Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response.

Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Pathways of thymidine hypermodification.

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.