Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry. The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry. Helicobacter pylori, the only Gram-negative bacterium capable of colonizing the human stomach, is the primary cause of active chronic gastritis in humans. Humans were already infected by H. pylori before their migrations from Africa and H. pylori has remained intimately associated with their human host populations ever since (1Moodley Y. Linz B. Yamaoka Y. Windsor H.M. Breurec S. Wu J.Y. Maady A. Bernhoft S. Thiberge J.M. Phuanukoonnon S. et al.The peopling of the Pacific from a bacterial perspective.Science. 2009; 323: 527-530Crossref PubMed Scopus (243) Google Scholar, 2Linz B. Balloux F. Moodley Y. Manica A. Liu H. Roumagnac P. Falush D. Stamer C. Prugnolle F. van der Merwe S.W. et al.An African origin for the intimate association between humans and Helicobacter pylori.Nature. 2007; 445: 915-918Crossref PubMed Scopus (711) Google Scholar). Similar to the cell surface structures of other Gram-negative bacteria, lipopolysaccharide (LPS) is a major component of H. pylori outer membrane. The H. pylori LPS consists of a lipid A region, a core region, and an O-chain polysaccharide (also known as the O-antigen) (3Moran A.P. Lipopolysaccharide in bacterial chronic infection: insights from Helicobacter pylori lipopolysaccharide and lipid A.Int. J. Med. Microbiol. 2007; 297: 307-319Crossref PubMed Scopus (46) Google Scholar). Lipid A, the hydrophobic moiety of LPS and a glucosamine-based saccharolipid, is the principal structural component responsible for the range of biological activities of LPS (4Rutten L. Mannie J.P. Stead C.M. Raetz C.R. Reynolds C.M. Bonvin A.M. Tommassen J.P. Egmond M.R. Trent M.S. Gros P. Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium.Proc. Natl. Acad. Sci. USA. 2009; 106: 1960-1964Crossref PubMed Scopus (37) Google Scholar, 5Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (943) Google Scholar). Generally, lipid A is a glucosamine disaccharide that carries phosphates at positions 1 and 4′ and usually has four primary (glucosamine-linked) hydroxyacyl chains and one or more secondary acyl chains (3Moran A.P. Lipopolysaccharide in bacterial chronic infection: insights from Helicobacter pylori lipopolysaccharide and lipid A.Int. J. Med. Microbiol. 2007; 297: 307-319Crossref PubMed Scopus (46) Google Scholar, 6Miller S.I. Ernst R.K. Bader M.W. LPS, TLR4 and infectious disease diversity.Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (770) Google Scholar, 7Trent M.S. Stead C.M. Tran A.X. Hankins J.V. Diversity of endotoxin and its impact on pathogenesis.J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar). However, H. pylori lipid A is different from that of other bacterial species, including both phosphorylation and acylation patterns (8Moran A.P. Lindner B. Walsh E.J. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.J. Bacteriol. 1997; 179: 6453-6463Crossref PubMed Google Scholar). The lipid A of H. pylori contains a phosphoethanolamine (PEtn) group directly linked to the 1-position of the disaccharide backbone. This is in contrast to the PEtn groups found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage (9Tran A.X. Karbarz M.J. Wang X. Raetz C.R. McGrath S.C. Cotter R.J. Trent M.S. Periplasmic cleavage and modification of the 1-phosphate group of Helicobacter pylori lipid A.J. Biol. Chem. 2004; 279: 55780-55791Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 10Tran A.X. Whittimore J.D. Wyrick P.B. McGrath S.C. Cotter R.J. Trent M.S. The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin.J. Bacteriol. 2006; 188: 4531-4541Crossref PubMed Scopus (92) Google Scholar). In addition, the predominant absence of ester-bound 4’-phosphate and the presence of tetraacyl lipid A with fatty acids of 16 to 18 carbons in length differentiate H. pylori lipid A from that of other Gram-negative bacteria. H. pylori synthesizes two types of lipid A molecules: hexaacyl- and tetraacyl-lipid A. Hexaacyl-lipid A has two phosphates or phosphoethanolamines on the lipid A disaccharide backbone, whereas tetraacyl-lipid A contains only one phosphate (8Moran A.P. Lindner B. Walsh E.J. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.J. Bacteriol. 1997; 179: 6453-6463Crossref PubMed Google Scholar). It has also been reported that H. pylori does not survive long before it is deacylated at the 3′ position from hexaacyl structure to form the tetraacyl major lipid A species (11Stead C.M. Beasley A. Cotter R.J. Trent M.S. Deciphering the unusual acylation pattern of Helicobacter pylori lipid A.J. Bacteriol. 2008; 190: 7012-7021Crossref PubMed Scopus (39) Google Scholar). The toxicity of H. pylori tetraacyl-lipid A on human monocytes is ∼4-fold lower than that of the hexaacyl form (12Ogawa T. Suda Y. Kashihara W. Hayashi T. Shimoyama T. Kusumoto S. Tamura T. Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506).Vaccine. 1997; 15: 1598-1605Crossref PubMed Scopus (59) Google Scholar). It has been suggested that the phosphorylation and acylation patterns in lipid A of H. pylori LPS are responsible for its low biological activity (13Muotiala A. Helander I.M. Pyhala L. Kosunen T.U. Moran A.P. Low biological activity of Helicobacter pylori lipopolysaccharide.Infect. Immun. 1992; 60: 1714-1716Crossref PubMed Google Scholar, 14Mattsby-Baltzer I. Mielniczuk Z. Larsson L. Lindgren K. Goodwin S. Lipid A in Helicobacter pylori..Infect. Immun. 1992; 60: 4383-4387Crossref PubMed Google Scholar). Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications (6Miller S.I. Ernst R.K. Bader M.W. LPS, TLR4 and infectious disease diversity.Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (770) Google Scholar). Pathogenic bacteria modify the lipid A portion of their LPS to help evade the host innate immune response. The variability of lipid A could have profound implications for disease, particularly in humans, owing to altered recognition by the Toll-like receptor-4 complex. Through binding to the specific receptors of the mammalian innate immune system, lipid A is recognized by immune cells as pathogen-associated molecular pattern and stimulates secretion of proinflammatory cytokines (6Miller S.I. Ernst R.K. Bader M.W. LPS, TLR4 and infectious disease diversity.Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (770) Google Scholar, 7Trent M.S. Stead C.M. Tran A.X. Hankins J.V. Diversity of endotoxin and its impact on pathogenesis.J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar, 14Mattsby-Baltzer I. Mielniczuk Z. Larsson L. Lindgren K. Goodwin S. Lipid A in Helicobacter pylori..Infect. Immun. 1992; 60: 4383-4387Crossref PubMed Google Scholar). Immune detection of lipid A is so sensitive and robust that a bloodstream infection can cause a severe complication called endotoxic shock (6Miller S.I. Ernst R.K. Bader M.W. LPS, TLR4 and infectious disease diversity.Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (770) Google Scholar). Therefore, an exquisitely sensitive analytical method is required to determine not only the major lipid A components, but also minor components directly from bacterial cells. Mass spectrometry has been widely used to gain knowledge about lipid A heterogeneity, i.e., the number of different species of the lipid A families and distribution of the fatty acids on each glucosamine group (11Stead C.M. Beasley A. Cotter R.J. Trent M.S. Deciphering the unusual acylation pattern of Helicobacter pylori lipid A.J. Bacteriol. 2008; 190: 7012-7021Crossref PubMed Scopus (39) Google Scholar, 15Aussel L. Therisod H. Karibian D. Perry M.B. Bruneteau M. Caroff M. Novel variation of lipid A structures in strains of different Yersinia species.FEBS Lett. 2000; 465: 87-92Crossref PubMed Scopus (51) Google Scholar, 16Casabuono A.C. D'Antuono A. Sato Y. Nonami H. Ugalde R. Lepek V. Erra-Balsells R. Couto A.S. A matrix-assisted laser desorption/ionization mass spectrometry approach to the lipid A from Mesorhizobium loti.Rapid Commun. Mass Spectrom. 2006; 20: 2175-2182Crossref PubMed Scopus (10) Google Scholar, 17El Hamadi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Jones J.W. Shaffer S.A. Ernst R.K. Goodlett D.R. Turecek F. Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach.Proc. Natl. Acad. Sci. USA. 2008; 105: 12742-12747Crossref PubMed Scopus (51) Google Scholar, 19Tirsoaga A. El Perry M.B. Caroff M. Novikov A. A for the structural characterization of lipid A to and of a new structural Lipid Res. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). have previously lipid A species through analysis of the LPS M. J. of from the using 2008; PubMed Scopus (10) Google Scholar). Lipid A is from the LPS by as or at for or as with the of at for 1 h M. A. L. of bacterial and of the of the phosphate present in the lipid of the Res. PubMed Scopus Google Scholar). at are required for a and of LPS, of lipid A is and El Hamadi et Hamadi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (133) Google a method based on a to lipid A directly from bacterial cells. This method the mass spectrometric in a with a detection of of cells. The relatively detection by the its to profiling lipid A structures from the material as as a single colony. In an to study the between the lipid A structure and the role of H. pylori LPS in we a and sensitive analytical method to lipid A based on microwave-assisted enzymatic digestion and mild used H. pylori and to the of the strains are of in H. pylori the of has been the is widely in a of H. pylori and mutant a core LPS structures have been Moran A.P. J. et structures of Helicobacter pylori strains and H. pylori H. pylori and H. pylori of H. pylori J. Biochem. 2000; PubMed Scopus Google Scholar). was from was from and were from and were from H. pylori was from R. at H. pylori was from A. of were at on in for h as previously K. J.W. V. A. H. et of a from Helicobacter Bacteriol. 2005; PubMed Scopus Google Scholar). was used for mutant strains in to K. J.W. V. A. H. et of a from Helicobacter Bacteriol. 2005; PubMed Scopus Google Scholar). The bacterial were and to an of of in were and of and were the for and at for in a or were in in with and to dried cells were in at a 1 were from the before each and to to cells were in of and for 2 h at in a The was in and for at The was dried by The sample was with of and at for the lipid A was with of a of and at for the was to and dried a of The cells were in of in and at for 1 The was dried by was by the with of and of by to by for The were with of and for The lipid A was from the with of a of and by at for The was to and dried a of The cells were in a and in of digestion was used to help cells. at the enzymatic digestion was for at The was for 1 h at The was at for The was The were with and at for the lipid A was and with of a of and by at for The was to and dried a of of each single colony were the and in a in with by the cells were in of the was at for 1 The was at for and the were with Lipid A was from the with of and at for the was to and dried a of Lipid A was using a in the were in a of and and with of in from which were on and were in were for each and were for each The was to were with of 1 and was used as the were and with The of lipid A directly from bacterial cells has been by using an Hamadi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 19Tirsoaga A. El Perry M.B. Caroff M. Novikov A. A for the structural characterization of lipid A to and of a new structural Lipid Res. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). first the H. pylori lipid A using this method and the mass from and of dried cells are in and C. in this method is for analysis of lipid A from H. pylori cells on as as μg. the other an method has been widely used for lipid A preparation from A. El Perry M.B. Caroff M. Novikov A. A for the structural characterization of lipid A to and of a new structural Lipid Res. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Novikov A. M. C. C. J.M. Caroff M. method for of for biological Microbiol. 2007; PubMed Scopus Google Scholar). we also used the method and found that it a of lipid A than that of The mass from and of H. pylori cells are in and D. It is that we observed the two for the preparation of lipid A not lipid A preparation using can be in one the of can be in which the of the lipid A In other we could the time for sample preparation the of could be The of microwave-assisted digestion and analysis has to C. in to determine the M. Liu X. A.P. van A. M. J. method for sensitive of in the from strains from and Microbiol. 2008; 46: PubMed Scopus Google Scholar). This method is not only rapid but also sensitive and as demonstrated by analysis using single colony to a rapid sample method to lipid A directly from bacterial cells based on microwave-assisted enzymatic digestion and detergent-free In this of was used to the cells. The can the enzymatic digestion of that the can be to only a with and The sample was using a detergent-free in which of was and the was at for 1 The for lipid A preparation from cells about 2 The mass for the lipid A from and 1 of dried cells are in The analysis that the of the method is than that of the previously reported The mass also suggested that the method could be used to the lipid A structures from than 1 of dried cells. and 1 of dried cells were also to digestion for of by in the microwave-assisted digestion can the of lipid A and the further the new we it to the lipid A from four strains of H. pylori and and The mass from H. pylori was to that from H. pylori The at to a lipid A with four and fatty residues and one PEtn group attached to the glucosamine backbone. The mass that the lipid A structures from H. pylori mutant in which the to a lipid A was also were different from of H. pylori and H. pylori In addition, the of at in the mass of lipid A from H. pylori it from H. pylori and its to the LPS at the the core and in an LPS structure that the O-chain polysaccharide and the outer core M. Liu X. A.P. van A. M. J. method for sensitive of in the from strains from and Microbiol. 2008; 46: PubMed Scopus Google Scholar). have previously that H. pylori a LPS was in its to the K. J.W. V. A. H. et of a from Helicobacter Bacteriol. 2005; PubMed Scopus Google Scholar). have also H. pylori mutant and demonstrated that this in of LPS the O-chain polysaccharide but the outer core J.W. of Helicobacter of a and of with altered Microbiol. 2000; PubMed Scopus Google Scholar). we have previously mutant a core LPS structure both the O-chain and the outer core and a single attached to by a PEtn The total fatty analysis of LPS from H. pylori strains and the mutant the presence of and as analysis of the fatty acids in LPS that both and were of were also present not mass spectrometry was for the of the lipid A The mass of the two lipid A and from H. pylori are in The mass for the at is in in which the was to the PEtn The at although of low to The at was as a at and were to the at one or one of both residues the at The major from the at are in The suggested that mass spectrometry is a for of lipid A However, this technique for of species, with structural In of to analysis or in with be required A. El Perry M.B. Caroff M. Novikov A. A for the structural characterization of lipid A to and of a new structural Lipid Res. 2007; Full Text Full Text PDF PubMed Scopus Google of the at The observed in the lipid A pattern of H. pylori mutant has to single colony of this the mass two at and colony is the in of the two from a different number of bacterial cells colony. the of H. pylori are it to of bacterial cells. we were to the major lipid A species in The from the analysis of single colony is detection of and heptaacyl lipid A species, and in It is that minor components have abundance in the mass for lipid A from single colony than from cell that in of to variation were responsible for the In other not the lipid A a the abundance of the species in a sample was lower than that in the colony. Furthermore, the in could also to the Therefore, the mass spectrometry were for the at and using the material from of the The lipid A species of was to be a hexaacyl previously in H. pylori mutant which the to an acyl (11Stead C.M. Beasley A. Cotter R.J. Trent M.S. Deciphering the unusual acylation pattern of Helicobacter pylori lipid A.J. Bacteriol. 2008; 190: 7012-7021Crossref PubMed Scopus (39) Google Scholar). structure was by the of in the which were also in the of the lipid A of was to be a hexaacyl form with an phosphate group at position 4′ of the disaccharide It has been reported in to the predominant tetraacyl lipid A, H. pylori smooth-form LPS also a minor a hexaacyl lipid that is from tetraacyl lipid A by or at and an phosphate group at (8Moran A.P. Lindner B. Walsh E.J. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.J. Bacteriol. 1997; 179: 6453-6463Crossref PubMed Google Scholar). of a relatively low the lipid A species of were in colony that this is a heptaacyl lipid A component the mass was not to the structure of the at to its low it could be by the detection of at and to phosphate and phosphoethanolamine and at and to and the heptaacyl form of lipid A has been in other Gram-negative bacteria Liu F. W. Bishop R.E. in the outer core in the of Escherichia Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google this study has for the first that H. pylori lipid A can heptaacyl structures for and heptaacyl lipid A The acylation and phosphorylation of lipid A has long been recognized as a of the toxicity of LPS or M.S. Stead C.M. Tran A.X. Hankins J.V. Diversity of endotoxin and its impact on pathogenesis.J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar, J. of the of and an the of human Immun. PubMed Scopus (37) Google Scholar). Lipid A is the component responsible for immune M.S. Stead C.M. Tran A.X. Hankins J.V. Diversity of endotoxin and its impact on pathogenesis.J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar). The of acylation is and species are known to be in proinflammatory F. S. S. A. Structural for LPS a biological role for LPS PubMed Scopus Google Scholar). The unusual structure of H. pylori lipid A is to be responsible for its low as with lipid A of Salmonella and Escherichia (12Ogawa T. Suda Y. Kashihara W. Hayashi T. Shimoyama T. Kusumoto S. Tamura T. Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506).Vaccine. 1997; 15: 1598-1605Crossref PubMed Scopus (59) Google Scholar). Generally, it a enzymatic to a hexaacyl lipid A (11Stead C.M. Beasley A. Cotter R.J. Trent M.S. Deciphering the unusual acylation pattern of Helicobacter pylori lipid A.J. Bacteriol. 2008; 190: 7012-7021Crossref PubMed Scopus (39) Google Scholar). However, the lipid A in H. pylori has only to the lipid A which an for H. pylori lipid A modifications M.S. Stead C.M. Tran A.X. Hankins J.V. Diversity of endotoxin and its impact on pathogenesis.J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar). The presence of heptaacyl in a minor of H. pylori lipid A has been known since the structure of lipid A was first but the in bacterial pathogenesis of lipid A has only R.E. The lipid A molecular and role in bacterial Microbiol. 2005; PubMed Scopus Google Scholar). it has been in lipid A species as an the activity of lipid A species with to the of and of cells T. S. H. J. of Helicobacter pylori cells a Immun. PubMed Scopus Google Scholar). in LPS F. S. S. A. Structural for LPS a biological role for LPS PubMed Scopus Google Scholar). In the modification the the of a to one of the primary linked acyl chains of in a heptaacyl lipid A structure Liu F. W. Bishop R.E. in the outer core in the of Escherichia Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The heptaacyl form observed in H. pylori mutant the that a in H. pylori. Furthermore, this is the first characterization of the lipid A structure from a single bacterial colony sample by mass spectrometry. have previously that the of of cells was for an H. pylori mutant as with the with an LPS structure V. A. J. J. of a mutant of Helicobacter pylori provides that an lipopolysaccharide structure has a role in the of Biol. 2007; PubMed Scopus Google Scholar). The role of heptaacyl species of H. pylori lipid A in is and further It is to that of the heptaacyl lipid A in could be linked to observed of H. pylori and the of H. pylori infection A. E.J. Caroff M. is a lipid A that is required for of the Microbiol. PubMed Scopus Google Scholar).

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