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DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers.

Zygmunt MS, Blasco JM, Letesson JJ, Cloeckaert A, Moriyón I - BMC Microbiol. (2009)

Bottom Line: Although most genes were highly conserved, species- and biovar-specific restriction patterns were found.Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization.The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, France. mzygmunt@tours.inra.fr

ABSTRACT

Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide.

Results: The polymorphism of O-polysaccharide genes wbkE, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manB(O-Ag) carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism.

Conclusion: The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

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Regions and genes encoding LPS biosynthetic enzymes in B. melitensis 16 M Region wbk contains genes coding for: (i), enzymes necessary for N-formylperosamine synthesis (gmd, per, wbkC); (ii), two O-PS glycosyltransferase (wbkE, wbkA); (iii), the ABC transporter (wzm, wzt); (iv) the epimerase/dehydratase necessary for the synthesis of an N-acetylaminosugar (wbkD); and (v), the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase enzyme that primes bactoprenol (wbkF). Genes manAO-Ag, manBO-Ag, manCO-Ag could be involved in the synthesis of mannose, the perosamine precursor. Restriction sites: A, AluI; AvI, AvaI; Av, AvaII; B, BglI; Bg, BglII; C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; Ha, HaeII; Hf, HinfI; P, PstI; Pv, PvuII; S, Sau3A; Sa, SaII; St, StyI.
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Figure 1: Regions and genes encoding LPS biosynthetic enzymes in B. melitensis 16 M Region wbk contains genes coding for: (i), enzymes necessary for N-formylperosamine synthesis (gmd, per, wbkC); (ii), two O-PS glycosyltransferase (wbkE, wbkA); (iii), the ABC transporter (wzm, wzt); (iv) the epimerase/dehydratase necessary for the synthesis of an N-acetylaminosugar (wbkD); and (v), the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase enzyme that primes bactoprenol (wbkF). Genes manAO-Ag, manBO-Ag, manCO-Ag could be involved in the synthesis of mannose, the perosamine precursor. Restriction sites: A, AluI; AvI, AvaI; Av, AvaII; B, BglI; Bg, BglII; C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; Ha, HaeII; Hf, HinfI; P, PstI; Pv, PvuII; S, Sau3A; Sa, SaII; St, StyI.

Mentions: Wild type B. melitensis, B. abortus, B. suis, B. neotomae, B. ceti, B. pinnipedialis and B. microti express a smooth (S)-type lipopolysaccharide (LPS) formed by an O-polysaccharide connected to a core oligosaccharide which, in turn, is linked to lipid A, the section embedded into the outer membrane. However, both B. ovis and B. canis lack the O-polysaccharide and, accordingly, their LPS is termed rough (R) (R-LPS). Brucella LPS is of great interest not only because of these species differences but also because it is the foremost diagnostic antigen and a major virulence factor [10]. Despite this, the structure and genetics of Brucella LPS is only partially understood. The O-polysaccharide is a homopolymer of N-formyl-perosamine in α (1–2) or in α (1–2) plus α (1–3) linkages [11], and these variations relate to the main serovars in Brucella S species (A dominant, related to the α (1–2) linkage; M dominant [α (1–2) plus α (1–3) in a 4:1 proportion]; or A = M [α (1–2) plus α (1–3) in a > 4:1 proportion]). Previous studies in B. melitensis 16 M and H38 (both biovar 1) have identified two genetic regions involved in O-polysaccharide synthesis and translocation (Figure 1)(reviewed in [12]). Region wbo encodes two putative glycosyltransferases (wboA and wboB) and region wbk contains the genes putatively involved in perosamine synthesis (gmd [GDP-mannose 4, 6 dehydratase] and per [perosamine synthetase]), its formylation (wbkC) and polymerization (glycosyltransferases) (wbkA and wbkE), as well as those for bactoprenol priming (wbkD and wbkF) and O-PS translocation (wzm and wzt). In addition, wbk contains genes (manAO-Ag, manBO-Ag, manCO-Ag) which may code for the enzymes that furnish mannose, the perosamine precursor. Intriguingly, wbkB and manBO-Ag do not generate R phenotypes upon disruption [12,13], and B. ovis and B. canis carry wbk genes despite the absence of the O-polysaccharide [14]. Much less is known on the Brucella core oligosaccharide. Reportedly, it contains 2-keto, 3-deoxyoctulosonic acid, mannose, glucose, glucosamine and quinovosamine [12,15] but the structure is unknown. Thus far, only three genes have been proved to be involved in core synthesis: pgm (phosphoglucomutase, a general biosynthetic function), manBcore (mannose synthesis) and wa** (putative glycosyltransferase) [12]. Obviously, genetic analysis encompassing a variety of strains could shed light on the differences behind the phenotypes of S and R species, confirm or rule out a role for known genes, and identify differences that could serve as serovar or biovar markers. With these aims, wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF, wkdD, wboA, wboB, wa** and manBcore were analyzed for polymorphism in the classical Brucella spp., B. ceti, and B. pinnipedialis.


DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers.

Zygmunt MS, Blasco JM, Letesson JJ, Cloeckaert A, Moriyón I - BMC Microbiol. (2009)

Regions and genes encoding LPS biosynthetic enzymes in B. melitensis 16 M Region wbk contains genes coding for: (i), enzymes necessary for N-formylperosamine synthesis (gmd, per, wbkC); (ii), two O-PS glycosyltransferase (wbkE, wbkA); (iii), the ABC transporter (wzm, wzt); (iv) the epimerase/dehydratase necessary for the synthesis of an N-acetylaminosugar (wbkD); and (v), the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase enzyme that primes bactoprenol (wbkF). Genes manAO-Ag, manBO-Ag, manCO-Ag could be involved in the synthesis of mannose, the perosamine precursor. Restriction sites: A, AluI; AvI, AvaI; Av, AvaII; B, BglI; Bg, BglII; C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; Ha, HaeII; Hf, HinfI; P, PstI; Pv, PvuII; S, Sau3A; Sa, SaII; St, StyI.
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Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Regions and genes encoding LPS biosynthetic enzymes in B. melitensis 16 M Region wbk contains genes coding for: (i), enzymes necessary for N-formylperosamine synthesis (gmd, per, wbkC); (ii), two O-PS glycosyltransferase (wbkE, wbkA); (iii), the ABC transporter (wzm, wzt); (iv) the epimerase/dehydratase necessary for the synthesis of an N-acetylaminosugar (wbkD); and (v), the polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferase enzyme that primes bactoprenol (wbkF). Genes manAO-Ag, manBO-Ag, manCO-Ag could be involved in the synthesis of mannose, the perosamine precursor. Restriction sites: A, AluI; AvI, AvaI; Av, AvaII; B, BglI; Bg, BglII; C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; Ha, HaeII; Hf, HinfI; P, PstI; Pv, PvuII; S, Sau3A; Sa, SaII; St, StyI.
Mentions: Wild type B. melitensis, B. abortus, B. suis, B. neotomae, B. ceti, B. pinnipedialis and B. microti express a smooth (S)-type lipopolysaccharide (LPS) formed by an O-polysaccharide connected to a core oligosaccharide which, in turn, is linked to lipid A, the section embedded into the outer membrane. However, both B. ovis and B. canis lack the O-polysaccharide and, accordingly, their LPS is termed rough (R) (R-LPS). Brucella LPS is of great interest not only because of these species differences but also because it is the foremost diagnostic antigen and a major virulence factor [10]. Despite this, the structure and genetics of Brucella LPS is only partially understood. The O-polysaccharide is a homopolymer of N-formyl-perosamine in α (1–2) or in α (1–2) plus α (1–3) linkages [11], and these variations relate to the main serovars in Brucella S species (A dominant, related to the α (1–2) linkage; M dominant [α (1–2) plus α (1–3) in a 4:1 proportion]; or A = M [α (1–2) plus α (1–3) in a > 4:1 proportion]). Previous studies in B. melitensis 16 M and H38 (both biovar 1) have identified two genetic regions involved in O-polysaccharide synthesis and translocation (Figure 1)(reviewed in [12]). Region wbo encodes two putative glycosyltransferases (wboA and wboB) and region wbk contains the genes putatively involved in perosamine synthesis (gmd [GDP-mannose 4, 6 dehydratase] and per [perosamine synthetase]), its formylation (wbkC) and polymerization (glycosyltransferases) (wbkA and wbkE), as well as those for bactoprenol priming (wbkD and wbkF) and O-PS translocation (wzm and wzt). In addition, wbk contains genes (manAO-Ag, manBO-Ag, manCO-Ag) which may code for the enzymes that furnish mannose, the perosamine precursor. Intriguingly, wbkB and manBO-Ag do not generate R phenotypes upon disruption [12,13], and B. ovis and B. canis carry wbk genes despite the absence of the O-polysaccharide [14]. Much less is known on the Brucella core oligosaccharide. Reportedly, it contains 2-keto, 3-deoxyoctulosonic acid, mannose, glucose, glucosamine and quinovosamine [12,15] but the structure is unknown. Thus far, only three genes have been proved to be involved in core synthesis: pgm (phosphoglucomutase, a general biosynthetic function), manBcore (mannose synthesis) and wa** (putative glycosyltransferase) [12]. Obviously, genetic analysis encompassing a variety of strains could shed light on the differences behind the phenotypes of S and R species, confirm or rule out a role for known genes, and identify differences that could serve as serovar or biovar markers. With these aims, wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF, wkdD, wboA, wboB, wa** and manBcore were analyzed for polymorphism in the classical Brucella spp., B. ceti, and B. pinnipedialis.

Bottom Line: Although most genes were highly conserved, species- and biovar-specific restriction patterns were found.Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization.The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, France. mzygmunt@tours.inra.fr

ABSTRACT

Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide.

Results: The polymorphism of O-polysaccharide genes wbkE, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manB(O-Ag) carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism.

Conclusion: The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

Show MeSH