Abstract
The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)[β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.
Highlights
These include the native R-LPS (LOS) obtained from a strain producing increased proportions of R-LPS [18] (BmEPR), a mutant in which the wbkD gene was disrupted (Bm_wbkD) and a mutant affected in the ManBcore protein (Bm_manBcore) as well as the double mutant strain Bm_wadC_per
The structural elucidation of the deacylated LOSs from BmEPR and Bm_wbkD, carried out using NMR spectroscopy, revealed that both mutants are capable of producing a complete core structure (Fig. 4B) as well as minor components lacking the terminal residue I (19 and 48%, respectively) and/or residue E (34 and 17%, respectively)
The pseudo-molecular ions observed in the mass spectra of the deacylated LOS from Bm_wbkD (Fig. 2B and Table 2) were in agreement with the two major components determined by NMR spectroscopy as follows: (i) a complete core decasaccharide with a molecular formula of C64H116N8O53P2 that is consistent with a structure containing two Hex (Glc and Man), four HexN (GlcN), two Kdo, and two HexNN residues bearing phosphomonoester substituents (GlcN3N1P and GlcN3N4P), and (ii) a nonasaccharide with a molecular formula of C58H104N7O49P2 that is consistent with the same components as above but having three GlcN residues instead of four
Summary
O-PS, O-specific polysaccharide; LOS, lipooligosaccharide; R-LPS, rough lipopolysaccharide; BMDC, bone marrow-derived dendritic cell; PE, phycoerythrin; Kdo, 3-deoxy-D-manno-oct-2ulosonic acid; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple-bond correlation; HR-MS, high resolution mass spectrometry. Can be due to mutation in genes encoding proteins involved in the biosynthesis of the monosaccharide components of the O-PS, its polymerization or transport, or the core oligosaccharide. The genes involved in the biosynthesis of the LPS of B. melitensis were screened, and several mutants affected in the biosynthesis of both the core oligosaccharide or O-PS were obtained [10]. To assign mutations to the biosynthetic pathways, the mutants were classified as R1 (complete core), R2 (defective core), and R3 (deep R core), respectively, according to the decrease in their LPS apparent molecular mass [10]. We report that deletion of B. melitensis wadC removes the core oligosaccharide section not linked to the O-PS. Despite carrying the O-PS essential in virulence, the lack of this core section results in a marked change in bacterial surface physicochemical properties, a more efficient detection by innate immunity, and attenuation in both cellular and animal models
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