Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large “snowflake”-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of β-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacAΔ6–27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacAΔ6–27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacAΔ301–328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.

Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.

Helicobacter pylori VacA is a pore-forming toxin that causes multiple alterations in human cells and contributes to the pathogenesis of peptic ulcer disease and gastric cancer. The toxin is secreted by H. pylori as an 88 kDa monomer (p88) consisting of two domains (p33 and p55). While an Xray crystal structure for p55 exists and p88 oligomers have been visualized by cryo-electron microscopy, a detailed analysis of p33 has been hindered by an inability to purify this domain in an active form. In this study, we expressed and purified a recombinant form of p33 under denaturing conditions and optimized conditions for the refolding of soluble protein. We show that refolded p33 can be added to purified p55 in trans to cause vacuolation of HeLa cells and inhibition of IL-2 production by Jurkat cells, effects identical to those produced by the p88 toxin from H. pylori. The p33 protein markedly enhances the cell-binding properties of p55. Size exclusion chromatography experiments suggest that p33 and p55 assemble into a complex consistent with the size of a p88 monomer. Electron microscopy of these p33/p55 complexes reveals small rod-shaped structures that can convert to oligomeric flower-shaped structures in the presence of detergent. We propose that the oligomerization observed in these experiments mimics the process by which VacA oligomerizes when in contact with membranes of host cells.Helicobacter pylori is a gram-negative bacterium that persistently colonizes the human stomach (1-4). Infection by H. pylori is associated with the development of peptic ulcer disease, gastric adenocarcinoma and gastric lymphoma (5-6). An important virulence factor in the pathogenesis of H. pylori infection is a secreted protein known as vacuolating cytotoxin (VacA) (7)(8)(9)(10)(11). In vivo studies have shown that VacA contributes to gastric damage in animal models (12-13), and specific vacA allelic forms are associated with an increased risk of disease in humans (14)(15). VacA causes a wide range of cellular alterations in vitro (9), including the formation of large cytoplasmic vacuoles (7-8), permeabilization of the plasma membrane (16), reduction of mitochondrial transmembrane potential and cytochrome c release (17-18), activation of mitogen-activated protein kinases (19), induction of autophagy (20), and inhibition of the activation and proliferation of T-* Address correspondence to Timothy L. Cover, Division of Infectious Diseases, A2200 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232; Phone (615) 322-2035; Fax (615) The vacA gene encodes a 140 kDa protein that undergoes proteolytic processing to yield an amino-terminal signal sequence, an 88 kDa secreted toxin, and a carboxyl-terminal autotransporter domain (13,(27)(28)(29). Partial proteolytic digestion in vitro of the 88 kDa secreted toxin yields two fragments, designated p33 and p55, which probably represent two domains of VacA (13,[30][31][32]. Cleavage of the p88 protein into these two fragments occurs at a site that is predicted...

e Previous human antibody studies have shown that the human V H 1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the V H 1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a V H 1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore. R otaviruses (RVs) are nonenveloped, triple-layered icosahedral viruses that belong to the Reoviridae family, and they are the leading cause of severe diarrheal illness in infants and young children worldwide (1). The inner capsid layer of virus protein 2 (VP2) encloses 11 segments of double-stranded RNA that each encode a single protein, except for segment 11, which codes for two proteins. The VP2 layer is surrounded by 780 molecules of virus protein 6 (VP6). VP6 is arranged into 260 trimers and forms the intermediate viral capsid layer. The VP2 and VP6 layers form the transcriptionally active double-layered particle (DLP). The outer capsid layer is composed of virus protein 7 (VP7) with spikes of virus protein 4 (VP4) and forms the transcriptionally inactive mature infectious virion particle (2-8).RV VP6 is folded into two distinct domains: an ␣-helical base domain with a triangular cross section and a jelly-roll head domain that forms a roughly hexagonal cross section. The Tϭ13 icosahedral symmetry of the VP6 and VP7 layers defines three types of channels that exist within the viral architecture: type I, II, and III channels. Twelve type I channels are located down the icosahedral 5-fold axes and serve as egress points of nascent viral mRNA during viral transcription (6). Sixty type II channels are arranged to surround the type I channels, and 60 type III channels are positioned around the icosahedral 3-fold axes (5, 9-12). W...