2597. Dolichos biflorus Agglutinin Binds to Pneumococcal Teichoic Acid and Lipoteichoic Acid

Background Dolichos biflorus agglutinin (DBA) is a lectin with a binding specificity toward α-linked N-acetylgalactosamine (α-GalNAc). While DBA is known to bind some, but not all, pneumococci, its target molecule has not been identified. Pneumococcus teichoic acid (TA) and lipoteichoic acid (LTA) have repeating units with α-GalNAc-(1→3)β-GalNAc decorated with phosphorylcholine (PC) at the O-6 positions. Two PC transferases, LicD1 and LicD2, mediate the attachment of PC to GalNAc residues while phosphorylcholine esterase (Pce) removes PCs attached to the terminal GalNAcs. We examined if DBA binds to the terminal α-GalNAc-(1→3)β-GalNAc created by Pce. Methods Fifteen pneumococcus strains expressing 14 different serotypes, including one non-encapsulated strain (R36A), were studied with flow cytometry (FC) and confocal fluorescence microscopy (CFM) for DBA binding. Pce enzyme activity was detected with a colorimetric assay using p-nitrophenyl-phosphorylcholine as the substrate. Mutant strains with pce knocked-out were constructed in R36A and D39 by replacing pce with Janus cassette. Both licD genes were sequenced for some of the strains. Results Ten of the 15 strains had Pce activity and all of them bound DBA (Table 1). When the pce gene was inactivated in two normally Pce-positive strains (R36A△pce and D39△pce), the strains did not show DBA binding by CFM (Figure 1). Thus, expression of Pce appears to be sufficient for expressing the DBA antigen. Of the five strains that had no Pce activity, two bound DBA. Sequencing of the licD genes in these two strains with positive DBA binding and negative Pce activity revealed one SNP in licD1 and four SNPs in licD2, resulting in a single amino acid difference each for LicD1 and LicD2, compared with R36A and D39. Conclusion DBA can bind to the terminal α-GalNAc-(1→3)β-GalNAc of pneumococcal TA and LTA, which is created by Pce. DBA binding is independent of capsule type. The unexpected binding of DBA to the two Pce-negative strains suggests that there is a Pce-independent mechanism for generating the target for DBA binding. Since LicD1 and LicD2 are involved in attaching PC to α-GalNAc-(1→3)β-GalNAc, we are now investigating their role in creating DBA targets independent of Pce. Disclosures All authors: No reported disclosures.

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro

Surface molecules of primitive male germ cells, gonocytes, are essential components for regulating cell adhesion and maintaining self-renewal in mammalian species. In domestic animals, the stage-specific glycan epitope α-N-acetylgalactosamine (GalNAc) is recognised by the lectin Dolichos biflorus agglutinin (DBA) and is found on the surface of gonocytes and spermatogonia. Gonocytes from bovine testis formed mouse embryonic stem-like cell colonies on plates that had been coated with DBA or extracellular matrix (ECM) components, such as gelatin (GN), laminin (LN) and poly-L-lysine (PLL). The number of colonies on the DBA-coated plate was significantly higher than that on the GN-, LN- and PLL-coated plates. Pretreating gonocytes with DBA to neutralise the terminal GalNAc residues strongly suppressed colony formation. Furthermore, expression of a germ cell-specific gene and pluripotency-related transcription factors was increased considerably on the DBA-coated plates. These results suggest that the GalNAc residues on gonocytes can recognise precoated DBA on plates and the resulting GalNAc–DBA complexes support germ cell and stem cell potentials of gonocytes in vitro. These glycan complexes, through the GalNAc epitope, may provide a suitable microenvironment for the adhesion and cell proliferation of gonocytes in culture.

The crypt cycle in mouse small intestinal epithelium

We have used a mutation-induced marker system in the intestine of mice heterozygous at the Dlb-1 locus, which determines the expression of binding sites for the lectin Dolichos biflorus agglutinin, and the frequency of clustering of mutated crypts with time as a means of investigating the frequency of the crypt fission process and the crypt cycle. Whole-mount preparations from heterozygous Dlb-1b/Dlb-1a mice were stained with a peroxidase conjugate of Dolichos biflorus agglutinin. Mutations at the Dlb-1b locus in crypt stem cells result in loss of DBA-Px binding in these cells and subsequently their progeny, which eventually results in a rare isolated single, unstained crypt. The subsequent development of pairs, triplets and clusters of negative staining crypts has been assumed to be the result of crypt fission. The frequency of these fission events has been measured in control untreated mice. These negative crypts are the result of spontaneous mutations. We have also looked at mutated crypts after treatment with N-nitroso-N-ethylurea or N-methyl-N'-nitro-N-nitrosoguanidine of young adult mice, which elevates the number of mutations. Our results suggest that the crypt cycle in control animals is very long, 187 +/- 44 weeks (3.6 years, i.e. essentially the life of a laboratory mouse). This implies that about a third of the crypts may divide once in the life of a mouse. After sufficient time for conversion of mixed crypts to monophenotypic crypts after mutagen treatment several clusters of negative crypts were seen.(ABSTRACT TRUNCATED AT 250 WORDS)