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Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.
Caston RR, Sierra JC, Foegeding NJ, Truelock MD, Campbell AM, Frick-Cheng AE, Bimczok D, Wilson KT, McClain MS, Cover TL
(2020) Infect Immun 88:
MeSH Terms: Amino Acid Sequence, Bacterial Proteins, Bacterial Toxins, Gene Expression Regulation, Bacterial, Genetic Variation, Helicobacter Infections, Helicobacter pylori, Humans, Protein Domains, Protein Multimerization, Protein Transport, Vacuoles
Show Abstract · Added April 15, 2020
colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
Copyright © 2020 American Society for Microbiology.
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12 MeSH Terms
Symmetrically substituted dichlorophenes inhibit -acyl-phosphatidylethanolamine phospholipase D.
Aggarwal G, Zarrow JE, Mashhadi Z, Flynn CR, Vinson P, Weaver CD, Davies SS
(2020) J Biol Chem 295: 7289-7300
MeSH Terms: Animals, Bacterial Proteins, Bithionol, Dichlorophen, Enzyme Inhibitors, HEK293 Cells, Hexachlorophene, Humans, Mice, Phospholipase D, Quinazolines, Streptomyces, Sulfonamides
Show Abstract · Added August 18, 2020
-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of -acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and -acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC = ∼2 μm) being symmetrically substituted dichlorophenes, hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD other tissue-associated lipases.
© 2020 Aggarwal et al.
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13 MeSH Terms
infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease.
Mileto SJ, Jardé T, Childress KO, Jensen JL, Rogers AP, Kerr G, Hutton ML, Sheedlo MJ, Bloch SC, Shupe JA, Horvay K, Flores T, Engel R, Wilkins S, McMurrick PJ, Lacy DB, Abud HE, Lyras D
(2020) Proc Natl Acad Sci U S A 117: 8064-8073
MeSH Terms: Animals, Bacterial Proteins, Bacterial Toxins, Cells, Cultured, Clostridioides difficile, Clostridium Infections, Colon, Disease Models, Animal, Female, Frizzled Receptors, Humans, Intestinal Mucosa, Mice, Organoids, Primary Cell Culture, Recombinant Proteins, Stem Cells
Show Abstract · Added March 24, 2020
Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, 8, e73204 (2013); S. Kozar , 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
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17 MeSH Terms
Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.
Maddur AA, Kroh HK, Aschenbrenner ME, Gibson BHY, Panizzi P, Sheehan JH, Meiler J, Bock PE, Verhamme IM
(2020) J Biol Chem 295: 5614-5625
MeSH Terms: Bacterial Proteins, Binding Sites, Coagulase, Humans, Models, Molecular, Protein Binding, Prothrombin, Staphylococcal Infections, Staphylococcus aureus, Substrate Specificity
Show Abstract · Added March 21, 2020
In -caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile-Val into the ProT Ile pocket, forming a salt bridge with ProT's Asp, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProT), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProT caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val-Val, Ile-Ala, and Leu-Val variants exhibiting ProT affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProT levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile pocket in ProT appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile-Val-Thr might emerge with similar ProT-activating efficiency.
© 2020 Maddur et al.
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10 MeSH Terms
Intestinal bile acids directly modulate the structure and function of TcdB toxin.
Tam J, Icho S, Utama E, Orrell KE, Gómez-Biagi RF, Theriot CM, Kroh HK, Rutherford SA, Lacy DB, Melnyk RA
(2020) Proc Natl Acad Sci U S A 117: 6792-6800
MeSH Terms: Bacterial Toxins, Bile Acids and Salts, Caco-2 Cells, Clostridioides difficile, Clostridium Infections, HCT116 Cells, Humans, Intestines, Receptors, Cell Surface
Show Abstract · Added March 24, 2020
Intestinal bile acids are known to modulate the germination and growth of Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact "balled up" conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in pathogenesis, these findings provide a framework for development of a mechanistic class of antitoxins.
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9 MeSH Terms
Structural elucidation of the transferase toxin reveals a single-site binding mode for the enzyme.
Sheedlo MJ, Anderson DM, Thomas AK, Lacy DB
(2020) Proc Natl Acad Sci U S A 117: 6139-6144
MeSH Terms: Bacterial Toxins, Clostridioides difficile, Cryoelectron Microscopy, Enterotoxins, Protein Conformation, beta-Strand, Protein Multimerization, Transferases
Show Abstract · Added March 24, 2020
is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the transferase toxin (CDT). Reported here is a 3.8-Å cryo-electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to β-barrel pore, although we also observe a single bound CDTa in the prepore and β-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.
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7 MeSH Terms
Gut Epithelial Metabolism as a Key Driver of Intestinal Dysbiosis Associated with Noncommunicable Diseases.
Shelton CD, Byndloss MX
(2020) Infect Immun 88:
MeSH Terms: Animals, Colon, Disease Susceptibility, Dysbiosis, Energy Metabolism, Enterobacteriaceae, Gastrointestinal Microbiome, Humans, Intestinal Mucosa, Noncommunicable Diseases, Obesity, Oxidation-Reduction, Risk Assessment, Risk Factors
Show Abstract · Added March 30, 2020
In high-income countries, the leading causes of death are noncommunicable diseases (NCDs), such as obesity, cancer, and cardiovascular disease. An important feature of most NCDs is inflammation-induced gut dysbiosis characterized by a shift in the microbial community structure from obligate to facultative anaerobes such as This microbial imbalance can contribute to disease pathogenesis by either a depletion in or the production of microbiota-derived metabolites. However, little is known about the mechanism by which inflammation-mediated changes in host physiology disrupt the microbial ecosystem in our large intestine leading to disease. Recent work by our group suggests that during gut homeostasis, epithelial hypoxia derived from peroxisome proliferator-activated receptor γ (PPAR-γ)-dependent β-oxidation of microbiota-derived short-chain fatty acids limits oxygen availability in the colon, thereby maintaining a balanced microbial community. During inflammation, disruption in gut anaerobiosis drives expansion of facultative anaerobic , regardless of their pathogenic potential. Therefore, our research group is currently exploring the concept that dysbiosis-associated expansion of can be viewed as a microbial signature of epithelial dysfunction and may play a greater role in different models of NCDs, including diet-induced obesity, atherosclerosis, and inflammation-associated colorectal cancer.
Copyright © 2020 American Society for Microbiology.
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14 MeSH Terms
Risk-stratified Management to Remove Low-Risk Penicillin Allergy Labels in the ICU.
Stone CA, Stollings JL, Lindsell CJ, Dear ML, Buie RB, Rice TW, Phillips EJ
(2020) Am J Respir Crit Care Med 201: 1572-1575
MeSH Terms: Amoxicillin, Anti-Bacterial Agents, Diagnostic Errors, Drug Hypersensitivity, Humans, Intensive Care Units, Penicillins, Risk Assessment, Skin Tests
Added March 30, 2020
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9 MeSH Terms
How to thrive in the inflamed gut.
Yoo W, Byndloss MX
(2020) Nat Microbiol 5: 10-11
MeSH Terms: Diet, Enterobacteriaceae, Intestinal Mucosa, Serine
Added March 30, 2020
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4 MeSH Terms
Penicillin Allergy.
Castells M, Khan DA, Phillips EJ
(2019) N Engl J Med 381: 2338-2351
MeSH Terms: Adult, Anaphylaxis, Anti-Bacterial Agents, Child, Desensitization, Immunologic, Drug Hypersensitivity, Humans, Immunologic Tests, Penicillins, Skin Tests
Added March 30, 2020
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MeSH Terms