Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Sheahan, Kerri Lynn; Cordero, Christina L.; Satchell, K.J.F.

doi:10.1038/sj.emboj.7601700

Cited by 82 publications

(121 citation statements)

References 37 publications

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“…2), indicating that autoprocessing is a defining feature of this family of toxins. Detailed alignments show that all MARTX toxin CPDs include the histidine and cysteine that comprise the catalytic dyad, indicating that all of them could be enzymatically functional (30). Thus, it seems that all MARTX toxins have the propensity for autocatalytic cleavage.…”

Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

“…Since autocleavage would not be expected until after translocation, it is not surprising that activation of the protease in vitro required addition of eukaryotic cell cytosol preparations. It was subsequently shown that binding, but not hydrolysis, of GTP was sufficient to stimulate processing, indicating that processing occurs after transit of the toxin to the eukaryotic cytoplasm with high GTP concentrations (30).…”

Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

“…Therefore, it was predicted that MARTX Vc would undergo proteolytic cleavage during translocation into host cells (31). A domain located at aa 3420 to 3526 has been shown to be an autocatalytic cysteine protease domain (CPD) whose activity is essential to the toxic action of MARTX Vc (30). Since autocleavage would not be expected until after translocation, it is not surprising that activation of the protease in vitro required addition of eukaryotic cell cytosol preparations.…”

Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

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MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

2007

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The repeats-in-toxin (RTX) exoproteins are a diverse collection of proteins exported via type I secretion by gram-negative bacteria. These proteins include the pore-forming RTX toxins typified by ␣-hemolysin of Escherichia coli and Bordetella pertussis adenylate cyclase toxin (ACT) and also secreted enzymes such as metalloproteases and lipases, S-layer proteins, a nodulation signaling factor, and the Fe-regulated FrpC "clipand-link" toxin of Neisseria meningitidis (24,27).In 1999, a new toxin from Vibrio cholerae was discovered that was initially categorized in the RTX toxin family and simply named "the RTX toxin" or VcRtxA (22). This toxin is produced by nearly all clinical and environmental isolates of V. cholerae, including the El Tor O1 strains responsible for the current cholera pandemic plus a broad selection of O139 and non-O1/non-O139 strains (7, 9, 10, 13). The toxin has been associated with increased epithelial cell damage in a mouse lung infection model (14) and has also been associated with increased virulence in a mouse gut infection model, although its role in intestinal disease is currently unclear (26). Recently, a related toxin from Vibrio vulnificus has been described and found to be important for virulence in mouse models (21, 23).On-going study of the mechanism of action of the V. cholerae toxin in vitro has revealed that it is a totally novel type of toxin with many features that distinguish it from other RTX toxins. In addition, release of genomic sequences from human, marine, and insect pathogens has indicated that the toxins from V. cholerae and V. vulnificus are not unique but rather are the first characterized members of a new family of the RTX exoproteins. Within this new family, the structural properties of the toxins are highly conserved, but the catalytic activities carried are predicted to vary since the toxins are assembled as mosaics of 10 activity domains assorted among the various toxins.In this review, the structure and function of the V. cholerae toxin are discussed in detail, revealing that this is a novel toxin distinct from the other RTX toxins. The structural features that define the new family and the mosaic structure of the activity domains are also discussed. Overall, it is shown that the V. cholerae toxin is a multifunctional autoprocessing RTX toxin, renamed MARTX Vc , that represents a larger group of MARTX toxins produced by at least eight gram-negative species. NOVEL REPEAT STRUCTURE OF MARTX TOXINSA common property of all RTX pore-forming toxins is their large size; their molecular masses range from 100 to 177 kDa (24). At the C terminus of these large proteins are "GD-rich" nonapeptide repeats (GGXGXDX[L/I/V/W/Y/F]X], where X is any amino acid), which are the defining characteristic of the RTX exoproteins (20). These repeats have been shown to fold to form a stable ␤-roll structure that binds Ca 2ϩ (3) and are thought to be involved in proper insertion of the toxins into the eukaryotic cytoplasmic membrane (29).Sequences of novel A, B, and C repeats. The MARTX V...

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Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

Section: Autoprocessing Of Martx Toxinsmentioning

confidence: 99%

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MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

2007

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“…The genome of R. insecticola 5.15 encodes eight putative proteins with significant NCBI Conserved Domain RPS-BLAST hits to RTX toxins and related Ca 2+ -binding proteins (COG2931). In H. defensa and both R. insecticola strains, putative RTX (COG2931) genes also encode cysteine protease effector domains toward the N terminus for both C80 and M10 families, as in Yersinia pseudotuberculosis (Sheahan et al 2007). R. insecticola 5.15 encodes several RTX toxins with homologs present in H. defensa or R. insecticola LSR1 (Table 4).…”

Section: Exotoxins and Type I Secretion Systems ( T1ss)mentioning

confidence: 99%

Genomic basis of endosymbiont-conferred protection against an insect parasitoid

Hansen¹,

Vorburger²,

Moran³

2011

Genome Res.

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Bacterial endosymbionts exert a variety of beneficial effects on insect hosts. In pea aphids (Acyrthosiphon pisum), several inherited endosymbiont species protect their hosts against parasitoid wasps, which are major natural enemies. However, strains of these symbiont species vary in their ability to confer protection against parasitoids, with some conferring almost complete protection and others conferring almost none. In this study, two strains of the endosymbiont Regiella insecticola (R. insecticola 5.15 and R. insecticola LSR1) were found to differ in ability to protect pea aphids attacked by the parasitoid Aphidius ervi. Parasitism trials reveal that R. insecticola 5.15, but not R. insecticola LSR1, significantly reduced parasitoid success and increased aphid survivorship. To address the potential genetic basis of protection conferred by R. insecticola 5.15 we sequenced the genome of this symbiont strain, and then compared its gene repertoire with that of the already sequenced nonprotective strain R. insecticola LSR1. We identified striking differences in gene sets related to eukaryote pathogenicity. The protective strain R. insecticola 5.15 encoded five categories of pathogenicity factors that were missing or inactivated in R. insecticola LSR1. These included genes encoding the O-antigen biosynthetic pathway, an intact Type 1 Secretion System and its secreted RTX toxins, an intact SPI-1 Type 3 Secretion System and its effectors, hemin transport, and the twocomponent system PhoPQ. These five pathogenicity factors and translocation systems are hypothesized to collectively play key roles in the endosymbiont's virulence against parasitoids, resulting in aphid protection. Mechanisms through which these factors may target parasitoids are discussed.

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“…This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria (14). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15).…”

mentioning

confidence: 99%

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

Pruitt¹,

Chagot

Cover³

et al. 2009

Journal of Biological Chemistry

101

137

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1.6 Å x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a ␤-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a -cation interaction within the ␤-flap that appear to be unique among the large clostridial cytotoxins.Clostridium difficile is a Gram-positive, spore-forming anaerobe that infects the colon and causes a range of disorders, including diarrhea, pseudomembranous colitis, and toxic megacolon (1, 2). Two large toxins, TcdA 2 and TcdB (308 and 270 kDa, respectively) are recognized as the main virulence factors of C. difficile, although their relative importance is the subject of on-going study (3,4). These proteins belong to a class of homologous toxins called large clostridial toxins (LCTs) and have been classified more broadly as AB toxins, wherein a B moiety is involved in the delivery of an enzymatic A moiety into the cytosol of a target cell. In LCTs, the A subunit is an N-terminal glucosyltransferase that inactivates small G-proteins, such as Rho, leading to cell rounding and apoptosis of the intoxicated cell (5, 6). The B subunit corresponds to the remainder of the toxin and is responsible for binding the target cell through a C-terminal receptor-binding domain (7-9) and forming the membrane pore needed for translocation of the A subunit (10, 11). Unlike other known AB toxins, the glucosyltransferase A domains of LCTs are released from the B subunits by an autoproteolytic cleavage event (12). Cleavage is triggered by host inositol phosphates and the reducing environment of the cytosol (12).In LCTs, autoproteolysis has been attributed to a cysteine protease activity located within the N-terminal region of the B subunit (13). This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria (14). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15). A recent crystal structure of VcRTx CPD bound to InsP6 suggests a novel mechanism of InsP6-induced allosteric activation (16). The CPDs of TcdA and VcRTx share only 19% sequence identity. To gain insight into the mechanistic commonalities between these entirely different toxins and to delineate the LCT-specific modes of InsP6-induced processing, we performed structural and functional analyses on the cysteine protease from TcdA. EXPERIMENTAL PROCEDURESPlasmid Construction and Point Mutants-The nucleotide sequence coding for amino acids 543-809 of TcdA (TcdA CPD) was amplified from C. difficile strain 10463 genomic DNA. The DNA was cloned into a modified pET27 vector such that the resulting protein contains an N-terminal His 10 tag followed by a 3C proteas...

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Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Cited by 82 publications

References 37 publications

MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

Genomic basis of endosymbiont-conferred protection against an insect parasitoid

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

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