Antigenic Variation among Bordetella
2010; Elsevier BV; Volume: 285; Issue: 35 Linguagem: Inglês
10.1074/jbc.m110.115121
ISSN1083-351X
AutoresEvgeny Vinogradov, Jerry D. King, Ashutosh K. Pathak, Eric T. Harvill, Andrew Preston,
Tópico(s)Pneumonia and Respiratory Infections
ResumoThe O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-α-l-GalNAc3NAcA-(4-β-d-GlcNAc3NAcA-4-α-l-GalNAc3NAcA-)5–6-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS. The O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-α-l-GalNAc3NAcA-(4-β-d-GlcNAc3NAcA-4-α-l-GalNAc3NAcA-)5–6-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS. IntroductionThe genus Bordetella currently comprises nine species of Gram-negative bacteria. The most extensively studied of these are the pathogens Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. B. pertussis infects only humans and is the causative agent of whooping cough in infants and persistent respiratory infections in adults (1Cherry J.D. J. Infect. Dis. 1996; 174: S259-S263Crossref PubMed Google Scholar). B. parapertussis exists as two separate lineages. One is adapted to the human host and causes whooping cough; the other is adapted to the ovine host, in which it can cause chronic pneumonia (2Porter J.F. Connor K. Donachie W. Microbiology. 1994; 140: 255-261Crossref PubMed Scopus (75) Google Scholar, 3Heininger U. Stehr K. Schmitt-Grohé S. Lorenz C. Rost R. Christenson P.D. Uberall M. Cherry J.D. Pediatr. Infect. Dis. J. 1994; 13: 306-309Crossref PubMed Scopus (148) Google Scholar). In contrast, B. bronchiseptica colonizes the respiratory tract of a large number of animals, and although it causes respiratory diseases in some farm, companion, and wild animals, most B. bronchiseptica infections are asymptomatic and chronic (4Goodnow R.A. Microbiol. Rev. 1980; 44: 722-738Crossref PubMed Google Scholar). B. bronchiseptica is occasionally isolated from the respiratory tract of humans and is probably acquired through contact with infected animals (5Gueirard P. Weber C. Le Coustumier A. Guiso N. J. Clin. Microbiol. 1995; 33: 2002-2006Crossref PubMed Google Scholar).Bordetella lipopolysaccharide (LPS) plays a number of different roles in Bordetella biology, including during infection (e.g. see Refs. 6Zhang X. Goebel E.M. Rodríguez M.E. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 5050-5058Crossref PubMed Scopus (15) Google Scholar, 7Zhang X. Rodríguez M.E. Harvill E.T. PLoS One. 2009; 4e6989Crossref PubMed Scopus (24) Google Scholar, 8Pilione M.R. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. Infect. Immun. 2004; 72: 2837-2842Crossref PubMed Scopus (39) Google Scholar, 9Harvill E.T. Preston A. Cotter P.A. Allen A.G. Maskell D.J. Miller J.F. Infect. Immun. 2000; 68: 6720-6728Crossref PubMed Scopus (93) Google Scholar). We and others have defined the structures of the LPS of these Bordetella (Fig. 1). All three synthesize a very similar LPS core. In B. bronchiseptica and B. pertussis, the core can be further substituted by a trisaccharide to produce the structure referred to as Band A LPS (10Peppler M.S. Infect. Immun. 1984; 43: 224-232Crossref PubMed Google Scholar, 11Caroff M. Brisson J. Martin A. Karibian D. FEBS Lett. 2000; 477: 8-14Crossref PubMed Scopus (80) Google Scholar). B. parapertussis and B. bronchiseptica, but not B. pertussis, synthesize O PSs. 2The abbreviations used are: O PSO chain polysaccharideGalNAc3NAcA2,3-diacetamido-2,3-dideoxygalacturonic acidHFhydrofluoric acidManNAc3NAcA2,3-diacetamido-2,3-dideoxymannuronic acidGlcNAc3NAcA2,3-diacetamido-2,3-dideoxy-glucuronic acidSDRshort chain dehydrogenase/reductaseaaamino acid(s)TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Initially, the O PSs of both species were reported to be identical and composed of linear polymers of 1,4-linked GalNAc3NAcA (12Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (75) Google Scholar), but later it was discovered that the terminal residue contained a number of modifications, that these modifications can vary between strains (13Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar), and that the O PS polymer is linked to the rest of the LPS molecule via an unusual 5-sugar linker (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To date, two types of modification, O1 and O2, have been described (13Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar, 15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar). O1 and O2 PSs both contain 2,3,4-triamino-2,3,4-trideoxy-α-galacturonamide derivatives at their non-reducing ends. In O1 PS, this residue is formylated at positions 3 and 4 and has N-formyl-l-alanyl or l-alanyl substituents at N-2. In O2 PS, the amino group at position 2 is acetylated, at position 3 it is formylated, and at position 4 it is modified with a 2-methoxypropionyl substituent. Importantly, antibody responses to O PS appear to be wholly directed against the modified terminal residue because antisera raised against O1 type O PS did not recognize O2 type O PSs and vice versa, and in mice, immunization with LPS of one type did not confer protection against strains with the other type (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar). This suggests that Bordetella O PSs display antigenic variation in order to help evade cross-reaction with existing immunity within hosts.We have also studied the genetics of O PS biosynthesis (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Preston A. Allen A.G. Cadisch J. Thomas R. Stevens K. Churcher C.M. Badcock K.L. Parkhill J. Barrell B. Maskell D.J. Infect. Immun. 1999; 67: 3763-3767Crossref PubMed Google Scholar). B. bronchiseptica and B. parapertussis each contain a locus, wbm, containing 24 genes that is responsible for biosynthesis of the linker, polymer repeat, modifications of the terminal residue, and export of the O PS from the cytoplasm. We proposed that the central region of the wbm locus contains the genes that direct biosynthesis of the terminal residue modifications, and supporting this, the gene content of this region correlates with the modification type (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A number of studies have used postgenomic technologies to investigate genetic diversity among the Bordetella (17Cummings C.A. Brinig M.M. Lepp P.W. van de Pas S. Relman D.A. J Bacteriol. 2004; 186: 1484-1492Crossref PubMed Scopus (117) Google Scholar, 18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). From these, the wbm locus emerged as one of the very few loci that exhibited clear variability among strains. In particular, these studies described a group of B. bronchiseptica strains (named Complex IV) that appeared more closely related to the human-restricted pathogen B. pertussis than other B. bronchiseptica. A majority of these B. bronchiseptica strains were isolated from cases of disease in humans and are thought to represent a lineage of human-adapted B. bronchiseptica (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). Using comparative genomic hybridization analyses, Complex IV strains appeared to contain polymorphic wbm loci (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). By these analyses, MO149 was identified as a B. bronchiseptica strain that expressed an O PS but contained homologues of just three previously characterized wbm genes, suggesting that it contained a novel wbm locus.In this report, we describe a novel O PS structure for strain MO149, confirm that it contains a novel wbm locus, and demonstrate that its O PS is very poorly immunogenic compared with type O1 or O2 O PSs. Our data support proposals that the wbm locus is one of the few regions of the Bordetella genome that undergoes extensive recombination and/or lateral gene transfer (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar, 18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar), that a greater number of O PS structures exist in the Bordetella population than was previously thought, and suggest that escape from host immunity might be a driver for the generation of this diversity.DISCUSSIONPrevious analyses suggested that many B. bronchiseptica strains had either O1 or O2 type O PSs (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar) and that variation among B. bronchiseptica and B. parapertussis O PS occurred at the terminal residue with the polymer backbone being highly conserved among strains (13Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar, 14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). One other variant O-antigen locus had been described, for the ovine B. parapertussis strain BPP5 (34Brinig M.M. Cummings C.A. Sanden G.N. Stefanelli P. Lawrence A. Relman D.A. J. Bacteriol. 2006; 188: 2375-2382Crossref PubMed Scopus (63) Google Scholar). Its wbm locus resembles that of the human B. parapertussis strain 12822 except for the presence of single nucleotide frameshift mutations in three genes, the absence of a wbmE and a wbmK homologue, and the presence of a unique methyltransferase-encoding gene. However, it is not known if this strain synthesizes an O-antigen or if these genetic differences lead to the biosynthesis of a different O PS. Our findings demonstrate that at least one other O PS type exists, that the extent of variability among wbm loci is greater than previously thought, and that variability is not confined to the terminal residue.Previously, we described the B. bronchiseptica RB50 and B. parapertussis 12822 wbm loci as being composed of a variable central portion that contained genes involved in synthesis of the strain variable terminal residue modifications flanked by highly conserved genes involved in synthesis of the highly conserved linker-polymer region (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). B. parapertussis BPP5 wbm also conforms to this model, containing the unique methlytransferase gene and other medium conserved genes flanked by very highly conserved wbm homologues. All of the MO149 wbm genes display homology to RB50 and/or 12822 wbm genes except for the putative methyltransferase that we propose as the sole terminal residue modification function in this locus. However, the gene arrangement in the MO149 locus is very different from that of the 12822/RB50 loci (Fig. 5), including multiple differences in regions that we had previously described as being part of the highly conserved flanking regions of the Bordetella wbm locus. The MO149 locus shows nearly complete DNA identity with the RB50/12822 loci at both ends, but there are clearly visible points at which divergence begins. At one end, this are 13 bp upstream of the wbmC stop codon (Fig. 7), resulting in MO149 WbmC containing a different C-terminal 5 amino acids compared with RB50/12822 WbmC, and at the other end it is 21 bp upstream of the BB0120 homologue start codon (Fig. 7), suggesting that in MO149, the BB0120 homologue may have a different promoter compared with RB50/12822 strains.FIGURE 7Alignment of the conserved ends of B. bronchiseptica RB50 and MO149 wbm loci. The junctions between highly conserved and divergent DNA sequences are clearly visible. The WbmC stop codons and BB0120 start codons are boxed in order to show the positions of these junctions relative to protein coding sequences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bordetella genomes have relatively high GC content genomes (Table 3). In all of the Bordetella strains for which sequence information is available, the GC contents of their wbm loci are considerably lower than their overall genome GC contents (Table 3). Interestingly, the variable regions have a lower GC content than the relatively conserved wbmA to -C genes, which in turn have a GC content different from either the variable region or the overall genome average. Even in the relatively distantly related Bordetella, Bordetella avium and Bordetella petrii, the wbm loci have low GC contents. These loci have not been functionally characterized but were designated as the wbm loci for these analyses on the basis that they are adjacent to the wlb locus in each strain and contain genes that encode proteins that are homologous to other O-antigen biosynthesis enzymes.TABLE 3The GC contents of Bordetella genomes compared with their wbm lociStrainGC content of genomeGC content of variable region of wbm locusGC content of wbmA to -C%%%B. bronchiseptica RB5068.0757.9864.64B. bronchiseptica M014968.4158.2164.85B. bronchiseptica 25367.6258.6464.57B. parapertussis 1282268.1058.6264.43B. parapertussis BPP567.8159.264.44B. avium 19761.5855aThe genes BAV0081–BAV0089, adjacent to the B. avium wlb locus and displaying homology to other O-antigen biosynthesis genes, were used as comprising the O-antigen biosynthesis locus in this strain.AbsentB. petrii DSMZ1280465.4856.24bThe genes BPE4829–BPE4837, adjacent to the B. petrii wlb locus and displaying homology to other O-antigen biosynthesis genes, were used as comprising the O-antigen biosynthesis locus in this strain.Absenta The genes BAV0081–BAV0089, adjacent to the B. avium wlb locus and displaying homology to other O-antigen biosynthesis genes, were used as comprising the O-antigen biosynthesis locus in this strain.b The genes BPE4829–BPE4837, adjacent to the B. petrii wlb locus and displaying homology to other O-antigen biosynthesis genes, were used as comprising the O-antigen biosynthesis locus in this strain. Open table in a new tab Low GC content is one of the signatures of horizontally acquired DNA. We propose that the Bordetella wbm loci are genomic islands, although they lack any obvious DNA mobility functions that are often associated with genomic islands. In the mammalian-adapted Bordetella, these islands are integrated at a highly conserved site and in all Bordetella are situated immediately adjacent to the wlb LPS biosynthesis locus. Our findings suggest that a repertoire of wbm genomic islands exists among the Bordetellae, conferring variability to the O PS of Bordetella LPS.There is little cross-reactivity between antibody responses generated to the previously characterized O1 and O2 PS (Fig. 6) (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar). This suggests that O PS diversity might represent a means of antigenic variation that allows escape from preexisting host immunity. The poor immunogenicity of MO149 PS suggests that some strains of B. bronchiseptica employ a different means by which they escape host immunity by avoiding the generation of strong anti-O PS responses. It is interesting that B. bronchiseptica MO149 is a Complex IV strain that was isolated from infection in a human. Complex IV strains are proposed to be more closely related to the human-adapted B. pertussis than they are to Complex I B. bronchiseptica strains that have been isolated mainly from non-human sources (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). B. pertussis does not express O PS because the wbm locus has been deleted from seemingly all B. pertussis strains (17Cummings C.A. Brinig M.M. Lepp P.W. van de Pas S. Relman D.A. J Bacteriol. 2004; 186: 1484-1492Crossref PubMed Scopus (117) Google Scholar). It is hypothesized that B. pertussis evolved from a human-adapted B. bronchiseptica (or B. bronchiseptica-like) ancestor and that avoidance of existing anti-B. bronchiseptica immunity in the human host was key to its emergence in this niche (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). Loss of expression of highly immunogenic O PS was probably part of this avoidance. The O PS confers protection on B. bronchiseptica against host immune mechanisms, such as complement-mediated killing (20Burns V.C. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. Infect. Immun. 2003; 71: 86-94Crossref PubMed Scopus (52) Google Scholar), but B. pertussis appears to have evolved alternative protection mechanisms to compensate for the loss of O PS. Previously, analysis of the gene content of a large number of B. bronchiseptica strains identified the O1 or O2 wbm locus in all non-Complex IV strains studied. However, 4 of 13 Complex IV strains contained variant loci, including apparent deletions that abrogated expression of the Band A/O PS LPS (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). Thus, it is possible that avoidance of a strong anti-O PS host immune response, either through a poorly immunogenic O PS, such as O3, or absence of O PS expression, is required for B. bronchiseptica strains to exist in human hosts and that this generates selection pressure for the generation of O PS diversity that appears to be a feature among Complex IV B. bronchiseptica strains (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). Interestingly, we have identified the O3 wbm cluster in a number of Complex IV strains but not in any of 96 Complex I strains analyzed, suggesting that the O3 PS is specific to Complex IV and thus either advantageous for some aspect of Complex IV biology or selected against in Complex I strains. 5S. Hester, L. Goodfield, and E. Harvill, manuscript in preparation. Interestingly, anti-MO149 recognized Band A LPS but gave a strong signal in Western blots only from strains deleted for wbm although MO149 and WT 1289 appeared to express high levels of Band A LPS. This suggests that the presence of O PS appears to reduce recognition of Band A LPS by anti-MO149 antisera. Thus, the wbm locus might modify Band A LPS in some subtle way that prevents efficient antibody binding. In turn, this could result in anti-MO149 antibodies recognizing B. pertussis that expresses high levels of Band A LPS, which would be unmodified due to the loss of wbm from B. pertussis and possibly result in cross-species immunity that would help eliminate B. pertussis from human hosts and promote survival of B. bronchiseptica strains in this niche. It is evident that the role of O PS in Bordetella infection is not yet fully understood. IntroductionThe genus Bordetella currently comprises nine species of Gram-negative bacteria. The most extensively studied of these are the pathogens Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. B. pertussis infects only humans and is the causative agent of whooping cough in infants and persistent respiratory infections in adults (1Cherry J.D. J. Infect. Dis. 1996; 174: S259-S263Crossref PubMed Google Scholar). B. parapertussis exists as two separate lineages. One is adapted to the human host and causes whooping cough; the other is adapted to the ovine host, in which it can cause chronic pneumonia (2Porter J.F. Connor K. Donachie W. Microbiology. 1994; 140: 255-261Crossref PubMed Scopus (75) Google Scholar, 3Heininger U. Stehr K. Schmitt-Grohé S. Lorenz C. Rost R. Christenson P.D. Uberall M. Cherry J.D. Pediatr. Infect. Dis. J. 1994; 13: 306-309Crossref PubMed Scopus (148) Google Scholar). In contrast, B. bronchiseptica colonizes the respiratory tract of a large number of animals, and although it causes respiratory diseases in some farm, companion, and wild animals, most B. bronchiseptica infections are asymptomatic and chronic (4Goodnow R.A. Microbiol. Rev. 1980; 44: 722-738Crossref PubMed Google Scholar). B. bronchiseptica is occasionally isolated from the respiratory tract of humans and is probably acquired through contact with infected animals (5Gueirard P. Weber C. Le Coustumier A. Guiso N. J. Clin. Microbiol. 1995; 33: 2002-2006Crossref PubMed Google Scholar).Bordetella lipopolysaccharide (LPS) plays a number of different roles in Bordetella biology, including during infection (e.g. see Refs. 6Zhang X. Goebel E.M. Rodríguez M.E. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 5050-5058Crossref PubMed Scopus (15) Google Scholar, 7Zhang X. Rodríguez M.E. Harvill E.T. PLoS One. 2009; 4e6989Crossref PubMed Scopus (24) Google Scholar, 8Pilione M.R. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. Infect. Immun. 2004; 72: 2837-2842Crossref PubMed Scopus (39) Google Scholar, 9Harvill E.T. Preston A. Cotter P.A. Allen A.G. Maskell D.J. Miller J.F. Infect. Immun. 2000; 68: 6720-6728Crossref PubMed Scopus (93) Google Scholar). We and others have defined the structures of the LPS of these Bordetella (Fig. 1). All three synthesize a very similar LPS core. In B. bronchiseptica and B. pertussis, the core can be further substituted by a trisaccharide to produce the structure referred to as Band A LPS (10Peppler M.S. Infect. Immun. 1984; 43: 224-232Crossref PubMed Google Scholar, 11Caroff M. Brisson J. Martin A. Karibian D. FEBS Lett. 2000; 477: 8-14Crossref PubMed Scopus (80) Google Scholar). B. parapertussis and B. bronchiseptica, but not B. pertussis, synthesize O PSs. 2The abbreviations used are: O PSO chain polysaccharideGalNAc3NAcA2,3-diacetamido-2,3-dideoxygalacturonic acidHFhydrofluoric acidManNAc3NAcA2,3-diacetamido-2,3-dideoxymannuronic acidGlcNAc3NAcA2,3-diacetamido-2,3-dideoxy-glucuronic acidSDRshort chain dehydrogenase/reductaseaaamino acid(s)TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Initially, the O PSs of both species were reported to be identical and composed of linear polymers of 1,4-linked GalNAc3NAcA (12Di Fabio J.L. Caroff M. Karibian D. Richards J.C. Perry M.B. FEMS Microbiol. Lett. 1992; 76: 275-281Crossref PubMed Scopus (75) Google Scholar), but later it was discovered that the terminal residue contained a number of modifications, that these modifications can vary between strains (13Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar), and that the O PS polymer is linked to the rest of the LPS molecule via an unusual 5-sugar linker (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To date, two types of modification, O1 and O2, have been described (13Vinogradov E. Peppler M.S. Perry M.B. Eur. J. Biochem. 2000; 267: 7230-7237Crossref PubMed Scopus (27) Google Scholar, 15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar). O1 and O2 PSs both contain 2,3,4-triamino-2,3,4-trideoxy-α-galacturonamide derivatives at their non-reducing ends. In O1 PS, this residue is formylated at positions 3 and 4 and has N-formyl-l-alanyl or l-alanyl substituents at N-2. In O2 PS, the amino group at position 2 is acetylated, at position 3 it is formylated, and at position 4 it is modified with a 2-methoxypropionyl substituent. Importantly, antibody responses to O PS appear to be wholly directed against the modified terminal residue because antisera raised against O1 type O PS did not recognize O2 type O PSs and vice versa, and in mice, immunization with LPS of one type did not confer protection against strains with the other type (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar). This suggests that Bordetella O PSs display antigenic variation in order to help evade cross-reaction with existing immunity within hosts.We have also studied the genetics of O PS biosynthesis (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Preston A. Allen A.G. Cadisch J. Thomas R. Stevens K. Churcher C.M. Badcock K.L. Parkhill J. Barrell B. Maskell D.J. Infect. Immun. 1999; 67: 3763-3767Crossref PubMed Google Scholar). B. bronchiseptica and B. parapertussis each contain a locus, wbm, containing 24 genes that is responsible for biosynthesis of the linker, polymer repeat, modifications of the terminal residue, and export of the O PS from the cytoplasm. We proposed that the central region of the wbm locus contains the genes that direct biosynthesis of the terminal residue modifications, and supporting this, the gene content of this region correlates with the modification type (14Preston A. Petersen B.O. Duus J.Ø. Kubler-Kielb J. Ben-Menachem G. Li J. Vinogradov E. J. Biol. Chem. 2006; 281: 18135-18144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A number of studies have used postgenomic technologies to investigate genetic diversity among the Bordetella (17Cummings C.A. Brinig M.M. Lepp P.W. van de Pas S. Relman D.A. J Bacteriol. 2004; 186: 1484-1492Crossref PubMed Scopus (117) Google Scholar, 18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). From these, the wbm locus emerged as one of the very few loci that exhibited clear variability among strains. In particular, these studies described a group of B. bronchiseptica strains (named Complex IV) that appeared more closely related to the human-restricted pathogen B. pertussis than other B. bronchiseptica. A majority of these B. bronchiseptica strains were isolated from cases of disease in humans and are thought to represent a lineage of human-adapted B. bronchiseptica (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). Using comparative genomic hybridization analyses, Complex IV strains appeared to contain polymorphic wbm loci (18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar). By these analyses, MO149 was identified as a B. bronchiseptica strain that expressed an O PS but contained homologues of just three previously characterized wbm genes, suggesting that it contained a novel wbm locus.In this report, we describe a novel O PS structure for strain MO149, confirm that it contains a novel wbm locus, and demonstrate that its O PS is very poorly immunogenic compared with type O1 or O2 O PSs. Our data support proposals that the wbm locus is one of the few regions of the Bordetella genome that undergoes extensive recombination and/or lateral gene transfer (15Buboltz A.M. Nicholson T.L. Karanikas A.T. Preston A. Harvill E.T. Infect. Immun. 2009; 77: 3249-3257Crossref PubMed Scopus (18) Google Scholar, 18Diavatopoulos D.A. Cummings C.A. Schouls L.M. Brinig M.M. Relman D.A. Mooi F.R. PLoS Pathog. 2005; 1: e45Crossref PubMed Scopus (213) Google Scholar), that a greater number of O PS structures exist in the Bordetella population than was previously thought, and suggest that escape from host immunity might be a driver for the generation of this diversity.
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