Abstract

srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis. Recently, SRF-3 was characterized as a nucleotide sugar transporter of the Golgi apparatus occurring exclusively in hypodermal seam cells, pharyngeal cells, and spermatheca. Based on the above observations, we hypothesized that srf-3 may have altered glyconjugates that may enable the mutant nematode to grow unaffected in the presence of the above pathogenic bacteria. Following analyses of N- and O-linked glycoconjugates of srf-3 and wild type nematodes using a combination of enzymatic degradation, permethylation, and mass spectrometry, we found in srf-3 a 65% reduction of acidic O-linked glycoconjugates containing glucuronic acid and galactose as well as a reduction of N-linked glycoconjugates containing galactose and fucose. These results are consistent with the specificity of SRF-3 for UDP-galactose and strongly suggest that the above glycoconjugates play an important role in allowing adhesion of M. nematophilum or Y. pseudotuberculosis biofilm to wild type C. elegans. Furthermore, because seam cells as well as pharyngeal cells secrete their glycoconjugates to the cuticle and surrounding surfaces, the results also demonstrate the critical role of these cells and their secreted glycoproteins in nematode-bacteria interactions and offer a mechanistic basis for strategies to block such recognition processes.

Highlights

  • The soil is the natural habitat for C. elegans, where it comes in contact with commensal and pathogenic bacteria

  • From the ‡Department of Molecular and Cell Biology, Boston University, Goldman School of Dental Medicine, Boston, Massachusetts 02118-2526 and the §Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118-2526 srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis

  • Rationale for Workup Strategy—Based on our knowledge that C. elegans srf-3 mutants are deficient in a Golgi apparatus transporter for UDP-Gal and UDP-GlcNAc, we decided to assess the structures of N- and O-glycans released from protein extracts of whole mixed stages of srf-3 and wild type, parental N2 Bristol strain nematodes

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Summary

EXPERIMENTAL PROCEDURES

Isolation of N-Glycans—The glycoprotein-rich fraction was isolated from 10 –15 g of C. elegans as previously described [16].

Golgi Nucleotide Sugar Transporters
RESULTS
DISCUSSION
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