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We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors. Italian Ministry of Education, University and Research Deutsche Forschungsgemeinschaft (DFG)

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape., Author Summary The only habitat of Helicobacter pylori is the human stomach, where it can promote stomach ulcers and cancer. Cells lining the stomach are protected from luminal acid by a thick layer of gastric mucus composed of polymerized gastric mucins. Gastric mucin undergoes a physical transition between a viscoelastic solution at neutral pH to a viscoelastic gel-like state at low pH. Helical rod shape in bacteria has been suggested to enhance swimming velocity in viscous solutions by a cork-screw mechanism, but H. pylori mutants lacking helical twist show normal swimming velocity in viscous polymer solutions used in prior studies comparing motility across bacterial species. These same mutants, however, show diminished colonization suggesting helical shape promotes stomach infection by another mechanism. Here we identified Csd4, a protease of cell wall tripeptides, which induces curvature in the cell body independently from the changes in cell wall crosslinking previously shown to promote helical twist. Cells lacking Csd4 form straight rods that also show colonization defects but normal velocity in several viscous polymer solutions. Upon examination of motility in gel-like media, however, we discovered that elimination or exaggeration of cell curvature perturbs motility. Thus H. pylori's helical shape may aid penetration of gel-like stomach mucus.

Publisher Summary This chapter provides an overview on the diversity and variability of the peptidoglycan structures found in various bacteria. It summarizes the biophysical properties of peptidoglycan and discusses its architecture. Peptidoglycan is responsible for the osmotic stability by encasing the cytoplasmic membrane of most bacteria. It is a unique, net-like polymer made of glycan strands that are cross-linked by unusual peptides. Although there are some common structural features of peptidoglycan, there is large variability in the structure of bacterial peptidoglycan due to differences in the amino acid sequence, types of cross-links, and the presence or absence of secondary modifications in both the glycan strands and peptides. The glycans are synthesized as oligo- ( N -acetylglucosamine- N -acetylmuramic acid) strands of variable length and become modified shortly after their synthesis in many species. The peptides contain L- and D-amino acids and have a species-specific amino acid composition, sequence, and type of cross-linkage. Maturation of peptidoglycan involves cleavage of amide bonds in the peptides resulting in a nonhomogeneous structure of the polymer. Introduction of secondary modifications and maturation are the reasons for the complex composition of mature peptidoglycan often manifesting in a species-specific fine structure.

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.