shut off the host inflammatory reaction. These results explain the ability of Bordetella species to avoid induction of the inflammatory response

Correction: BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0148387.]

BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages.

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis

<i>B</i>. <i>bronchiseptica</i> infection with cytochalasin D treatment.

<p>A. L2 cells were treated with cytochalasin D (cyto. D), latrunculin B (lat. B), MG-132, or DMSO for 1 hour. The inhibitors were washed (+) or not washed (-) with the cell culture media. The cells then infected with the wild-type or BteA-deficient strain (ΔBteA) of <i>B</i>. <i>bronchiseptica</i>. At 1 hour post-infection, the amount of LDH released into the extracellular medium was measured. The error bars represent the standard error of the mean (SEM) from triplicate experiments. An asterisk (*) and NS show a significant difference (<i>p</i> < 0.05) and no significant difference, respectively. Representative data from one of three independent experiments were shown. B. Actin polymerization inhibitor-treated L2 cells or DMSO-treated cells (control) were fixed after 1 hour of infection with the wild-type <i>B</i>. <i>bronchiseptica</i>. The infected bacteria (green) and F-actin (red) were stained with anti-<i>B</i>. <i>bronchiseptica</i> antisera and rhodamine phalloidin, respectively. The objectives of 10x (numerical aperture: 0.30) and 63x (numerical aperture: 1.40) were used to acquire the images. The upper six photos show the cells infected with the wild-type <i>B</i>. <i>bronchiseptica</i> and the lower six photos show uninfected cells. Scale bars in photos obtained with 10x or 63x objective show 100 or 10μm, respectively. Representative data from one of three independent experiments.</p

Gentamicin protection assay of J774A.1 cells with <i>B</i>. <i>bronchiseptica</i> strains.

<p>A. J774A.1 cells cultured in 24-well plates were infected with <i>B</i>. <i>bronchiseptica</i> for 1 hour and extracellular bacteria were then treated with gentamicin for 30 minutes. Phagocytosed bacteria were recovered and plated on LB agar plates in order to grow a single bacterial particle to a visible colony. The number of colonies of each strain was calculated relative to the number of colonies by the wild-type strain infection, which was set as 1.0. Asterisk (*) and NS show a significant difference (<i>p</i> < 0.05) and no significant difference, respectively. The error bars represent SEM from triplicate experiments. Representative data from one of three independent experiments were shown. B. Immunofluorescence microscopy of DC2.4 cells infected with the indicated strains for 30 minutes. Infected bacteria (green) and F-actin (red) were stained with anti-<i>B</i>. <i>bronchiseptica</i> serum and rhodamine phalloidin, respectively. Arrows indicate intracellular bacteria. Scale bar shows 10 μm. Data are representative of three independent experiments. C. The percentage of intracellular bacteria was scored for each of 50 cells. Amounts of intracellular bacteria of ΔBteA or ΔT3SS strain were significantly higher than those of the wild-type strain at each time point (<i>p</i> < 0.05). The experiment was performed in tripricate. Representative data from one of three independent experiments were shown.</p

Time-lapse analyses of morphological changes of <i>B</i>. <i>bronchiseptica</i>-infected mammalian cells.

<p>The pEGFP-C1 plasmid encoding EGFP was introduced into L2 cells to look at the morphologies of the cells under fluorescent microscope. The transfected L2 cells were infected with <i>B</i>. <i>bronchiseptica</i> strains in a glass-bottomed dish. The infected cells were analyzed under a confocal laser scanning microscope equipped with a time-lapse CCD camera. A representative cell of each type at the indicated time post-infection is shown. Arrows and arrowheads indicate membrane ruffles and blebs, respectively. Scale bar shows 10 μm. Data are representative of three independent experiments.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

BteA C-terminal moiety interacts with BteA N-terminal and the C-terminal moiety itself.

<p>The recombinant full length (FL), N-terminal moiety (amino acid region 1–312; N), and C-terminal moiety (amino acid region 313–658; C) of BteA, and BtcA were purified as FLAG- or Strep-tagged protein. Each FLAG-tagged protein was mixed with Strep-tagged proteins. Streptactin beads were added to the tubes in order to pull-down Strep-tagged proteins and interacting FLAG-tagged proteins. After 1 hour incubation, the supernatant was transferred to new eppendolf tubes to prepare supernatant fractions (S). The resulting streptactin beads were washed and used for preparation of precipitated fractions (P). One sixth (12.5%) of total input was loaded to each lane of 12.5% of SDS-PAGE gel. The upper panel and lower panel show the results of Western blot with anti-FLAG antibodies (Sigma-Aldrich F3165) and anti-Strep antibodies (GenScript A00626-40), respectively. Data are representative of three independent experiments.</p