A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

Margarit, S.M.; Davidson, Walter; Frego, Lee; Stebbins, C. Erec

doi:10.1016/j.str.2006.05.022

Cited by 78 publications

(91 citation statements)

References 66 publications

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“…We did not determine the structure of apo actin-Ia or the postreaction ADPR-actin-Ia complex. However, actin assumes the same conformation in the monomeric ATP form (1IJJ) in our complex and in the ADPR-actin form (32). This finding suggests that no structural changes occur during the ADPribosylation reaction.…”

Section: Common Reaction Mechanisms Based On Structural Relations Withsupporting

confidence: 51%

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

Tsuge

Nagahama

Oda

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

109

144

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The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens -toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog ␤TAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60 -Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADPribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.complex ͉ crystal structure ͉ protein recognition

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Section: Common Reaction Mechanisms Based On Structural Relations Withsupporting

confidence: 51%

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

Tsuge

Nagahama

Oda

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

109

144

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“…We identified arginine 177 as the modification site for C/SpvB within the actin homolog Act88F (Drosophila indirect flight muscle actin) (8). Our finding was confirmed by a recent publication (9) reporting that arginine 177 is the modification side in mammalian actin for SpvB. S. enterica is a Gram-negative facultative intracellular pathogen, which causes diseases ranging from mild gastroenteritis to severe systemic infections in humans (10,11).…”

supporting

confidence: 73%

A Cell-permeable Fusion Toxin as a Tool to Study the Consequences of Actin-ADP-ribosylation Caused by the Salmonella enterica Virulence Factor SpvB in Intact Cells

Pust

Hochmann

Kaiser

et al. 2007

Journal of Biological Chemistry

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The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N-and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.Several pathogenic bacteria produce toxins and effector proteins, which attack the cytoskeleton of eukaryotic cells by mono-ADP-ribosylation of actin. In past years, we focused on the mode of action of the Clostridium botulinum C2 toxin as the prototype of binary actin-ADP-ribosylating toxins (1). The enzyme component of the C2 toxin (C2I) 3 ADP-ribosylates G-actin at arginine 177 (2). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding/translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (3). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. For translocation of the C2I protein through the lumen of these pores, a partial unfolding of C2I is required (4). The subsequent refolding of C2I in the cytosol is facilitated by the host cell chaperone Hsp90 (5). The interaction of C2I with C2IIa is mediated by the N-terminal domain of the C2I protein (C2IN, amino acid residues 1-225). C2IN, which is enzymatically inactive and does not induce cell rounding when applied in combination with C2IIa to cells, was successfully used as an adaptor for the C2IIa-mediated delivery of different proteins into the cytosol of eukaryotic cells (6).The SpvB protein from Salmonella enterica was identified as a new member of bacterial actin-ADP-ribosylating enzymes...

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“…The C-terminal domain possesses ADPribosyltransferase activity that covalently modifies G-actin monomers at arginine-177 and prevents their polymerization into F-actin filaments [308][309][310][311]. SpvB inhibits the formation of vacuole-associated actin polymerizations (VAPs) in HeLa cells [312] and negatively regulates the formation of SIFs [313].…”

Section: Spvb and Spvcmentioning

confidence: 99%

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

Ramos‐Morales

2012

ISRN Cell Biology

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Type III secretion systems are molecular machines used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, directly into eukaryotic host cells. These proteins manipulate host signal transduction pathways and cellular processes to the pathogen's advantage. Salmonella enterica possesses two virulence-related type III secretion systems that deliver more than forty effectors. This paper reviews our current knowledge about the functions, biochemical activities, host targets, and impact on host cells of these effectors. First, the concerted action of effectors at the cellular level in relevant aspects of the interaction between Salmonella and its hosts is analyzed. Then, particular issues that will drive research in the field in the near future are discussed. Finally, detailed information about each individual effector is provided.

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A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

Cited by 78 publications

References 66 publications

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

A Cell-permeable Fusion Toxin as a Tool to Study the Consequences of Actin-ADP-ribosylation Caused by the Salmonella enterica Virulence Factor SpvB in Intact Cells

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

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