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• W2097738501 abstract "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 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 (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (629) Google Scholar, 2Balandreau J. Viallard V. Cournoyer B. Coenye T. Laevens S. Vandamme P. Appl. Environ. Microbiol. 2001; 67: 982-985Crossref PubMed Scopus (124) Google Scholar). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (629) Google Scholar) 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) 5The abbreviations used are: APantimicrobial peptideDQF-COSYdouble quantum filtered correlation spectroscopyd-QuiNd-quinovosamineHMBCheteronuclear multiple bond correlationHSQCheteronuclear single quantum coherenceKdo3-deoxy-d-manno-octulosonic acidKod-glycero-d-talo-octulosonic acidl-Ara4N4-amino-4-deoxy-l-arabinoseLPSlipopolysaccharidel-Rhal-rhamnoseMALDImatrix-assisted laser desorption ionizationMSmass spectrometryNOEnuclear Overhauser effectOSoligosaccharidePmBpolymyxin BTOCSYtotal correlation spectroscopyTOFtime-of-flightT-ROESYtransverse rotating-frame Overhauser enhancement spectroscopyDMEMDulbecco’s modified Eagle’s mediumFBSfetal bovine serumdansyl5-dimethylaminonaphthalene-1-sulfonylTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBcCVB. cenocepacia-containing vacuole. 5The abbreviations used are: APantimicrobial peptideDQF-COSYdouble quantum filtered correlation spectroscopyd-QuiNd-quinovosamineHMBCheteronuclear multiple bond correlationHSQCheteronuclear single quantum coherenceKdo3-deoxy-d-manno-octulosonic acidKod-glycero-d-talo-octulosonic acidl-Ara4N4-amino-4-deoxy-l-arabinoseLPSlipopolysaccharidel-Rhal-rhamnoseMALDImatrix-assisted laser desorption ionizationMSmass spectrometryNOEnuclear Overhauser effectOSoligosaccharidePmBpolymyxin BTOCSYtotal correlation spectroscopyTOFtime-of-flightT-ROESYtransverse rotating-frame Overhauser enhancement spectroscopyDMEMDulbecco’s modified Eagle’s mediumFBSfetal bovine serumdansyl5-dimethylaminonaphthalene-1-sulfonylTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBcCVB. cenocepacia-containing vacuole. (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (629) Google Scholar, 3Aaron S.D. Ferris W. Henry D.A. Speert D.P. Macdonald N.E. Am. J. Respir. Crit. Care Med. 2000; 161: 1206-1212Crossref PubMed Scopus (201) Google Scholar).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 (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar). 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 (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar). The lipid A also anchors the LPS molecule to the outer leaflet of the outer membrane and accounts for the endotoxic activity of LPS (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar, 6Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (895) Google Scholar). 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′ (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar). 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 (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar). 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 (7Isshiki Y. Kawahara K. Zähringer U. Carbohydr. Res. 1998; 313: 21-27Crossref PubMed Scopus (55) Google Scholar). 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) (8Silipo A. Molinaro A. Cescutti P. Bedini E. Rizzo R. Parrilli M. Lanzetta R. Glycobiology. 2005; 15: 561-570Crossref PubMed Scopus (48) Google Scholar) 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 (7Isshiki Y. Kawahara K. Zähringer U. Carbohydr. Res. 1998; 313: 21-27Crossref PubMed Scopus (55) Google Scholar, 9Isshiki Y. Zähringer U. Kawahara K. Carbohydr. Res. 2003; 338: 2659-2666Crossref PubMed Scopus (28) Google Scholar). Although this is also the case for the inner core OS of B. cenocepacia J2315 (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (56) Google Scholar), 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 (11Molinaro A. De Castro C. Lanzetta R. Evidente A. Parrilli M. Holst O. J. Biol. Chem. 2002; 277: 10058-10063Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).Burkholderia species, including B. cenocepacia, are intrinsically resistant to human and non-human APs such as these produced by airway epithelial cells (12Baird R.M. Brown H. Smith A.W. Watson M.L. Immunopharmacology. 1999; 44: 267-272Crossref PubMed Scopus (29) Google Scholar, 13Devine D.A. Mol. Immunol. 2003; 40: 431-443Crossref PubMed Scopus (72) Google Scholar), human Β-defensin 3 (14Sahly H. Schubert S. Harder J. Rautenberg P. Ullmann U. Schröder J. Podschun R. Antimicrob. Agents Chemother. 2003; 47: 1739-1741Crossref PubMed Scopus (67) Google Scholar), human neutrophil peptides (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (107) Google Scholar), and polymyxin B (PmB) (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (107) Google Scholar, 16Burtnick M.N. Woods D.E. Antimicrob. Agents Chemother. 1999; 43: 2648-2656Crossref PubMed Google Scholar). 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 (13Devine D.A. Mol. Immunol. 2003; 40: 431-443Crossref PubMed Scopus (72) Google Scholar). 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 (17Cox A.D. Wilkinson S.G. Mol. Microbiol. 1991; 5: 641-646Crossref PubMed Scopus (82) Google Scholar), 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 (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (107) Google Scholar). 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.DISCUSSIONWe have identified gene loci responsible for the biosynthesis of the core OS moiety in B. cenocepacia K56-2. This allowed us to create a set of core OS-deficient mutants, three of which were used to determine the structure of the core OS. The mutant XOA3 has an insertional mutation in the wbxE gene that encodes a glycosyltransferase involved in O antigen synthesis, resulting in the production of lipid A-core OS and a partial O antigen unit (34Ortega X. Hunt T.A. Loutet S. Vinion-Dubiel A.D. Datta A. Choudhury B. Goldberg J.B. Carlson R. Valvano M.A. J. Bacteriol. 2005; 187: 1324-1333Crossref PubMed Scopus (61) Google Scholar). This mutation recreates the same LPS phenotype as observed in strain J2315, whose structure has been recently reported (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (56) Google Scholar). In J2315, the spontaneous insertion of the IS402 element in wbxE causes the formation of a lipid A-core OS with a partial O antigen repeat that cannot be polymerized (34Ortega X. Hunt T.A. Loutet S. Vinion-Dubiel A.D. Datta A. Choudhury B. Goldberg J.B. Carlson R. Valvano M.A. J. Bacteriol. 2005; 187: 1324-1333Crossref PubMed Scopus (61) Google Scholar). The core OS structures in J2315 and XOA3 strains are identical, except for the presence of α-galactose instead of α-Glc linked to the outer core branched 3,7-disubstituted Hep. The galactose in the J2315 strain has an additional α-glucose at the O-6 position. The mutant XOA7, which has an inactivated waaL gene, lacks the terminal Rha-QuiNAc disaccharide found in the outer core OS of strains XOA3 and J2315 (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (56) Google Scholar). From these data, we conclude that the Rha-QuiNAc disaccharide is a remnant of the interrupted O antigen in these strains. The O antigen in B. cenocepacia K56-2 is synthesized via the ABC export pathway (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar, 34Ortega X. Hunt T.A. Loutet S. Vinion-Dubiel A.D. Datta A. Choudhury B. Goldberg J.B. Carlson R. Valvano M.A. J. Bacteriol. 2005; 187: 1324-1333Crossref PubMed Scopus (61) Google Scholar). This particular mode of O antigen synthesis requires an adaptor sugar bound to undecaprenyl-PP, to which the remainder of the O antigen repeating units become attached (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar). Based on our structural information, combined with the mutagenesis data, we conclude that the QuiNAc residue is the adaptor sugar for the O antigen synthesis in B. cenocepacia K56-2. Furthermore, our data support the conclusion that the Β-d-QuiNAc-(1→7)-α-ld-Hep linkage is made by the WaaL O antigen ligase, explaining why the Rha-QuiNAc disaccharide is absent in strain XOA7. The terminal rhamnose in the core OS of the XOA3 mutant, is likely the first sugar of the repeating O unit, which we have previously established as a Rha-GalNAc-GalNAc trisaccharide (34Ortega X. Hunt T.A. Loutet S. Vinion-Dubiel A.D. Datta A. Choudhury B. Goldberg J.B. Carlson R. Valvano M.A. J. Bacteriol. 2005; 187: 1324-1333Crossref PubMed Scopus (61) Google Scholar), but which cannot be completed because of the mutation in the WbxE glycosyltransferase. Therefore, our data also suggest that WbxE encodes a GalNAc transferase. Current work in our laboratories is under way to resolve the complete biosynthesis pathway of the O antigen component of the B. cenocepacia LPS.The structure of the core OS in the XOA8 strain revealed a major truncation, consistent with the migration pattern of the LPS in SDS-PAGE. The mutated wabO gene in XOA, encodes a putative glycosyltransferase, and based on the elucidated structure and the short lipid A-core OS band produced by the mutant strain, we predict that WabO protein is the glucosyltransferase responsible for the glucosylation of HepI (Fig. 4C). Also, the structural data suggest that glucosylation of HepI may be a requirement for the glucosylation of HepII and the continuation of the extension of the lipid A-core OS. Analysis of the LPS structure of XOA17, currently in progress in our laboratories, would be required to unequivocally support this conclusion.The composition of the lipid A moiety of B. cenocepacia K56-2 was identical to that of B. cepacia (8Silipo A. Molinaro A. Cescutti P. Bedini E. Rizzo R. Parrilli M. Lanzetta R. Glycobiology. 2005; 15: 561-570Crossref PubMed Scopus (48) Google Scholar) and B. cenocepacia J2315 (10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (56) Google Scholar), and the inner core OS is also composed of the trisaccharide Kdo-Ko-l-Ara4N, as described before (7Isshiki Y. Kawahara K. Zähringer U. Carbohydr. Res. 1998; 313: 21-27Crossref PubMed Scopus (55) Google Scholar, 9Isshiki Y. Zähringer U. Kawahara K. Carbohydr. Res. 2003; 338: 2659-2666Crossref PubMed Scopus (28) Google Scholar, 10Silipo A. Molinaro A. Ieranó T. De Soyza A. Sturiale L. Garozzo D. Aldridge C. Corris P.A. Khan C.M. Lanzetta R. Parrilli M. Chemistry. 2007; 13: 3501-3511Crossref PubMed Scopus (56) Google Scholar). l-Ara4N is found as a nonstoichiometric substitution in the core OS of several bacteria such as Proteus penneri (43Holst O. FEMS Microbiol. Lett. 2007; 271: 3-11Crossref PubMed Scopus (111) Google Scholar). The presence of l-Ara4N as a component of the core OS is unusual as this positively charged sugar is commonly found as a modification of the lipid A in response to specific environmental signals (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar), but it is rarely found as a component of the core OS. In a previous study, we demonstrated that the synthesis of l-Ara4N is essential for the viability of B. cenocepacia (19Ortega X.P. Cardona S.T. Brown A.R. Loutet S.A. Flannagan R.S. Campopiano D.J. Govan J.R. Valvano M.A. J. Bacteriol. 2007; 189: 3639-3644Crossref PubMed Scopus (84) Google Scholar), and more recently we have observed the same requirement for a B. cenocepacia mutant defective in the production of UDP-glucuronic acid, a precursor for UDP-l-Ara4N (44Loutet S.A. Bartholdson S.J. Govan J.R.W. Campopiano D.J. Valvano M.A. Microbiology. 2009; 155: 2029-2039Crossref PubMed Scopus (25) Google Scholar), and speculated that the presence of l-Ara4N in the core OS may be critical for the completion of the synthesis or the assembly of the LPS. The presence of l-Ara4N linked to Ko is not universal to all Burkholderia, as it has been recently shown that a heptose residue is located at this position in the core OS of “B. cepacia” serotype O4 (45Masoud H. Perry M.B. Brisson J.R. Uhrin D. Li J. Richards J.C. Glycobiology. 2009; 19: 462-471Crossref PubMed Scopus (9) Google Scholar). This strain also has a very different core OS structure than the ones we have determined for B. cenocepacia ET12 strains. Unfortunately, given the heterogeneity of the genus Burkholderia (46Coenye T. Vandamme P. Environ. Microbiol. 2003; 5: 719-729Crossref PubMed Scopus (647) Google Scholar), the lack of a detailed taxonomical assignment for B. cepacia serotype O4 makes it difficult to compare with other strains. The presence of the Kdo analog Ko is also unusual. Ko has also been found as a substitute for Kdo II in the LPS of Yersinia pestis and Serratia marcescens and for Kdo I in the LPS of Acinetobacter haemolyticus (5Caroff M. Karibian D. Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar), but the function of such substitution and its biosynthesis are unknown (43Holst O. FEMS Microbiol. Lett. 2007; 271: 3-11Crossref PubMed Scopus (111) Google Scholar). On the other hand, the LPS of other Burkholderia species such as B. caryophylli do not contain Ko (11Molinaro A. De Castro C. Lanzetta R. Evidente A. Parrilli M. Holst O. J. Biol. Chem. 2002; 277: 10058-10063Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 47Molinaro A. Lindner B. De Castro C. Nolting B. Silipo A. Lanzetta R. Parrilli M. Holst O. Chemistry. 2003; 9: 1542-1548Crossref PubMed Scopus (29) Google Scholar).A heptoseless mutant of B. cepacia has been reported as a result of a mutation of the heptosyltransferase I waaC (48Gronow S. Noah C. Blumenthal A. Lindner B. Brade H. J. Biol. Chem. 2003; 278: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The structure of the core OS for this mutant is the same as the one we have determined for the B. cenocepacia K56-2 heptoseless mutant SAL1 6S. A. Loutet and M. A. Valvano, unpublished observations. (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (107) Google Scholar). Thus, the inner core OS is highly conserved in Burkholderia species.The panel of isogenic strains with gradual truncations in their lipid A-core OS allowed us to investigate the relationship between LPS and the extraordinary resistance of B. cenocepacia against cationic APs. Although, as it was shown previously with B. cepacia (49Moore R.A. Hancock R.E. Antimicrob. Agents Chemother. 1986; 30: 923-926Crossref PubMed Scopus (98) Google Scholar), PmB binds to P. aeruginosa much better than to B. cenocepacia K56-2, progressive truncation of the lipid A-core OS leads to increased PmB binding. These findings support the notion that the cell envelope of B. cenocepacia has unusual characteristics that enable it to act as a barrier against APs. Although significantly more sensitive than the wild type strain to PmB, our core mutants are still much more resistant to PmB than other organisms such as Salmonella and E. coli with intact core OS. Thus B. cenocepacia must possess additional mechanisms that make these bacteria extremely resistant to APs.Taking advantage of the set of isogenic mutants in B. cenocepacia K56-2 that range from the formation of a full-length LPS O antigen (parental strain) to a mutant producing heptoseless lipid A-core OS (CCB1), we also investigated the biological role of LPS in adhesion to epithelial cells and intracellular survival in macrophages. Our results demonstrated that O antigen production by B. cenocepacia prevents bacterial adhesion to epithelial cells. This suggests that the O antigen in these bacteria masks bacterial surface molecules that can interact with epithelial cell receptors, or alternatively, the exposed core OS residues are themselves ligands for binding. We considered the latter hypothesis less likely given that all the core OS mutants with progressive truncations showed increased adhesion, suggesting that no specific sugar residue is required for adhesion, in contrast to recent observations in other bacteria (50Hoare A. Bittner M. Carter J. Alvarez S. Zaldívar M. Bravo D. Valvano M.A. Contreras I. Infect. Immun. 2006; 74: 1555-1564Crossref PubMed Scopus (54) Google Scholar). It has been previously shown that Bcc isolates can survive intracellularly within amoebae (51Marolda C.L. Hauröder B. John M.A. Michel R. Valvano M.A. Microbiology. 1999; 145: 1509-1517Crossref PubMed Scopus (112) Google Scholar), respiratory epithelial cells (38Burns J.L. Jonas M. Chi E.Y. Clark D.K. Berger A. Griffith A. Infect. Immun. 1996; 64: 4054-4059Crossref PubMed Google Scholar), and macrophages (39Saini L.S. Galsworthy S.B. John M.A. Valvano M.A. Microbiology. 1999; 145: 3465-3475Crossref PubMed Scopus (111) Google Scholar, 40Lamothe J. Huynh K.K. Grinstein S. Valvano M.A. Cell. Microbiol. 2007; 9: 40-53Crossref PubMed Scopus (83) Google Scholar). Others have reported that the LPS O antigen plays an essential role in internalization and survival of the related bacterium B. pseudomallei in macrophages (52Arjcharoen S. Wikraiphat C. Pudla M. Limposuwan K. Woods D.E. Sirisinha S. Utaisincharoen P. Infect. Immun. 2007; 75: 4298-4304Crossref PubMed Scopus (50) Google Scholar). Our data investigating the ability of the various mutants with defects in lipid A-core OS production to survive intracellularly in macrophages revealed that only the heptoseless mutant CCB1 is impaired for survival. These results are somewhat surprising and indicate that the ability of B. cenocepacia to survive in macrophages does not correlate with the level of truncation of the core OS. B. cenocepacia can resist oxidative (53Keith K.E. Hynes D.W. Sholdice J.E. Valvano M.A. Microbiology. 2009; 155: 1004-1015Crossref PubMed Scopus (36) Google Scholar, 54Bylund J. Burgess L.A. Cescutti P. Ernst R.K. Speert D.P. J. Biol. Chem. 2006; 281: 2526-2532Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and nonoxidative (55Speert D.P. Bond M. Woodman R.C. Curnutte J.T. J. Infect. Dis. 1994; 170: 1524-1531Crossref PubMed Scopus (132) Google Scholar) intracellular killing mechanisms, and the latter mainly depend on APs. Therefore, the intracellular survival of the other mutants with core OS truncations, despite their increased sensitivity to PmB in vitro, suggest that either the AP concentration in BcCVs is not enough to compromise the viability of these mutants or other factors are involved. It is possible that the ability of B. cenocepacia to survive intracellularly is highly dependent on the stability of the outer membrane cell envelope, which may be only seriously perturbed in the presence of a drastically truncated lipid A-core OS. This is in agreement with other observations indicating that B. cenocepacia heptoseless mutants have defects in motility and increased permeability to other hydrophobic compounds in addition to antimicrobial peptides (15Loutet S.A. Flannagan R.S. Kooi C. Sokol P.A. Valvano M.A. J. Bacteriol. 2006; 188: 2073-2080Crossref PubMed Scopus (107) Google Scholar).6In conclusion, we have identified the genes involved in the biosynthesis of the core OS in B. cenocepacia, performed the structural analysis of the core OS, and assigned function to most of the genes of the core OS loci. We also demonstrated that progressive truncations of core OS are associated with a dramatic reduction in the resistance to PmB, which inversely correlates with increasing binding of this peptide to the bacterial cell envelope of the mutant strains. Finally, we also show that the majority of the core OS is expendable for intracellular survival of B. cenocepacia in macrophages, whereas the O antigen contributes to prevent bacterial adhesion to epithelial cells. Further investigations are underway in our laboratories to better elucidate the characteristic of the outer membrane and the LPS molecules that contribute to the extraordinary resistance of B. cenocepacia to a wide range of antimicrobial molecules, including APs. Burkholderia cenocepacia is a Gram-negative opportunistic pathogen ubiquitously found in the environment (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (629) Google Scholar, 2Balandreau J. Viallard V. Cournoyer B. Coenye T. Laevens S. Vandamme P. Appl. Environ. Microbiol. 2001; 67: 982-985Crossref PubMed Scopus (124) Google Scholar). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (629) Google Scholar) 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) 5The abbreviations used are: APantimicrobial peptideDQF-COSYdouble quantum filtered correlation spectroscopyd-QuiNd-quinovosamineHMBCheteronuclear multiple bond correlationHSQCheteronuclear single quantum coherenceKdo3-deoxy-d-manno-octulosonic acidKod-glycero-d-talo-octulosonic acidl-Ara4N4-amino-4-deoxy-l-arabinoseLPSlipopolysaccharidel-Rhal-rhamnoseMALDImatrix-assisted laser desorption ionizationMSmass spectrometryNOEnuclear Overhauser effectOSoligosaccharidePmBpolymyxin BT" @default.
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• W2097738501 date "2009-08-01" @default.
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• W2097738501 title "Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide" @default.
• W2097738501 cites W1841321987 @default.
• W2097738501 cites W1844223772 @default.
• W2097738501 cites W1855513913 @default.
• W2097738501 cites W1965250197 @default.
• W2097738501 cites W1995622181 @default.
• W2097738501 cites W2008602270 @default.
• W2097738501 cites W2009032652 @default.
• W2097738501 cites W2010683661 @default.
• W2097738501 cites W2014117083 @default.
• W2097738501 cites W2017056768 @default.
• W2097738501 cites W2021208459 @default.
• W2097738501 cites W2021599177 @default.
• W2097738501 cites W2034483565 @default.
• W2097738501 cites W2036125189 @default.
• W2097738501 cites W2039811006 @default.
• W2097738501 cites W2040581338 @default.
• W2097738501 cites W2049745094 @default.
• W2097738501 cites W2049839212 @default.
• W2097738501 cites W2059417131 @default.
• W2097738501 cites W2082389567 @default.
• W2097738501 cites W2091447158 @default.
• W2097738501 cites W2093188199 @default.
• W2097738501 cites W2095885982 @default.
• W2097738501 cites W2097377990 @default.
• W2097738501 cites W2098942474 @default.
• W2097738501 cites W2099925167 @default.
• W2097738501 cites W2101266397 @default.
• W2097738501 cites W2102484565 @default.
• W2097738501 cites W2106832324 @default.
• W2097738501 cites W2111842378 @default.
• W2097738501 cites W2117541427 @default.
• W2097738501 cites W2118512529 @default.
• W2097738501 cites W2123219111 @default.
• W2097738501 cites W2123905657 @default.
• W2097738501 cites W2126006315 @default.
• W2097738501 cites W2126290991 @default.
• W2097738501 cites W2126636554 @default.
• W2097738501 cites W2127435259 @default.
• W2097738501 cites W2128654130 @default.
• W2097738501 cites W2131392955 @default.
• W2097738501 cites W2132666490 @default.
• W2097738501 cites W2144369716 @default.
• W2097738501 cites W2145253648 @default.
• W2097738501 cites W2147985190 @default.
• W2097738501 cites W2151201851 @default.
• W2097738501 cites W2151906718 @default.
• W2097738501 cites W2151999391 @default.
• W2097738501 cites W2154392123 @default.
• W2097738501 cites W2158937196 @default.
• W2097738501 cites W2165955930 @default.
• W2097738501 cites W2171780940 @default.
• W2097738501 cites W2317729118 @default.
• W2097738501 cites W4234317413 @default.
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