Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide

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 (661) Google Scholar, 2Balandreau J. Viallard V. Cournoyer B. Coenye T. Laevens S. Vandamme P. Appl. Environ. Microbiol. 2001; 67: 982-985Crossref PubMed Scopus (129) 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 (661) 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. (1Mahenthiralingam E. Urban T.A. Goldberg J.B. Nat. Rev. Microbiol. 2005; 3: 144-156Crossref PubMed Scopus (661) 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 (213) Google Scholar). antimicrobial peptide double quantum filtered correlation spectroscopy d-quinovosamine heteronuclear multiple bond correlation heteronuclear single quantum coherence 3-deoxy-d-manno-octulosonic acid d-glycero-d-talo-octulosonic acid 4-amino-4-deoxy-l-arabinose lipopolysaccharide l-rhamnose matrix-assisted laser desorption ionization mass spectrometry nuclear Overhauser effect oligosaccharide polymyxin B total correlation spectroscopy time-of-flight transverse rotating-frame Overhauser enhancement spectroscopy Dulbecco’s modified Eagle’s medium fetal bovine serum 5-dimethylaminonaphthalene-1-sulfonyl N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine B. cenocepacia-containing vacuole. 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 (3530) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. PubMed Scopus Google Scholar). The O antigen as a barrier and serum whereas the core OS and the lipid to the of the outer (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar, 5Caroff M. Karibian D. Carbohydr. Res. PubMed Scopus Google Scholar). The lipid the to the outer of the outer and for the of (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar, C.R. Annu. Rev. Biochem. PubMed Scopus Google Scholar). is a with at and and at and (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar). The core OS the inner core and outer The inner core OS consists of or 3-deoxy-d-manno-octulosonic acid linked to the lipid and three linked to the (4Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google Scholar). The outer core OS in bacteria consists of linked to heptose II of the inner a of the lipid and core the bacterial surface a an in the of the bacterial surface with such as which are that bacteria to a of such as or high bacteria modified that in a the binding of and resistance to these that Burkholderia in Burkholderia and Burkholderia and the between and lipid is resistant to acid in Carbohydr. Res. PubMed Scopus Google Scholar). B. 4-amino-4-deoxy-l-arabinose is to the lipid a at of the P. E. M. 2005; PubMed Scopus Google Scholar) and is as a component of the core Also, of the B. core OS and the d-glycero-d-talo-octulosonic acid which is with a with Carbohydr. Res. PubMed Scopus Google Scholar, Carbohydr. Res. PubMed Scopus Google Scholar). Although is the for the inner core OS of B. cenocepacia T. D. C. M. PubMed Scopus Google is not a for the core OS in all the inner core of Burkholderia consists of and not C. M. J. 2002; PubMed Scopus Google Scholar). Burkholderia B. are intrinsically resistant to and such as these PubMed Scopus Google Scholar, D.A. PubMed Scopus Google S. J. P. J. PubMed Scopus Google peptides C. J. PubMed Scopus Google and polymyxin B C. J. PubMed Scopus Google Scholar, PubMed Google Scholar). The for some of these peptides in Burkholderia is which these of the D.A. PubMed Scopus Google Scholar). that the resistance of B. to binding to the outer as a of the of and in the lipopolysaccharide Microbiol. PubMed Scopus Google a of the of resistance in B. cenocepacia and Burkholderia is We that a heptoseless B. cenocepacia mutant is the parental strain K56-2 to C. J. PubMed Scopus Google Scholar). mutant a truncated inner core and the outer that a complete core OS is required for resistance of B. cenocepacia to the of of the B. cenocepacia core OS in resistance is not we report the structure of the core OS for B. cenocepacia strain K56-2 and isogenic mutants and which core OS The with to to the of the genes in core OS biosynthesis and of the O antigen and to that the of truncation of the core OS with increased binding and bacterial sensitivity to PmB in vitro and bacterial in and used in are in B. cenocepacia strain K56-2 was at in medium as with and strain is a that to the as the strain E. Coenye T. Speert D.P. P. Vandamme P. J. Microbiol. 2000; PubMed Google Scholar). at in medium with or as mutants at in medium with and or glucose as J. 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Google Scholar) using E. the PubMed Scopus Google Scholar). with the E. and and for of K56-2 for the using a to a of the and to of the was using a to a the used for The of the used for are in the and the genes using The with and and which was with the E. the PubMed Scopus Google and with The using and which to the of and of the the B. cenocepacia mutant as as J. J. PubMed Google Scholar). was in using a and the of the was bacteria in at for the of and in Scholar) of B. cenocepacia and The of the was as the core OS acid in for at as T. D. C. M. PubMed Scopus Google Scholar). The of their and as well as the of total and their all as T. D. C. M. 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PubMed Scopus Google Scholar) and in with the we the genes as and The gene of the is in the to the are the of and is to a The of and these three genes a Also, that the genes are a of a of these gene which we are that the and are because to the E. and of the The of and with was as the of the in O antigen and a which and Microbiol. PubMed Scopus Google Scholar). the of results in of O antigen surface in B. cenocepacia K56-2 for lipid OS in a to the O antigen gene T.A. S. B. Goldberg J.B. J. 2005; PubMed Scopus Google Scholar). a and a Also, is a gene between and that to all of these with an gene in the B. cenocepacia strain which is the as E. Coenye T. Speert D.P. P. Vandamme P. J. 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PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of bacteria that the bacterial the of the K56-2 of and not in to the bacterial of K56-2 a of such as and a a of their not Together, these that the mutant the ability to survive whereas mutants core OS or a complete core OS with O antigen are not in We gene for the biosynthesis of the core OS moiety in B. cenocepacia K56-2. to a of core three of which used to the structure of the core The mutant an in the gene that a in O antigen in the of lipid OS and a O antigen T.A. S. B. Goldberg J.B. J. 2005; PubMed Scopus Google Scholar). the as in strain structure T. D. C. M. PubMed Scopus Google Scholar). the of the in the of a lipid OS with a O antigen that T.A. S. B. Goldberg J.B. J. 2005; PubMed Scopus Google Scholar). The core OS in and are for the of of linked to the outer core The in the strain an at the The mutant which an the terminal found in the outer core OS of and T. D. C. M. 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Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3530) Google is found as a component of the core a we demonstrated that the of is for the of B. cenocepacia J. PubMed Scopus Google and we the for a B. cenocepacia mutant in the of acid, a for PubMed Scopus Google and that the of in the core OS for the of the or the of the The of linked to is not to all as that a heptose is at in the core OS of D. J. PubMed Scopus Google Scholar). strain a very core OS structure the we for B. cenocepacia the of the Burkholderia T. Vandamme P. Environ. Microbiol. PubMed Scopus Google the of a for B. difficult to with The of the is found as a for II in the of and and for in the of M. Karibian D. Carbohydr. Res. PubMed Scopus Google the of such and biosynthesis are Microbiol. PubMed Scopus Google Scholar). the the of Burkholderia such as B. not C. M. J. 2002; PubMed Scopus Google Scholar, B. C. B. M. PubMed Scopus Google Scholar). heptoseless mutant of B. as a of a of the S. C. B. J. PubMed Scopus Google Scholar). The structure of the core OS for mutant is the as the we for the B. cenocepacia K56-2 heptoseless mutant and M. C. J. PubMed Scopus Google Scholar). the inner core OS is in Burkholderia The panel of isogenic with in their lipid OS to the between and the resistance of B. cenocepacia as was with B. PubMed Scopus Google PmB to P. to B. cenocepacia truncation of the lipid OS to increased PmB the that the cell of B. cenocepacia that to as a barrier Although the strain to our core mutants are resistant to PmB such as and E. with intact core B. cenocepacia that these bacteria resistant to of the of isogenic mutants in B. cenocepacia K56-2 that the of a O antigen to a mutant producing heptoseless lipid OS we the of in to and in results demonstrated that O antigen B. cenocepacia bacterial to cells. that the O antigen in these bacteria bacterial surface that with cell or the core OS are for We the that all the core OS mutants with increased that is required for in to in bacteria M. J. S. M. D. PubMed Scopus Google Scholar). that survive B. PubMed Scopus Google M. PubMed Google and macrophages PubMed Scopus Google Scholar, J. S. Microbiol. PubMed Scopus Google Scholar). that the O antigen an in and of the B. in macrophages S. C. M. S. P. 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PubMed Scopus Google we the genes in the biosynthesis of the core OS in B. the of the core and to of the genes of the core OS We demonstrated that of core OS are with a in the resistance to which with binding of peptide to the bacterial cell of the mutant we that the of the core OS is for of B. cenocepacia in whereas the O antigen to prevent bacterial to cells. are in our to the of the outer and the that to the resistance of B. cenocepacia to a of antimicrobial We and D. for C. for and S. and C. for of the with

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