The binding mechanism of the virulence factor Streptococcus suis adhesin P subtype to globotetraosylceramide is associated with systemic disease.

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.

Glycosphingolipids Recognized by Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer) and lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAcß3Galß4Glcß1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as "lactosylceramide-binding". Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.

Porcine intestinal glycosphingolipids recognized by F6-fimbriated enterotoxigenic Escherichia coli.

One important virulence factor of enterotoxigenic Escherichia coli is their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. Here, the potential glycosphingolipid receptors of enterotoxigenic F6-fimbriated E. coli were examined by binding of purified F6 fimbriae, and F6-expressing bacteria, to glycosphingolipids on thin-layer chromatograms. When intestinal mucosal non-acid glycosphingolipids from single pigs were assayed for F6 binding capacity, a selective interaction with two glycosphingolipids was observed. The binding-active glycosphingolipids were isolated and characterized as lactotriaosylceramide (GlcNAcß3Galß4Glcß1Cer) and lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer). Further binding assays using a panel of reference glycosphingolipids showed a specific interaction between the F6 fimbriae and a number of neolacto core chain (Galß4GlcNAc) glycosphingolipids. In addition, an occasional binding of the F6 fimbriae to sulfatide, galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide was obtained. From the results we conclude that lactotriaosylceramide and lactotetraosylceramide are major porcine intestinal receptors for F6-fimbriated E. coli.