RESUMO
The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.
Assuntos
Neoplasias Pulmonares , Anticorpos de Domínio Único , ATPases Vacuolares Próton-Translocadoras , Animais , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/imunologia , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Feminino , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Linhagem Celular Tumoral , Neoplasias da Mama/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Camundongos Endogâmicos BALB C , Humanos , Invasividade NeoplásicaRESUMO
Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.
Assuntos
Proteínas Ativadoras de GTPase , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/química , Coativadores de Receptor Nuclear/metabolismo , Coativadores de Receptor Nuclear/química , Ligação Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas MitocondriaisRESUMO
Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors' optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.
Assuntos
Nanoporos , Peptídeos , Proteínas/análise , Eletricidade , Nanotecnologia/métodosRESUMO
Vacuolar ATPases (V-ATPases, V1 Vo -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V1 -ATPase disengages from Vo proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V1 and Vo subcomplexes.
Assuntos
Proteínas de Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Animais , Saccharomyces cerevisiae/metabolismo , Amor , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Mamíferos/metabolismoRESUMO
The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
Assuntos
Proteínas Mitocondriais/química , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Mutagênese , Ligação Proteica , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme's isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain-containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismoRESUMO
In this issue of Molecular Cell, Wang et al. (2020a) present near-atomic resolution cryoEM structures of a proton-pumping vacuolar ATPase from human cells, illuminating the glycosylation of integral subunits and identifying a novel and conserved glycolipid ligand.
Assuntos
Açúcares , ATPases Vacuolares Próton-Translocadoras , Microscopia Crioeletrônica , Humanos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V1 and Vo, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase in vitro Here, using the model organism Saccharomyces cerevisiae, we show that a key impediment for binding of V1 to Vo is the conformation of the inhibitory C-terminal domain of subunit H (HCT). Using biolayer interferometry and biochemical analyses of purified mutant V1-ATPase and Vo proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of HCT's V1-binding site facilitates assembly of a functionally coupled and stable V1Vo-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting HCT's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molecular events underlying regulation of V-ATPase activity by reversible disassembly.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Mutação , Domínios Proteicos , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V1 However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full-length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H or HNT with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Conformação Proteica , Subunidades ProteicasRESUMO
The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.
Assuntos
ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/ultraestrutura , Animais , Remodelação Óssea/fisiologia , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Endocitose/fisiologia , Homeostase/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Transporte Proteico/fisiologia , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.
Assuntos
Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Difosfato de Adenosina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
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.
RESUMO
Vacuolar 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 V0 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 (C(head)). The structures show EG heterodimer folded in a noncanonical coiled coil that is stabilized at its N-terminal ends by binding to C(head). 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.
Assuntos
Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , ATPases Vacuolares Próton-Translocadoras/químicaRESUMO
Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V(1)-ATPase and V(o)-proton channel domains. We found previously that the head domain of the single copy C subunit (C(head)) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654-24664). Here we generated a water-soluble construct of the N-terminal domain of the V(o) "a" subunit composed of amino acid residues 104-372 (a(NT(104-372))). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that a(NT(104-372)) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the a(NT(104-372)) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that a(NT(104-372)) binds the C(foot) and EG heterodimer with dissociation constants of 22 and 33 µM, respectively. We speculate that the spatial closeness of the a(NT), C(foot), and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the a(NT(104-372))-C(foot) or a(NT(104-372))-EG interaction by an as-yet unknown signaling mechanism.
Assuntos
Trifosfato de Adenosina/metabolismo , Subunidades Proteicas/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Calorimetria , Dicroísmo Circular , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genomeâ¼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI â¼2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (â¼10(11) total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (â¼99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (â¼99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants.
RESUMO
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 V(1) sector disengages from the membrane integral V(o), and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V(1) subunit, C, is released into the cytosol. The affinities of the other V(1) and V(o) 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 (C(head) and C(foot)) 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 V(1)-V(o) interface where the subunit is in contact with two of the three peripheral stators (subunit EG heterodimers): one via C(head) and one via C(foot) (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 C(head) and C(foot) and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C(head). In addition, we provide evidence that the EGC(head) interaction greatly stabilizes EG heterodimer.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , ATPases Vacuolares Próton-Translocadoras/fisiologia , Calorimetria/métodos , Cromatografia em Gel/métodos , Dicroísmo Circular/métodos , Cristalografia por Raios X/métodos , Citosol/metabolismo , Dimerização , Espectrometria de Massas/métodos , Modelos Biológicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Temperatura , ATPases Vacuolares Próton-Translocadoras/químicaRESUMO
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.