Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide: TRUNCATION OF THE CORE OLIGOSACCHARIDE LEADS TO INCREASED BINDING AND SENSITIVITY TO POLYMYXIN B*

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.Burkholderia cenocepacia is a Gram-negative opportunistic pathogen ubiquitously found in the environment (1, 2). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1) such as those with cystic fibrosis and chronic granulomatous disease. Infected cystic fibrosis patients commonly develop chronic lung infections that are very difficult to treat because these bacteria are intrinsically resistant to virtually all clinically useful antibiotics as well as antimicrobial peptides (APs)⁵ (1, 3).Lipopolysaccharide (LPS) is the major surface component of Gram-negative bacteria and consists of lipid A, core oligosaccharide (OS), and in some bacteria O-specific polysaccharide or O antigen (4, 5). The O antigen acts as a protective barrier against desiccation, phagocytosis, and serum complement-mediated killing, whereas the core OS and the lipid A contribute to maintain the integrity of the outer membrane (4, 5). The lipid A also anchors the LPS molecule to the outer leaflet of the outer membrane and accounts for the endotoxic activity of LPS (4, 6). Lipid A is a bisphosphorylated β-1,6-linked glucosamine disaccharide substituted with fatty acids ester-linked at positions 3 and 3′ and amide-linked at positions 2 and 2′ (4). The core OS can be subdivided into the inner core and outer core. The inner core OS typically consists of one or two 3-deoxy-d-manno-octulosonic acid (Kdo) residues linked to the lipid A and three l-glycero-d-manno-heptose residues linked to the first Kdo (4). The outer core OS in enteric bacteria typically consists of 8–12 branched sugars linked to heptose II of the inner core. As a result of phosphate groups on the lipid A and core OS, the bacterial surface has a net negative charge. This plays an important role in the interaction of the bacterial surface with positively charged compounds such as cationic APs, which are cationic amphipathic molecules that kill bacteria by membrane permeabilization. In response to a series of environmental conditions such as low magnesium or high iron, bacteria can express modified LPS molecules that result in a less negative surface. This reduces the binding of APs and promotes resistance to these compounds. Previous studies have shown that Burkholderia LPS molecules possess unique properties. For example, Kdo cannot be detected by classic colorimetric methods in LPS from Burkholderia pseudomallei and Burkholderia cepacia, and the covalent linkage between Kdo and lipid A is more resistant to acid hydrolysis than in conventional LPS molecules (7). In B. cepacia, 4-amino-4-deoxy-l-arabinose (l-Ara4N) is bound to the lipid A by a phosphodiester linkage at position 4 of the nonreducing glucosamine (GlcN II) (8) and is also present as a component of the core OS. Also, instead of two Kdo molecules, the B. cepacia core OS has only one Kdo and the unusual Kdo analog, d-glycero-d-talo-octulosonic acid (Ko), which is nonstoichiometrically substituted with l-Ara4N forming a 1→8 linkage with α-Ko (7, 9). Although this is also the case for the inner core OS of B. cenocepacia J2315 (10), it is not a common feature for the core OS in all Burkholderia. For example, the inner core of Burkholderia caryophylli consists of two Kdo residues and does not possess l-Ara4N (11).Burkholderia species, including B. cenocepacia, are intrinsically resistant to human and non-human APs such as these produced by airway epithelial cells (12, 13), human β-defensin 3 (14), human neutrophil peptides (15), and polymyxin B (PmB) (15, 16). The minimum inhibitory concentration determined for some of these peptides in several Burkholderia species is greater than 500 μg/ml, which could aid these microorganisms during colonization of the respiratory epithelia (13). It has been proposed that the resistance of B. cepacia to cationic APs stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide (17), but a systematic analysis of the molecular basis of AP resistance in B. cenocepacia and other Burkholderia is lacking. We have previously reported that a heptoseless B. cenocepacia mutant (SAL1) is significantly more sensitive than the parental clinical strain K56-2 to APs (15). This mutant has a truncated inner core and lacks the outer core, suggesting that a complete core OS is required for resistance of B. cenocepacia to APs.Apart from heptoses, the role of other sugar moieties of the B. cenocepacia core OS in AP resistance is not known. In this study, we report the structure of the core OS for B. cenocepacia strain K56-2 and its isogenic mutants XOA3, XOA7, and XOA8, which carry various core OS truncations. The structural analysis, combined with mutagenesis, allowed us to assign function to the majority of the genes involved in core OS biosynthesis and ligation of the O antigen and to establish that the degree of truncation of the core OS correlates with increased binding and bacterial sensitivity to PmB in vitro and reduced bacterial intracellular survival in macrophages.