Identification of mammalian proteins that collaborate with type III secretion system function: involvement of a chemokine receptor in supporting translocon activity.

UNLABELLED: The type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, including Yersinia pseudotuberculosis. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from a Ccr5(-/-) knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis. IMPORTANCE: Many Gram-negative bacteria require type III secretion systems (T3SS) for host survival, making these highly specialized secretion systems good targets for antimicrobial agents. After the bacterium binds to host cells, T3SS deposit proteins into the cytosol of host cells through a needle-like appendage and a protein translocon channel. Translocation of proteins via this system is highly regulated, and the contribution of the host cell in promoting assembly and insertion of the channel into the plasma membrane, folding of the bacterial proteins, and trafficking of these substrates are all poorly characterized events. In this study, we identified host cell proteins important for activity of YopE, a Yersinia pseudotuberculosis T3SS-delivered protein. The results demonstrate that insertion and assembly of the translocon are complex processes, requiring a variety of membrane trafficking and cytoskeletal processes, as well as a surprising role for cell surface signaling molecules in supporting proper function.

Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases.

Actin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACD(Vc)) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP- and Mg/Mn(2+)-dependent manner. In vivo, cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACD(Vc) catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACD(Vc) required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACD(Vc) activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.

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.

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.

Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin.

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae.

FAK blunts adenosine-homocysteine-induced endothelial cell apoptosis: requirement for PI 3-kinase.

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130(Cas) cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.