Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells

Craven, Ryan E.; Lacy, D. Borden

doi:10.1128/msphere.00012-15

Cited by 11 publications

(13 citation statements)

References 41 publications

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“…3H), suggesting that the Rac1 effector function likely plays a role in its MAVS-inhibiting activity. To further explore this, MAVS −/− iBMDMs or those reconstituted with wild-type MAVS (MAVS −/− + MAVS) were pretreated with the clostridial glucosylation toxin TcsL that specifically inactivates Rac and Cdc42 effector function by modifying their switch region ( 31 ). Since Rac2 and Cdc42 are not involved in MAVS signaling (Fig.…”

Section: Resultsmentioning

confidence: 99%

Control of antiviral innate immune response by protein geranylgeranylation

et al. 2019

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The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS’ interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIPL to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.

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Section: Resultsmentioning

confidence: 99%

Control of antiviral innate immune response by protein geranylgeranylation

et al. 2019

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“…rN-TcdA (applied at a relatively high concentration of 100 nM) has been shown to glucosylate a broader profile of substrate GTPases (including Ras subtype GTPases), while full-length TcdA fails to do so (Genth et al, 2014); (2) Complementation of the cell-free system with membrane-containing lysates: The substrate GTPases of the LCGTs are anchored to membranes through their C-terminally located polybasic domain and the isoprenyl residue. The 4-helix-bundle at the very N-terminus of the glucosyltransferase domain of the LCGTs mediates membrane anchoring as well (Varela Chavez et al, 2015, 2016; Craven and Lacy, 2016). Membrane anchoring of both substrate GTPases and the glucosyltransferase domain of the LCGTs facilitates enhanced glucosylation as compared with the soluble components, as substrate GTPase glucosylation catalyzed by either rN-TcdA (Figure 5B), rN-TcdB (Figure 5C), and rN-TcsL (Figure 6) was enhanced in a cell-free system upon addition of membrane-containing lysates.…”

Section: Discussionmentioning

confidence: 99%

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018

et al. 2018

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Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

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“…Indeed, HeLa cells expressing fluorescent TcsL 4HBM show a decoration of the plasma membrane, whereas mutations R18A in loop 1 or R68A in loop 3 abolish the membrane localisation (Geissler et al, 2012). The impaired membrane localisation of the TcsL mutants F17N and R18A was further confirmed by liposome binding (Craven & Lacy, 2015). TcsL 1–93 region as well as TcsL‐cat in fusion with the green fluorescent protein directly transfected into HeLa cells co‐localise to the cytoplasmic face of the plasma membrane.…”

Section: Lesson From Clostridium Sordellii Lethal Toxin: a Toxin Addrmentioning

confidence: 95%

“…The 4HBM structure shows two protruding loops (loop 1 connecting the helices 1 and 2, and loop 3 connecting the helices 3 and 4). The importance of these loops in lipid binding was confirmed by the mutations of F17N in loop 1 and R18A in loop 2 which impair TcsL cytotoxicity as well as binding to liposomes (Craven & Lacy, 2015).…”

Section: Lesson From Clostridium Sordellii Lethal Toxin: a Toxin Addrmentioning

confidence: 99%

“…These mutants lacking the membrane binding are no longer cytotoxic via Ras glucosylation (Varela Chavez et al, 2016). In addition, whole TcsL harbouring F17N and R18A mutations in loop 1 yields non‐toxic molecules (Craven & Lacy, 2015). This further supports the catalytic domain translocates into the cytosol and then localises to the plasma membrane as a prerequisite step for the enzymatic modification of the membrane anchored GTPases and subsequent cellular effects.…”

Section: Lesson From Clostridium Sordellii Lethal Toxin: a Toxin Addrmentioning

confidence: 99%

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Bacterial intracellularly active toxins: Membrane localisation of the active domain

Varela-Chavez

Blondel

Popoff

2020

Cellular Microbiology

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Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine. K E Y W O R D S Bacterial virulence factors, cell membrane, large clostridial glucosylating toxins, MARTX toxins, Pasteurella multocida toxin, phospholipids, Rho/Ras-GTPases

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Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells

Cited by 11 publications

References 41 publications

Control of antiviral innate immune response by protein geranylgeranylation

Control of antiviral innate immune response by protein geranylgeranylation

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018

Bacterial intracellularly active toxins: Membrane localisation of the active domain

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