Crystal Structure of the Yeast Vacuolar ATPase Heterotrimeric EGChead Peripheral Stalk Complex

SummaryVacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V1-ATPase and Vo proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit “a” and three “peripheral stalks,” each made of a heterodimer of E and G subunits. Here, we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (Chead). The structures show EG heterodimer folded in a noncanonical coiled coil that is stabilized at its N-terminal ends by binding to Chead. The coiled coil is disrupted by a bulge of partially unfolded secondary structure in subunit G and we speculate that this unique feature in the eukaryotic V-ATPase peripheral stalk may play an important role in enzyme structure and regulation by reversible dissociation

Identification of a novel variant of LMP-1 of EBV in patients with endemic Burkitt lymphoma in western Kenya

BACKGROUND: Epstein Barr virus (EBV) is a gammaherpesvirus that is associated with nasopharyngeal carcinoma (NPC) and endemic Burkitt lymphoma (eBL). EBV carries several latent genes that contribute to oncogenesis including the latent membrane protein 1 (LMP-1), a known oncogene and constitutively active CD40 homolog. Variation in the C terminal region of LMP-1 has been linked to NPC pathogenesis, but little is known regarding LMP-1 variation and eBL. RESULTS: In the present study, peripheral blood samples were obtained from 38 eBL patients and 22 healthy controls in western Kenya, where the disease is endemic. The LMP-1 C-terminal region from these samples was sequenced and analyzed. The frequency of a 30 base pair deletion of LMP-1 previously linked to NPC was not associated with eBL compared to healthy controls. However a novel LMP-1 variant was identified, called K for Kenya and for the G318K mutation that characterizes it. The K variant LMP-1 was found in 40.5% of eBL sequences and 25.0% of healthy controls. All K variant sequences contained mutations in both of the previously described minimal T cell epitopes in the C terminal end of LMP-1. These mutations occurred in the anchor residue at the C-terminal binding groove of both epitopes, a pocket necessary for MHC loading. CONCLUSIONS: Overall, our results suggest that there is a novel K variant of LMP-1 in Kenya that may be associated with eBL. Further studies are necessary to determine the functional implications of the LMP-1 variant on early events in eBL genesis

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

Aim: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50 000 head commercial beef feedlot. Methods and Results: Escherichia coli O157 was detected in ∼27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (∼97%) contained phage that infected E. coli B or O157:H7. Conclusions: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. Significance and Impact of the Study: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.Fil: Oot, Rebecca. The Evergreen State College; Estados UnidosFil: Raya, Raul Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Callaway, Todd R.. College Station. Food and Feed Safety Research Unit. Agricultural Research Service; Estados UnidosFil: Edrington, Tom S.. College Station. Food and Feed Safety Research Unit. Agricultural Research Service; Estados UnidosFil: Kutter, Elizabeth M.. The Evergreen State College; Estados UnidosFil: Brabban, Andrew D.. The Evergreen State College; Estados Unido

Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G*

The proton pumping activity of the eukaryotic vacuolar ATPase (V-ATPase) is regulated by a unique mechanism that involves reversible enzyme dissociation. In yeast, under conditions of nutrient depletion, the soluble catalytic V1 sector disengages from the membrane integral Vo, and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V1 subunit, C, is released into the cytosol. The affinities of the other V1 and Vo subunits for subunit C are therefore of particular interest. The C subunit crystal structure shows that the subunit is elongated and dumbbell-shaped with two globular domains (Chead and Cfoot) separated by a flexible helical neck region (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1148–1152). We have recently shown that subunit C is bound in the V1-Vo interface where the subunit is in contact with two of the three peripheral stators (subunit EG heterodimers): one via Chead and one via Cfoot (Zhang, Z., Zheng, Y., Mazon, H., Milgrom, E., Kitagawa, N., Kish-Trier, E., Heck, A. J., Kane, P. M., and Wilkens, S. (2008) J. Biol. Chem. 283, 35983–35995). In vitro, however, subunit C binds only one EG heterodimer (Féthière, J., Venzke, D., Madden, D. R., and Böttcher, B. (2005) Biochemistry 44, 15906–15914), implying that EG has different affinities for the two domains of the C subunit. To determine which subunit C domain binds EG with high affinity, we have generated Chead and Cfoot and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is Chead. In addition, we provide evidence that the EGChead interaction greatly stabilizes EG heterodimer