Evaluation of Azido 3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Analogues for Click Chemistry-Mediated Metabolic Labeling of Myxococcus xanthus DZ2 Lipopolysaccharide.

Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N3) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N3-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N3-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N3-Kdo and 7-N3-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N3-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N3-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N3-Kdo is indeed a substrate of the enzyme, 7-N3-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N3-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N3-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

Total Synthesis of 6-Amino-2,6-dideoxy-α-Kdo from d-Mannose.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) biosynthetic pathway is a promising target in antibacterial drug discovery. Herein, we report the total synthesis of 6-amino-2,6-dideoxy-α-Kdo in 15 steps from d-mannose as a potential inhibitor of Kdo-processing enzymes. Key steps of the synthetic sequence involve a Horner-Wadsworth-Emmons reaction for the two-carbon chain homologation followed by either a 6-exo-trig Pd-catalyzed reductive cyclization or a tandem Staudinger/aza-Wittig reaction with concomitant α-iminoester reduction, enabling the α-stereoselective formation of the Kdo-like six-membered azacyclic ring.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.

Polysaccharides from Burkholderia species as targets for vaccine development, immunomodulation and chemical synthesis.

Covering: up to 2018 Burkholderia species are a vast group of human pathogenic, phytopathogenic, and plant- or environment-associated bacteria. B. pseudomallei, B. mallei, and B. cepacia complex are the causative agents of melioidosis, glanders, and cystic fibrosis-related infections, respectively, which are fatal diseases in humans and animals. Due to their high resistance to antibiotics, high mortality rates, and increased infectivity via the respiratory tract, B. pseudomallei and B. mallei have been listed as potential bioterrorism agents by the Centers for Disease Control and Prevention. Burkholderia species are able to produce a large network of surface-exposed polysaccharides, i.e., lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, which are virulence factors, immunomodulators, major biofilm components, and protective antigens, and have crucial implications in the pathogenicity of Burkholderia-associated diseases. This review provides a comprehensive and up-to-date account regarding the structural elucidation and biological activities of surface polysaccharides produced by Burkholderia species. The chemical synthesis of oligosaccharides mimicking Burkholderia polysaccharides is described in detail. Emphasis is placed on the recent research efforts toward the development of glycoconjugate vaccines against melioidosis and glanders based on synthetic or native Burkholderia oligo/polysaccharides.