Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog

Abstract

Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

FIG. 1.

MALDI negative-ion mass spectra of O-deacylated B. bronchiseptica strain 4650 batch 2 lipid A: direct time-of-flight spectrum (a), PSD spectrum of the 1,113-m/z precursor ions (b), and PSD spectrum of the 1,274-m/z precursor ions (c). (d) Negative-ion mass spectrum of N-acetylated (+Ac), O-deacylated B. bronchiseptica lipid A.

FIG. 2.

(A) Schematic representation of the genomic organization of the glycosylation loci BP0399 and BP0398 in B. pertussis strain Tohama I. Orthologous genes are found in other bordetellae, including B. bronchiseptica RB50 (locus tags BB4269 and BB4268), B. parapertussis hu 12822 (locus tags BPP3825 and BPP3824), and B. avium 197N (locus tags BAV2928 and BAV2927) (54, 63). BP0399 and BP0398 encode putative glycosyl transferases belonging to CAZy families 2 and 83, respectively (1). BP0397 and BP0396 encode putative proteins of as-yet-unknown function. #, characters in capital indicate imperfect heptads identified by the BvgA binding site motif, and characters in lowercase indicate a base between the heptads (19). (B) Results of semiquantitative RT-PCR analysis (boxed) and PCR controls of BP0399, BP0398, and vag8 with B. pertussis strain BP338 (wild type) and its isogenic mutant BP347 (bvgS::Tn5) after growth in SS broth and on BG agar. †, RT-PCRs using cDNA from 100 ng total RNA as a template; ‡, control PCRs using 100 to 200 ng total RNA as a template; +, control PCRs using 100 ng genomic DNA as a template; −, control PCRs without a template.

FIG. 3.

Comparison of negative-ion MALDI mass spectra of B. pertussis LPS and lipid A. Results are shown for LPS isolated from the mutant BPM2859 (a) and the wild-type BP338 (b) strains and lipid A isolated from the mutant BPM2859 (c) and the wild-type BP338 (d) strains. The crossed peaks at m/z values of 1,349 in panels c and d correspond to a contaminant.

FIG. 4.

PSD MALDI negative-ion mass spectra of the extra-HexN-containing molecular species of lipid A isolated from the B. pertussis wild-type strain BP338: 1,494-m/z (a), 1,720-m/z (b), and 1,881-m/z (c) precursor ions.

FIG. 5.

MALDI negative-ion mass spectra of O-deacylated lipid A from BPM2859 (a) and BP338 (b) strains. (c) PSD MALDI spectrum of the precursor ion from spectrum b at an m/z of 1,274.

FIG. 6.

Amino acid analyzer elution profiles of hydrofluoric acid-released lipid A substituents. (a) Standard GlcN; (b) standard GalN; (c) derivatives released from wild-type strain BP338 lipid A; (d) derivatives released from mutant strain BPM2859. R_t, retention time.

FIG. 7.

Proposed structure for strain BP338 B. pertussis lipid A penta-acyl molecular species representing both phosphate groups substituted with GlcNs. Dotted-line bonds indicate incompleteness of the substitutions, leading to structures with one or no substituting GlcNs (m/z = 1,720 or 1,559, respectively). MW, molecular weight.

FIG. 8.

Model for the catalytic reaction mediated by the gene products of BP0399 and BP0398 of B. pertussis Tohama I derivatives, representative of the orthologous proteins of other bordetellae. Hypothesized reactions are based on amino acid sequence similarities of the gene products of BP0399 and BP0398 to ArnC/GtrB and ArnT proteins, respectively. GlcN is symbolized by a hexagon. PP, periplasm; IM, inner membrane; CP, cytoplasm; P_i, phosphate (inorganic).