Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux

A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis.

Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen's virulence

The <i>Salmonella</i> Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members

<div><p><i>Salmonella</i> Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of <i>Salmonella</i> Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a <i>Salmonella</i> mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms.</p></div

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response

Proposed model for SopA action.

<p>After its delivery by the <i>S</i>. Typhimurium type III secretion system, SopA targets TRIM56 and/or TRIM65 and through ubiquitination, enhances their ability to modulate RIG-I and/or MDA-5-dependendant signal transduction pathways leading to pro-inflammatory cytokine production. The modulatory activity of SopA may require the prior stimulation of RIG-I and/or MDA-5 by other agonists such as nucleic acids.</p

TRIM65 interacts with MDA5 and enhances MDA5-stimulated interferon-β expression.

<p>(<b>A</b>) FLAG-epitope-tagged RIG-I (2CARD) (the two CARD domains of RIG-I), RIG-I (full length), MDA5 (2CARD) (the two CARD domains of MDA5), or MDA5 (full length) were transiently expressed in HEK 293T cells together with M45-epitope-tagged TRIM65. Protein interactions were analyzed by immunoprecipitation with anti-FLAG and immunoblotting with anti-M45 and anti-FLAG antibodies. This experiment was repeated 3 independent times with similar results. (<b>B</b>-<b>E</b>) HEK293T cells were co-transfected with plasmids expressing TRIM65 or the catalytically-deficient TRIM65^C15A mutant along with plasmids expressing RIG-I (20 ng), MDA5 (50 ng), MDA5(2CARD) (10 ng) or MAVS (50 ng) as indicated, and the reporter plasmids expressing firefly luciferase under the control of interferon-β promoter and renilla luciferase (to standardize the transfection efficiency). The activation of the interferon-β promoter was analyzed 18 h after transfection by Dual Luciferase Reporter Assay System (Promega). Values represent the mean +/- standard deviation of the relative levels of normalized firefly luciferase from 3 independent experiments. The normalized firefly luciferase activity relative to mock-transfected cells was 10.5 ±2.9 for MDA5 only transfected cells (<b>C</b>), 11.1 ± 4.8 for RIG-I only transfected cells (<b>D</b>), 30.6 ± 17.8 for MAVS only transfected cells (<b>E</b>), and 15.4 ± 3.3 for MDA5(2CARD) only transfected cell (<b>F</b>). *: indicates statistically significant difference (p < 0.05) vs. mock transfected cells (<b>B</b>), vs. MDA5-transfected cells (<b>C</b>), vs. RIG-I-transfected cells (<b>D</b>), vs. MAVS-transfected cells (<b>E</b>) and vs. MDA5(2CARD)-transfected cells (<b>F</b>).</p

Absence of the PipA-family of effector proteins increases the ability of <i>S</i>. Typhimurium to stimulate pro-inflammatory cytokine expression in the mouse intestine.

<p>(<b>A</b> and <b>B</b>) C57/BL6 <i>nramp</i>^+/+ mice were orally infected with wild type (n = 5) or <i>ΔpipA ΔgogA ΔgtgA</i> (n = 5) <i>S</i>. Typhimurium strains and 24 (<b>A</b>) or 48 (<b>B</b>) hours after infection the relative levels of the indicated cytokines in the intestine were measured by quantitative PCR. Data were normalized to the levels of GAPDH and are expressed relative to uninfected control animals (n = 5). The data shown were compiled from two independent experiments of three measurements each. <i>p</i> values of the indicated differences determined by the Student <i>t</i> test are shown.</p

SopA enhances TRIM65 ubiquitination.

<p>(<b>A</b>) Purified FLAG-epitope-tagged TRIM65 was incubated with SopA or its catalytic mutant SopA^C753S in the presence of ubiquitin, ATP, E1 (UBE1) and E2 (UbcH5b) and TRIM65 ubiquitination was detected by its mobility shift in Western blot analysis with anti-FLAG antibody. The chromatographic profiles of the SopA preparations used in the assays are shown. (<b>B</b>) HEK293T cells were co-transfected with plasmids encoding HA-epitope-tagged ubiquitin, and either FLAG-epitope-tagged TRIM65, or the mutants TRIM65^C15A (catalytically deficient) and TRIM65^T24K (autoubiquitination deficient) along with plasmids encoding either SopA or its catalytic mutant SopA^C753S. Cell lysates were evaluated for the levels of protein expression and TRIM65 ubiquitination by immunoprecipitation with anti-FLAG and Western immunoblot analysis with anti-FLAG, anti-HA, and anti-M45 antibodies, respectively. This experiment was repeated 2 independent times with similar results. (<b>C</b>) HEK293T cells were co-transfected with plasmids encoding HA-epitope-tagged ubiquitin and FLAG-epitope tagged TRIM65 and 18 hours after transfection, cells were infected with an <i>S</i>. Typhimurium strain expressing either wild type SopA or its catalytic mutant SopA^C753S. Five hours after infection, cell lysates were evaluated for the levels of protein expression and TRIM65 ubiquitination by immunoprecipitation and Western immunoblot analysis with anti-FLAG, anti-HA, and anti-M45 antibodies, respectively. This experiment was repeated 3 independent times with similar results.</p

Absence of the PipA-family of effector proteins increases the mouse virulence of <i>S</i>. Typhimurium without increasing bacterial loads.

<p>(<b>A</b> and <b>B</b>) C57/BL6 <i>nramp</i>^+/+ mice were orally (<b>A</b>) or intraperitoneally (<b>B</b>) infected with wild-type S. <i>Typhimurium</i> or the <i>ΔpipA ΔgogA ΔgtgA</i> mutant and bacterial loads in the indicated tissues enumerated 7 days after infection. Each circle represents the bacterial load for an individual animal and horizontal bars indicate geometric means. The results are the combination of two independent experiments. (<b>C</b> and <b>D</b>) Survival of animals orally (<b>C</b>) or intraperitoneally (<b>D</b>) infected with wild-type <i>S</i>. Typhimurium (n = 7) or the <i>ΔpipA ΔgogA ΔgtgA</i> mutant (n = 8) strains 6 days after infection. The <i>p</i> values of the difference in the survival of animals infected with wild type or mutant strains determined by the log-rank test are shown. The results are the combination of two independent experiments.</p