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by
John R. Horton |
Total Records (
2 ) for
John R. Horton |
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Patricia A. Spears
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M. Mitsu Suyemoto
,
Angela M. Palermo
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John R. Horton
,
Terri S. Hamrick
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Edward A. Havell
and
Paul E. Orndorff
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A Listeria monocytogenes bacteriophage was used to identify a phage-resistant Tn917 insertion mutant of the mouse-virulent listerial strain F6214-1. The mutant was attenuated when it was inoculated orally into female A/J mice and failed to replicate efficiently in cultured mouse enterocytes. Phage binding studies indicated that the mutant had a cell surface alteration that precluded phage attachment. All phenotypes associated with the mutation could be complemented in trans by a single open reading frame (ORF) that corresponded to the ORF interrupted by the Tn917 insertion. The complementation effected was, in all cases, at a level indistinguishable from that of the parent. The Tn917 insertion interrupted a gene that is predicted to encode a group 2 glycosyl transferase (provisionally designated glcV). A similar glcV gene is present in Listeria welshimeri and Listeria innocua and in some serotypes of L. monocytogenes. We speculate that the loss of the glcV product results in a defective phage receptor and that this alteration coincidentally influences a feature of the normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, did not alter the mutant`s ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appeared to alter a subsequent step in intracellular replication measured by a reduction in plaque-forming efficiency and plaque size. In vivo, the mutant was undetectable in the liver and spleen 48 h after oral inoculation. The mutation is significant in part because it is one of the few that produce attenuation when the mutant is delivered orally. |
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Sean R. Stowell
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Connie M. Arthur
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Kristin A. Slanina
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John R. Horton
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David F. Smith
and
Richard D. Cummings
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Human galectins have distinct and overlapping biological roles in immunological homeostasis. However, the underlying differences among galectins in glycan binding specificity regulating these functions are unclear. Galectin-8 (Gal-8), a tandem repeat galectin, has two distinct carbohydrate recognition domains (CRDs) that may cross-link cell surface counter receptors. Here we report that each Gal-8 CRD has differential glycan binding specificity and that cell signaling activity resides in the C-terminal CRD. Full-length Gal-8 and recombinant individual domains (Gal-8N and Gal-8C) bound to human HL60 cells, but only full-length Gal-8 signaled phosphatidylserine (PS) exposure in cells, which occurred independently of apoptosis. Although desialylation of cells did not alter Gal-8 binding, it enhanced cellular sensitivity to Gal-8-induced PS exposure. By contrast, HL60 cell desialylation increased binding by Gal-8C but reduced Gal-8N binding. Enzymatic reduction in surface poly-N-acetyllactosamine (polyLacNAc) glycans in HL60 cells reduced cell surface binding by Gal-8C but did not alter Gal-8N binding. Cross-linking and light scattering studies showed that Gal-8 is dimeric, and studies on individual subunits indicate that dimerization occurs through the Gal-8N domain. Mutations of individual domains within full-length Gal-8 showed that signaling activity toward HL60 cells resides in the C-terminal domain. In glycan microarray analyses, each CRD of Gal-8 showed different binding, with Gal-8N recognizing sulfated and sialylated glycans and Gal-8C recognizing blood group antigens and polyLacNAc glycans. These results demonstrate that Gal-8 dimerization promotes functional bivalency of each CRD, which allows Gal-8 to signal PS exposure in leukocytes entirely through C-terminal domain recognition of polyLacNAc glycans. |
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