ABSTRACT
Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI(+) bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein from Neisseria meningitidis MC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of RNA-binding proteins, but appears to lack the characteristic nucleic acid-binding motif of this family. Sequence homology suggests that the cognate CdiA-CT is related to the eukaryotic EndoU family of RNA-processing enzymes. A homology model is presented of the CdiA-CT based on the structure of the XendoU nuclease from Xenopus laevis. Molecular-docking simulations predict that the CdiA-CT toxin active site is occluded upon binding to the CdiI immunity protein. Together, these observations suggest that the immunity protein neutralizes toxin activity by preventing access to RNA substrates.
Subject(s)
Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/chemistry , Escherichia coli Proteins/chemistry , Neisseria meningitidis/chemistry , Amino Acid Motifs , Animals , Antibiosis/immunology , Bacterial Toxins/immunology , Contact Inhibition/immunology , Crystallography, X-Ray , Endoribonucleases/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Gene Expression , Molecular Docking Simulation , Molecular Sequence Data , Neisseria meningitidis/immunology , Neisseria meningitidis/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Structural Homology, Protein , Xenopus Proteins/chemistry , Xenopus laevis/metabolismABSTRACT
OBJECTIVES: To assess for stem cell migration to liver and lung after transplantation in injured rat anal sphincters. To evaluate histological findings of unanticipated ectopic foci of growth. METHODS: This is a prospective study involving 33 female virginal Sprague-Dawley rats. Anal sphincters were transected and repaired under sterile technique. Animals received injections of 5.0 × 10 myogenic stem cells (24 rats) or sham control (9 rats) and were killed on day 30. Liver and lung samples were obtained. Upon encountering abnormal foci of growth, further staining protocols were employed. Enzyme-linked immunosorbent assay studies evaluated stem cell media for in vitro growth factor secretion. RESULTS: No evidence of cell migration to liver or lung was found at the time of euthanasia in any study animal. Ectopic foci of growth were noted in 2 transplant rats. Further histological evaluations of these growths were consistent with benign tumors: no nuclear abnormalities and no evidence of proliferation at day 30. Enzyme-linked immunosorbent assay studies demonstrated positive secretion of vascular endothelial growth factor and insulin growth factor into the media of cultured rat myogenic stem cells. CONCLUSIONS: Whereas distant migration was not encountered in the liver or lung, 2 transplanted rats developed abnormal foci of growth, that is, tumors, from the external anal sphincter-raising further safety questions. Additional evaluation of these foci seemed benign. Possible explanations include cell trapping, stem cell overgrowth, and/or paracrine factors. The lack of cell migration supports that future investigation of safety parameters could focus locally.
