Phosphoethanolamine addition to the Heptose I of the Lipopolysaccharide modifies the inner core structure and has an impact on the binding of Polymyxin B to the Escherichia coli outer membrane.

Phosphoethanolamine (pEtN) decoration of E. coli Lipopolysaccharide (LPS) provides resistance to the antimicrobial Polymyxin B (PolB). While EptA and EptB enzymes catalyze the addition of pEtN to the Lipid A and Kdo (pEtN-Kdo-Lipid A), EptC catalyzes the pEtN addition to the Heptose I (pEtN-HeptI). In this study, we investigated the contribution of pEtN-HeptI to PolB resistance using eptA/eptB and eptC deficient E. coli K12 and its wild-type parent strains. These mutations were shown to decrease the antimicrobial activity of PolB on cells grown under pEtN-addition inducing conditions. Furthermore, the 1-N-phenylnapthylamine uptake assay revealed that in vivo PolB has a reduced OM-permeabilizing activity on the ΔeptA/eptB strain compared with the ΔeptC strain. In vitro, the changes in size and zeta potential of LPS-vesicles indicate that pEtN-HeptI reduce the PolB binding, but in a minor extent than pEtN-Kdo-Lipid A. Molecular dynamics analysis revealed the structural basis of the PolB resistance promoted by pEtN-HeptI, which generate a new hydrogen-bonding networks and a denser inner core region. Altogether, the experimental and theoretical assays shown herein indicate that pEtN-HeptI addition promote an LPS conformational rearrangement, that could act as a shield by hindering the accession of PolB to inner LPS-targets moieties.

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone.

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.

Intestinal passive absorption of water-soluble compounds by sparrows: effect of molecular size and luminal nutrients.

We tested predictions that: (1) absorption of water-soluble probes decreases with increasing molecular size, consistent with movement through effective pores in epithelia, and (2) absorption of probes is enhanced when measured in the presence of luminal nutrients, as predicted for paracellular solvent drag. Probes (L-arabinose, L-rhamnose, perseitol, lactulose; MW 150.1-342.3 Da) were gavaged in nonanesthetized House sparrows ( Passer domesticus), or injected into the pectoralis, and serially measured in plasma. Bioavailability was calculated as F=AUC by gavage/AUC by injection, where AUC is the area under the curve of plasma probe concentration vs. time. Consistent with predictions, F declined with probe size by 75% from the smallest to the largest probe, and absorption of probes increased by 40% in the presence of luminal glucose or food compared to a mannitol control. Absorption of water-soluble probes by sparrows is much higher than in humans, which is much higher than in rats. These differences seem mainly attributable to differences in paracellular solvent flux and less to differences in effective paracellular pore size.