Characterization of Lipopolysaccharides of Polymyxin‐Resistant and Polymyxin‐Sensitive Klebsiella pneumoniae O3

Abstract

Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniue 0 3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-~-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-~-arabinopyranose. In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-I .Keywords: Klebsiella pneumoniae ; lipopolysaccharide; lipid A ; structure ; polymyxin.In gram-negative enteric bacteria such as Escherichia coli and Salmonella typhimurium, resistance to the polycationic cyclic peptide antibiotic polymyxin B is associated with extensive cationic substitution of phosphate groups of the cell surface lipopolysaccharide (LPS) [l -61. Phosphate groups occur in LPS in the lipid part (termed lipid A) and in the proximal inner core oligosaccharide [7]. Such capping of LPS phosphates by 2-aminoethanol and 4-amino-4-deoxy-~-arabinopyranose (~-Arap4N) results in a shift of the electrostatic net charge of the LPS molecule towards cationicity, which decreases the polymyxin-binding tendency of the LPS and, as a consequence, that of the bacterial cell [I, 2, 8-10]. The degree of substitution of LPS phosphates appears to be genetically regulated. In S. typhimurium, capping of LPS phosphate takes place as a result of mutations in the prnr gene locus, and structural investigation of the wild-type and resistant-type lipopolysaccharides by phosphorus NMR has revealed that the ester-linked 4'-phosphate of lipid A becomes almost stoichiometrically esterified with ~-Arap4N, and the glycosidically linked diphosphate of lipid A becomes esterified with 2-aminoethanol in the LPS of polymyxin resistant-type pmrA 166, FIN-00300 Helsinki, FinlandA: in the LPS of polymyxin-resistant mutants, ~-Arap4N and 2-aminoethanol were shown to be present in lipid A in linkages similar to those found in S. typhimurium [6]. Capping of LPS phosphates, which is expected to affect the surface charge of bacteria, thus seems to be a general phenomenon, which may have important implications to bacterial virulence.Whereas E. coli and S. typhimurium differ very little in their lipid A and inner co...

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

“…Ion m/z 1,797 has been previously detected in Klebsiella lipid A and is consistent with two glucosamines, two phosphates, four R-3-hydroxymyristoyl primary acyl chains, and one myristate (C 14 ) and one laureate (C 12 ) as the secondary acyl chains (Fig. 2) (15,16,20,21).…”

“…The fact that the lipid A from the lpxM mutant contained 2-hydroxymyristate both in vivo and in vitro (m/z 1,630 and m/z 1,866) strongly suggests that Klebsiella LpxO (KpLpxO) hydroxylates the myristate (C 14 ) on the primary 2′-linked R-3-hydroxymyristoyl group. Mass spectrometry analysis has previously shown that the nonreducing glucosamine of Klebsiella lipid A is substituted with only one (amide-linked) R-3-hydroxymyristoyl group further acylated with myristate (C 14 ) or 2-hydroxymyristate (C 14:OH ) (20,21). In good agreement, we show that KpLpxO hydroxylated Yersinia enterocolitica O:8 myristate (C 14 ) on the primary 2′-linked R-3-hydroxymyristoyl group (peaks m/z 1,404 and m/z 1,813; SI Appendix, Fig.…”

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…These molecules often act via a combination of their positive charge, which enables them to interact with anionic molecules on the bacterial surface, in combination with their hydrophobic character, which results in a disruption of the bacterial membrane (54,55). The LPS is the target molecule of defensins in Gram-negative bacteria, and bacteria that are resistant acquire this resistance by modification to their LPS structures (56 -60); perhaps, one of the best known examples is the resistance acquired through the addition of aminoarabinose and ethanol amine groups to the LPS of S. typhimurium (57)(58)(59)(60). Because of the structural alteration of the LPS during bacteroid formation, bacteria and bacteroids were compared for their resistance to poly-L-lysine, polymyxin B, and melittin.…”

Structural Basis of a Physical Blockage Mechanism for the Interaction of Response Regulator PmrA with Connector Protein PmrD from Klebsiella pneumoniae

“…A PmrA-PmrB TCS, dealing with cationic antimicrobial peptides and polymyxin resistance in pathogens (6), is responsible for sensing the external stimuli caused directly or indirectly by excess Fe 3ϩ , low Mg 2ϩ , and mild acidic environments (7)(8)(9). Under this TCS, pathogens can alter the composition of their cell walls to resist neutralization of polymyxin for drug resistance and allow bacterial survival within macrophages by reducing the affinity with cationic antimicrobial peptides (10,11).…”