Karla J. F. Satchell

Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.

The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and β-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into ∼250/210- and ∼63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1–955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415–419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.

Structure and Function of MARTX Toxins and Other Large Repetitive RTX Proteins

The Repeats-in-Toxins (RTX) family of proteins classically consists of cytolysins and hemolysins. Over the past decade, genome sequencing revealed the existence of very large members of this family. These are all repetitive proteins ranging in size from 200 to 900 kDa that function as toxins or adhesins. Many are exported by Type I secretion. One major new subfamily is the large repetitive RTX adhesins and biofilm-associated proteins. These are characterized by 80- to 300-amino-acid repeats ordered in tandem, although the sequence and number of the repeats vary by protein. The second major new subfamily is the multifunctional-autoprocessing RTX toxins, which are associated with cytotoxicity and pathogenesis. These proteins are in turn distantly related to Yersinia hypothetical RTX proteins that may autoprocess by a similar mechanism. This review discusses current knowledge regarding the structure and function of these new subfamilies of RTX proteins.

MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

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 α-hemolysin of Escherichia coli and Bordetella pertussis adenylate cyclase toxin (ACT) and also

The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.

Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.

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

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin‐like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.

Hemolysin and the Multifunctional Autoprocessing RTX Toxin Are Virulence Factors during Intestinal Infection of Mice with Vibrio cholerae El Tor O1 Strains

ABSTRACT The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.

Prolonged Colonization of Mice by Vibrio cholerae El Tor O1 Depends on Accessory Toxins

ABSTRACT Cholera epidemics caused by Vibrio cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of cholera in adult C57BL/6 mice wherein V. cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10- to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. cholerae to prevent clearance and establish this prolonged colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require cholera toxin or toxin-coregulated pili. The defect in colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic colonization-proficient V. cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.

Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection

Vibrio vulnificus is a food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly because of consumption of contaminated seafood. The ability of V. vulnificus to cause disease is linked to the production of a large cytotoxin called the “multifunctional-autoprocessing RTX” (MARTXVv) toxin, a factor shown here to be an important virulence factor by the intragastric route of infection in mice. In this study, we examined genetic variation of the rtxA1 gene that encodes MARTXVv in 40 V. vulnificus Biotype 1 strains and found four distinct variants of rtxA1 that encode toxins with different arrangements of effector domains. We provide evidence that these variants arose by recombination either with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum. Contrary to expected results, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency and is distinct from the toxin produced by strains isolated from market oysters. These results indicate that an important virulence factor of V. vulnificus is undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment. This finding would imply further that in the future on-going genetic variation of the MARTXVv toxins could result in the emergence of novel strains with altered virulence in humans.

Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae

Many bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin cross‐linking. However, identification and subsequent deletion of the actin cross‐linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548‐amino‐acid region of RTX was identified to be responsible for the toxin‐induced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho‐modifying bacterial toxins.

The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LFNACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LFNACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg2+ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.