Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae

The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an α-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., Månsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both α-2,3- and α-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance. The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an α-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., Månsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both α-2,3- and α-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance. Sialylation of lipopolysaccharide (LPS) 3The abbreviations used are: LPS, lipopolysaccharide; NTHi, non-typeable H. influenzae; Neu5Ac, N-acetylneuraminic acid; Kdo, 2-keto-3-deoxyoctulosonic acid; Hep, l-glycero-d-mannoheptose; PEtn, phosphoethanolamine; Neu5Ac, N-acetylneuraminic acid; PCho, phosphocholine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MS, mass spectroscopy; Mes, 4-morpholineethanesulfonic acid; Lac, lactose; FCHASE, 6-(5-fluorescein-carboxamido)-hexanoic acid succinimidyl ester. is a widely conserved structural modification among mucosal pathogens, with a reported role in virulence in a number of organisms. In Haemophilus influenzae LPS sialylation has been demonstrated to be important in resistance to the killing effects of normal human serum. A significant decrease in bacterial survival in human serum was observed in sialylation-deficient mutants, in which the CMP-Neu5Ac synthetase gene (siaB) had been disrupted, compared with the wild-type organisms (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). Furthermore, the role of sialylation as a critical virulence factor in the pathogenesis of experimental otitis media has been demonstrated, Neu5Ac-deficient mutants of non-typeable H. influenzae (NTHi) were profoundly attenuated in a chinchilla model of infection (3Bouchet V. Hood D.W. Li J. Brisson J.R. Randle G.A. Martin A. Li Z. Goldstein R. Schweda E.K. Pelton S.I. Richards J.C. Moxon E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8898-8903Crossref PubMed Scopus (176) Google Scholar). Related to this, it has been proposed that LPS sialylation may be implicated in the formation of biofilms (4Swords W.E. Moore M.L. Godzicki L. Bukofzer G. Mitten M.J. VonCannon J. Infect. Immun. 2004; 72: 106-113Crossref PubMed Scopus (111) Google Scholar, 5Greiner L.L. Watanabe H. Phillips N.J. Shao J. Morgan A. Zaleski A. Gibson B.W. Apicella M.A. Infect. Immun. 2004; 72: 4249-4260Crossref PubMed Scopus (98) Google Scholar, 6Jurcisek J. Greiner L. Watanabe H. Zaleski A. Apicella M.A. Bakaletz L.O. Infect. Immun. 2005; 73: 3210-3218Crossref PubMed Scopus (117) Google Scholar). These studies provide evidence that Neu5Ac-containing LPS promotes bacterial persistence in vivo, potentially indicating a multi-faceted role for Neu5Ac in bacterial colonization and disease. Virtually all NTHi strains tested include Neu5Ac in their LPS (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar, 7Bauer S.H. Månsson M. Hood D.W. Richards J.C. Moxon E.R. Schweda E.K. Carbohydr. Res. 2001; 335: 251-260Crossref PubMed Scopus (34) Google Scholar), but the patterns of sialylation and the repertoire of putative sialyltransferases differ between strains. Two common sialylated species have been identified to date in H. influenzae LPS, sialylated lactose and sialylated lacto-N-neotetrose (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar, 8Cox A.D. Hood D.W. Martin A. Makepeace K.M. Deadman M.E. Li J. Brisson J.R. Moxon E.R. Richards J.C. Eur. J. Biochem. 2002; 269: 4009-4019Crossref PubMed Scopus (37) Google Scholar, 9Jones P.A. Samuels N.M. Phillips N.J. Munson Jr., Zaleski A. Gibson B.W. Apicella M.A. J. 2002; PubMed Scopus Google Scholar). Lic3A, the only biochemically characterized sialyltransferase in H. influenzae, is responsible for the addition of Neu5Ac in a to a lactose acceptor the LPS of H. influenzae D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). the of the of the Moxon E.R. J. PubMed Google Scholar), and the sialyltransferase is to In strain terminal expressed as of a is to in H. influenzae by the sialyltransferase gene and J. Greiner L. Watanabe H. Zaleski A. Apicella M.A. Bakaletz L.O. Infect. Immun. 2005; 73: 3210-3218Crossref PubMed Scopus (117) Google Scholar, 9Jones P.A. Samuels N.M. Phillips N.J. Munson Jr., Zaleski A. Gibson B.W. Apicella M.A. J. 2002; PubMed Scopus Google Scholar, D.W. Deadman M.E. H. Martin A. Brisson J.R. R. J.C. Richards J.C. Moxon E.R. Mol. Microbiol. PubMed Scopus Google Scholar, D.W. Randle G. A.D. Makepeace K. Li J. Schweda E.K. Richards J.C. Moxon E.R. J. 2004; PubMed Scopus (37) Google Scholar). In a of NTHi acid residues have been detected in a of LPS glycoforms (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar), indicating a role for sialyltransferase in strains to Neu5Ac as an a sialyltransferase of both may in H. influenzae strains as has been reported in M. Brisson J.R. J. N.M. J. PubMed Scopus Google Scholar), a sialyltransferase has been The findings from the provide the evidence that a sialyltransferase in H. and NTHi isolates used in this and were from the were from the of with otitis media and as of the of H. influenzae analysis D. S. Deadman M.E. V. Infect. 2003; PubMed Scopus Google Scholar). H. influenzae isolates were used in this Moxon E.R. J. H. W. J. G. M. M. K. Li J. J.C. M.L. Infect. PubMed Scopus Google Scholar). H. influenzae was at in with and were with and with and of or Neu5Ac Escherichia coli strain was used to and was at in J. A with or of lic3B and analysis identified a which with a from a of a to the was of was from the and purified was into the were by J. A a gene The of the was confirmed by the and an The containing the was The lic3B gene from NTHi was by with and of a resistance from by with to mutants, the gene D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google was with by of a resistance from to into E. all mutant strains were confirmed by and of of strains were in NTHi isolates and by the M. J. PubMed Google Scholar). were confirmed by the media and by of both the and lic3B strain had been D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google Scholar), and to a was with lic3B mutants, containing the lic3B was used to NTHi isolates and In this were confirmed by a a mutant strain was with a strain was with from strain strain has been (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). a containing the by of a resistance (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google was used to NTHi of lic3B into a lic3B-deficient resistance was into the gene the gene of in to as a was with and a resistance from was at the was by and used to NTHi from was used to strains and D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). in were confirmed by of LPS by were from with Neu5Ac, in to a with an of at were with and by W.E. Apicella M.A. J. PubMed Scopus Google and with The purified from terminal to Structural of of LPS was as A.D. Hood D.W. Martin A. Makepeace K.M. Deadman M.E. Li J. Brisson J.R. Moxon E.R. Richards J.C. Eur. J. Biochem. 2002; 269: 4009-4019Crossref PubMed Scopus (37) Google Scholar). LPS was by with at for were in the by a The to an mass a The were in containing A of was at the and a was at a of and of Lic3A and and lic3B were by from NTHi The the is in and the is in were used to the The were used to the is in and the is in The were with and and in containing the the E. coli the and the E. coli strains containing from NTHi in or from NTHi in were in J. A containing and The were at with and at The were an and the and were purified by affinity the sialyltransferase were at for The α-2,3-sialyltransferase activity was CMP-Neu5Ac as and as acceptor in containing The α-2,8-sialyltransferase activity was CMP-Neu5Ac as and as in containing The activity was at for as and as acceptor in Mes, containing the were by the addition of The purified were to between and of the into the indicated The were by as Mol. 2003; Google Scholar). of the was by of the the The sialyltransferase and expressed in of of purified to in with Neu5Ac were in organisms were and in normal human serum for were by and A a to of in a of NTHi isolates by analysis identified a homologue Deadman M.E. Schweda E.K. Moxon E.R. Hood D.W. Mol. Microbiol. 2005; PubMed Scopus Google Scholar). NTHi isolates two that to both an and a The from NTHi that which in with the was to of the an a of with to Lic3A was Lic3A homologue was by a with to Li A. A. Mol. Microbiol. PubMed Scopus Google Scholar), and by a synthetase is to the to which responsible for the and of the expressed the LPS of a of and NTHi strains D.W. Randle G. A.D. Makepeace K. Li J. Schweda E.K. Richards J.C. Moxon E.R. J. 2004; PubMed Scopus (37) Google Scholar). second of has been and the lic3B were used to by of and that in the and between of NTHi isolates tested an of indicating a gene that of strain in of the isolates a of was indicating an of in this of the all of the to a indicating that the is conserved between strains not A of the of lic3B in H. influenzae by indicated that lic3B was present in a of H. influenzae strains observed with NTHi The lic3B gene was to be from the and strains tested and was present in of a of of and of strains tested not The gene is expressed to a the M. D. P. J. Moxon R. J. PubMed Google Scholar). Lic3B a at the to the The of the and lic3B in the NTHi isolates was and the number of was to from to in and from to in both two of the to for of gene from and of the The acid of Lic3A number and Lic3B number from NTHi and of lic3B of the of the gene from NTHi was by of a resistance used to NTHi isolates and that only lic3B had been and that had not been and with analysis was used to the of the not NTHi was as a second strain background in which to the of Lic3B the of the LPS has been and Neu5Ac the as in which is to the of lactose to the M. Hood D.W. Li J. Richards J.C. Moxon E.R. Schweda E.K. Eur. J. Biochem. 2002; 269: PubMed Scopus Google Scholar). In disialylated species have been detected in the LPS of this M. Hood D.W. Li J. Richards J.C. Moxon E.R. Schweda E.K. Eur. J. Biochem. 2002; 269: PubMed Scopus Google Scholar). LPS sialylation in a in two NTHi mutant strains in which D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google and the CMP-Neu5Ac synthetase had been were with a from wild-type isolates and and mutant strains were to LPS LPS from the wild-type and and lic3B mutant strains of NTHi and was and the were A.D. Hood D.W. Martin A. Makepeace K.M. Deadman M.E. Li J. Brisson J.R. Moxon E.R. Richards J.C. Eur. J. Biochem. 2002; 269: 4009-4019Crossref PubMed Scopus (37) Google Scholar). The LPS of wild-type the two and indicated were upon that with and disialylated species M. Hood D.W. Li J. Richards J.C. Moxon E.R. Schweda E.K. Eur. J. Biochem. 2002; 269: PubMed Scopus Google Scholar). In and mutant strains only sialylated in was the sialylated evident in wild-type LPS is from the LPS of the In the mutant strain all sialylated glycoforms Structural analysis of the LPS of that glycoforms containing only a Neu5Ac present in this whereas analysis of LPS identified disialylated species The of the sialylated in the LPS of strain may be to of in this mutant strain compared with and proposed of LPS from H. influenzae strains wild-type and and and mass were used for of proposed as Hep, PEtn, PCho, acid were from the of the of the and as PCho, PCho, PCho, PCho, PCho, PCho, PCho, PCho, PCho, PCho, PCho, PCho, were from the of the of the and as in a a LPS the of which is a that in is to the disialylated LPS species present in this The whereas a in LPS. monosialylated LPS species and a of disialylated species (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). In the the observed with wild-type LPS was not indicating the of disialylated species in this a sialylation was evident upon this in the of the LPS that mono-sialylated species present in this mutant strain. In the mutant LPS sialylation is that observed in of this LPS resulted in the of the The of LPS is from that of wild-type this was and be sialylation was observed of the LPS in the or mutant strains not to findings with NTHi mass spectrometric analysis of the LPS of that glycoforms containing only a Neu5Ac present in this whereas analysis of LPS identified disialylated species In from analysis of LPS of the acid with Neu5Ac to Neu5Ac in the LPS not of of and Lic3B from NTHi were expressed in E. coli with the and purified In both the was the to the of in the recombinant The in the acid and their resulted in the of and residues at the of Lic3A and Lic3B, We the sialyltransferase activity of purified recombinant Lic3A and Lic3B with fluorescent model Lic3A and Lic3B acid to lactose whereas only Lic3B to These of that Lic3A can acid only to terminal whereas Lic3B can acid to a terminal or to a terminal Neu5Ac We have that Lic3B can form acid a terminal by two Neu5Ac residues CMP-Neu5Ac as a we observed a of and or not increase the of sialylated which that the an between the acceptor and the sialylated Lic3A activity with as a significant of the of a second acid was activity is it is in the of Lic3A has significant activity and the in the of sialyllactose and is that is a of in the CMP-Neu5Ac used as and this we observed of sialyllactose we Lic3A for α-2,8-sialyltransferase activity is significant in the of Lic3B of the activity of the Lic3A and Lic3B sialyltransferases from NTHi Lic3A and Lic3B were expressed in E. coli as with the in the and purified α-2,3-sialyltransferase activity was CMP-Neu5Ac as and as The acid was to the α-2,8-sialyltransferase activity was CMP-Neu5Ac as and as The acid was to the Neu5Ac activity was as and as The α-2,3-sialyltransferase activity was CMP-Neu5Ac as and as The acid was to the The α-2,8-sialyltransferase activity was CMP-Neu5Ac as and as The acid was to the Neu5Ac The activity was as and as acceptor in a of lic3B into a lic3B-deficient the of Lic3B, the lic3B gene was into a lic3B-deficient strain strain to a sialylation to lic3B be resistance was into the gene of to as a The of mutants were to that inactivation of had the LPS compared with not containing the lic3B in strain increased LPS sialylation compared with the parent strain. in the LPS of wild-type strain the of which is as by the that with the of this The decrease in of this with that a non-sialylated with the sialylated LPS in this strain In the mutants the of the is with a decrease in of two observed upon this strain containing a in the gene the was used as this for the lactose acceptor and the formation of non-sialylated has been to increase the of observed sialylated glycoforms D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). Two sialylated glycoforms evident in the mutants that both upon These findings with that the introduction of lic3B into strain the and of LPS sialylation in that strain. Mass spectrometric analysis of LPS from strain evidence of glycoforms containing and acid residues These glycoforms were present in glycoforms a Neu5Ac were upon analysis of LPS from strain and proposed of LPS from H. influenzae strains wild-type and mass were used for of proposed as Hep, PEtn, PCho, PEtn, PEtn, PCho, PEtn, PCho, PEtn, PCho, PCho, PCho, PCho, in a of Neu5Ac is to the resistance of organisms to the killing of human (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). The of sialyltransferase mutants in NTHi were to to the of inactivation of and of putative sialyltransferase upon biological activity of the LPS. The wild-type strain with the mutant was in for and were NTHi the strain was and the mutant to the killing of human The mutant was to serum to mutants resistance a significant between strains acid in their LPS and compared with strains or acid residues in their LPS The in serum resistance was not significant between the and lic3B mutant strains. a significant was between wild-type and the and lic3B mutants, indicating that of of sialyltransferase serum resistance The in the serum resistance with the LPS by the The sialylated strains to the and the strains to LPS to the acid has been implicated as a virulence factor in bacterial The by which sialylation of LPS to both the and of NTHi be to the pathogenesis of by this Sialylation of LPS is a critical virulence factor in for NTHi to otitis media in a chinchilla model of infection (3Bouchet V. Hood D.W. Li J. Brisson J.R. Randle G.A. Martin A. Li Z. Goldstein R. Schweda E.K. Pelton S.I. Richards J.C. Moxon E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8898-8903Crossref PubMed Scopus (176) Google Scholar). H. influenzae the for of Neu5Ac and an of the as a for E. S. Mol. Microbiol. PubMed Scopus Google or for modification of LPS a number of the and of LPS sialylation NTHi strains. In this we the of the sialyltransferase Lic3B, by a gene identified as a close homologue of lic3A. the only biochemically characterized α-2,3-sialyltransferase for sialylation of lactose in H. influenzae LPS (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). Lic3B and Lic3A that have from and the role of Lic3B as a with α-2,3- and α-2,8-sialyltransferase activity to both terminal of lactose and Neu5Ac of sialyllactose as acceptor The recombinant purified Lic3A activity sialyllactose D.W. A.D. M. Makepeace K. S. Deadman M.E. A. Martin A. Månsson M. Schweda E.K. Brisson J.R. Richards J.C. Moxon E.R. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). Mass spectrometric analysis of LPS from and lic3B mutant strains of NTHi demonstrated that upon inactivation of lic3B only monosialylated species were detectable, whereas inactivation of disialylated species were Furthermore, the introduction of lic3B into a lic3B-deficient strain background a significant increase in LPS The of disialylated species was confirmed in this strain. sialyltransferase have been in mucosal and M. Brisson J.R. J. N.M. J. PubMed Scopus Google Scholar, D. Li J. Brisson J.R. N.M. M. J. 2001; PubMed Scopus Google Scholar). In the gene an and the gene a α-2,3-sialyltransferase or a with α-2,3- and α-2,8-sialyltransferase the of M. Brisson J.R. J. N.M. J. PubMed Scopus Google Scholar). Lic3A and Lic3B of H. influenzae and and of in the of as by the indicating the of In the gene an with α-2,3-sialyltransferase activity in strain and a with α-2,3- and activity in strain D. Li J. Brisson J.R. N.M. M. J. 2001; PubMed Scopus Google Scholar). The from can be into a by of a acid D. Li J. Brisson J.R. N.M. M. J. 2001; PubMed Scopus Google Scholar). is to the that of the Lic3B sialyltransferase A between the of H. influenzae LPS that to have biological and upon gene Lic3A and Lic3B both expressed in a all to the terminal lactose the as an acceptor as is which the terminal of the lactose the sialyllactose or the is potentially of the LPS expressed by an to the organism in Neu5Ac a in LPS These may to in or between is the of of the and lic3B but an of may be by the that both have two In it has been that can to of of the gene Moxon E.R. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). be of LPS sialylation the of both of The gene is present in all strains whereas lic3B is present in only a is not lic3B by or by the of the of lic3B to the it is that isolates to the has of the of with organisms containing it is that two be the has been demonstrated in organisms that can of sialyltransferase D. Li J. Brisson J.R. N.M. M. J. 2001; PubMed Scopus Google Scholar). be to that gene have been to of the Lic3A and Lic3B the of the the Two putative sialyltransferases and can be present in NTHi but the The gene was both and in with the and lic3B sialyltransferase sialylation was observed was in NTHi that Lic3A and Lic3B responsible for sialylation of and may be in sialylation of the expressed by NTHi isolates D.W. Randle G. A.D. Makepeace K. Li J. Schweda E.K. Richards J.C. Moxon E.R. J. 2004; PubMed Scopus (37) Google Scholar). The findings from that of the and of LPS sialylation in NTHi is isolates The of a in the mutant be by the activity of Lic3A in In addition to this inactivation of putative sialyltransferase this strain resulted in and in the LPS between that were not to A of in the sialylation of mutant strains was reported by P.A. Samuels N.M. Phillips N.J. Munson Jr., Zaleski A. Gibson B.W. Apicella M.A. J. 2002; PubMed Scopus Google upon of the putative and in strain be that the observed in LPS between in NTHi were to in LPS by an of gene A is that in the of sialyltransferase be form of of the In this a was between of Neu5Ac LPS and serum resistance of NTHi, as observed (2Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). be that Neu5Ac from human serum in an to a potentially the by NTHi in the of Neu5Ac their is by the of NTHi strains the sialyltransferase compared with strains of H. influenzae and the that NTHi isolates Neu5Ac in their LPS S.H. Månsson M. Hood D.W. Richards J.C. Moxon E.R. Schweda E.K. Carbohydr. Res. 2001; 335: 251-260Crossref PubMed Scopus (34) Google Scholar). The by which acid to serum resistance to be of LPS sialylation may be in of LPS or by by an or by Sialylation of LPS a the bacterial potentially from in the has been proposed that NTHi a in with increased sialylated LPS glycoforms compared with in organisms L.L. Watanabe H. Phillips N.J. Shao J. Morgan A. Zaleski A. Gibson B.W. Apicella M.A. Infect. Immun. 2004; 72: 4249-4260Crossref PubMed Scopus (98) Google Scholar, J.C. J. Scopus Google Scholar, J.C. 2001; PubMed Scopus Google Scholar, R. J.C. J. 2002; PubMed Scopus Google Scholar). has been evidence of an of the of a the sialylated LPS glycoforms of organisms in implicated in pathogenesis studies in a chinchilla model of otitis media (3Bouchet V. Hood D.W. Li J. Brisson J.R. Randle G.A. Martin A. Li Z. Goldstein R. Schweda E.K. Pelton S.I. Richards J.C. Moxon E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8898-8903Crossref PubMed Scopus (176) Google Scholar, 6Jurcisek J. Greiner L. Watanabe H. Zaleski A. Apicella M.A. Bakaletz L.O. Infect. Immun. 2005; 73: 3210-3218Crossref PubMed Scopus (117) Google Scholar). The of of a sialylated LPS in NTHi isolates may be the of LPS glycoforms expressed at in in addition to the of patterns of LPS glycoforms expressed by NTHi by mucosal that has been it that this be a factor in the of between and between We and for and and the for the of strains used in this with

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