Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.     

Structural Analysis of Toxin-Neutralizing,Single-Domain Antibodies that Bridge Ricin’s A-B Subunit Interface

Ricin toxin kills mammalian cells with notorious efficiency. The toxin’s B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin’s A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin’s individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (V_HHs) that span the RTA-RTB interface, including four Tier 1 V_HHs with IC₅₀ values <1 nM. Crystal structures of each V_HH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA’s F-G loop (mode 1), RTB’s subdomain 2γ (mode 2) or both (mode 3). V_HHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 V_HHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin’s cytotoxic pathway.     

Transmembrane TNF-dependent uptake of anti-TNF antibodies

TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside ‘reverse’ signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.     

Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells

We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH(2) (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 microM, 0.1U/ml, and 1 microM, respectively. Ionomycin elevated [Ca(2+)](i) in a concentration-dependent manner. However, AYPGKF-NH(2) and thrombin induced no [Ca(2+)](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca(2+)](i) elevation induced by 1 microM ionomycin, while it had no significant effect on the AYPGKF-NH(2)-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH(2)-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca(2+) signal and also that G(i/o) is involved in such NO production in vascular endothelial cells.     

Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

A型肉毒毒素联合眼袋整形术对眼周皮肤松弛患者的疗效观察

目的:探讨A型肉毒毒素联合眼袋整形术对眼周皮肤松弛患者的治疗效果和对患者满意度的影响,为临床提供参考。方法:选取2015年6月至2016年6月间因眼周皮肤松弛来我院治疗的患者60例。患者根据自身意愿选择治疗方式,按其选择的治疗方式分组为对照组(n=28)和治疗组(n=32)。对照组采用单纯眼袋整形术进行治疗,治疗组采用A型肉毒毒素联合眼袋整形术治疗。治疗后对两组患者均随访6个月。观察记录患者手术后鱼尾纹的改善效果、眼袋减轻的情况和眼周皮肤光泽的改善效果。观察患者并发症的发生情况。随访结束时采用自制的满意度调查问卷对患者满意度进行调查。结果:经过治疗,治疗组鱼尾纹改善率为100.00%,明显高于对照组的60.71%;治疗组眼袋改善率为96.88%,明显高于对照组的32.14%;治疗组皮肤光泽改善率为87.50%,明显高于对照组的53.57%,差异均具有统计学意义(均P0.05)。治疗后,两组患者切口红肿、少量分泌物、小血肿的发生率比较无明显差异(P0.05),治疗组术眼红肿与外眦部切口瘀痕发生率明显少于对照组(P0.05)。治疗组总满意率为93.75%(30/32),明显高于对照组的67.86%(19/28),差异具有统计学意义(P0.05)。结论:A型肉毒毒素与眼袋整形术联合应用在眼周皮肤松弛的治疗上有较好的疗效和安全性,能明显降低患者术眼红肿与外眦部切口瘀痕的发生,并提高患者满意率,值得在临床上推广应用。     

Host response to intravenous injection of epsilon toxin in mouse model: A proteomic view

Epsilon toxin (ETX) is an extremely potent pore‐forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE‐MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets.