Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

Abstract

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharide (LPS) molecules, O-LPS and A-LPS. We have recently shown that P. gingivalis strain HG66 lacks A-LPS. Here, we found that introduction of a wild-type wbpB gene into strain HG66 restored formation of A-LPS. Sequencing of the wbpB gene from strain HG66 revealed the presence of a nonsense mutation in the gene. The wbpB gene product is a member of the Wbp pathway, which plays a role in the synthesis of UDP-ManNAc(3NAc)A in Pseudomonas aeruginosa; UDP-ManNAc(3NAc)A is sequentially synthesized by the WbpA, WbpB, WbpE, WbpD and WbpI proteins. We then determined the effect of the PGN_0002 gene, a wbpD homolog, on the biosynthesis of A-LPS. A PGN_0002-deficient mutant demonstrated an A-LPS biosynthesis deficiency. Taken together with previous studies, the present results suggest that the final product synthesized by the Wbp pathway is one of the sugar substrates necessary for the biosynthesis of A-LPS.

Figure 1. Pigmentation and hemagglutination and gingipain activities of Porphyromonas gingivalis.

(A) Colony pigmentation of Porphyromonas gingivalis cells on blood agar plates. (B) The hemagglutination activities of P. gingivalis strains were measured. (C) The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of W83, HG66, HG66/WbpB+ and ATCC 33277 were measured. The mean of each protease activity of W83 was regarded as 100%. Asterisks indicate significant differences in enzymatic activity between W83 and various strains (P < 0.01). The bars are expressed as the means ± standard deviation for triplicate samples from one of two independent experiments.

Figure 2. Immunoblot and LPS analyses of Porphyromonas gingivalis strain HG66.

(A) Immunoblot analyses of cell lysates from various P. gingivalis strains were performed with anti-A-LPS (mAb 1B5), anti-O-LPS (mAb TDC-5-2-1), anti-HBP35 or anti-Rgp antibodies. Three sets of HG66/WbpB+ strains were obtained from each clone. (B) The LPS fraction from P. gingivalis HG66 or HG66/WbpB+ strain was stained by silver staining. The cropped silver stained gels were run under a same SDS-PAGE gel. Immunoblot analyses were performed with anti-A-LPS (mAb 1B5) or anti-O-LPS (mAb TDC-5-2-1) antibodies. (C) Physical map of the area around the wbpB gene in P. gingivalis. The arrowhead indicates the nonsense mutation in HG66.

Figure 3. Wbp pathway for the biosynthesis of UDP-ManNAc(3NAc)A.

Abbreviations were seen in Supplemental Text 3. Gene homologs of the Wbp pathway in Porphyromonas gingivalis are represented in colored PGN_ numbers.

Figure 4. Pigmentation and hemagglutination and gingipain activities of various Porphyromonas gingivalis strains.

(A) Colony pigmentation of Porphyromonas gingivalis cells on blood agar plates. (B) The hemagglutination activities of P. gingivalis strains were measured. (C) The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of ATCC 33277 (wild-type), ugdA, wbpB, porR, PGN_0002, PGN_0002/PGN_0002+ and PGN_0002/Pseudomonas aeruginosa (P. a) WbpD+ were measured. The mean of each protease activity of ATCC 33277 was regarded as 100%. Asterisks indicate significant differences in enzymatic activity between ATCC 33277 and various strains (P < 0.01). The bars are expressed as the means ± standard deviation for triplicate samples from one of two independent experiments.

Figure 5. Immunoblot and LPS analyses of various Porphyromonas gingivalis strains.

(A) Immunoblot analyses of cell lysates from various P. gingivalis strains were performed with various antibodies. (B) The LPS fraction from various P. gingivalis strains was stained by silver staining. Immunoblot analyses were performed with antibodies for LPS.

Figure 6. Gene context for representative gene homologs of the Wbp pathway in Porphyromonas gingivalis ATCC 33277, Thermus thermophiles HB27, Zobellia galactanivorans, Odoribacter splanchnicus, Wolinella succinogenes, Sulfurihydrogenibium azorense, Xenorhabdus bovienii and Photorhabdus asymbiotica.

The lengths of the genes are not drawn to scale. Each homologous set of sequences is represented by one color.