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

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.

a putative transaminase 4 . However, the exact substrate of the PorR protein has not been identified. As A-LPS is assumed to be a P. gingivalis virulence factor, genes involved in A-LPS biosynthesis have been recently identified. To date, vimA, vimE, vimF 14,15 , wbpB 16 , waaL 11 ugdA, rfa 17 , wzy 9 , gtfB 18 , PGN_0242, PGN_0663 19 wbaP, wzx, wzzP 13 and porR have been demonstrated to be involved in A-LPS biosynthesis. The PorR, WbpB and UgdA proteins are predicted to participate in the initial synthesis of structural sugar(s) in the APS. The Rfa protein is involved in the synthesis of the core oligosaccharide of LPS molecules. WbaP (initial phosphoryl glycosyl transferase onto undecaprenyl monophosphate), Wzx (O-antigen flippase), Wzy (O-antigen polymerase), WzzP (O-antigen chain length regulator) and WaaL (O-antigen ligase) are involved in the biosynthesis of both O-antigen and A-antigen. The VimA, VimE and VimF proteins are acetyltransferase, hypothetical protein and galactosyltransferase proteins, respectively [20][21][22] .
P. gingivalis has approximately 30 proteins (called CTD proteins) that contain a conserved C-terminal domain 23 . Some of these proteins are secreted onto the cell surface via the PorSS/T9SS system and are then bound to A-LPS or secreted in the culture supernatant 6,7,19,24 . RgpB and HBP35 are used as model proteins to analyze the PorSS/ T9SS secretion system in P. gingivalis because these proteins exhibit diffuse bands on a gel, which is indicative of the A-LPS bound form 19,25,26 .
Whereas wild-type P. gingivalis strains possess membrane associated gingipain activities on the cell surface, P. gingivalis strain HG66 has significantly reduced gingipain activities on the cell surface but still retains the activities in the culture supernatant 27 . Some CTD proteins, such as RgpB 28 , peptidyl arginine deaminase 29 , periodontain 30 and CPG70 31 , have been purified from the culture supernatant of strain HG66. We have recently shown that the latter strain possesses O-LPS, but lacks A-LPS, similar to the porR mutant 13 . One aim of this study was to determine the reason for the A-LPS deficiency in strain HG66.
Our study revealed that the A-LPS deficiency of strain HG66 was the result of a nonsense mutation in the wbpB gene, and likewise, Wbp pathway gene mutants were A-LPS deficient.

Results
Identification of the gene mutation responsible for A-LPS deficiency in strain HG66. We have recently shown that HG66 possesses O-LPS, but not A-LPS 13 . When expressed in a wild-type background, porR (PGN_1236), vimA (PGN_1056), vimE (PGN_1055), wbpB (PGN_0168), ugdA (PGN_0613), and wbaP (PGN_1896) mutants retain O-LPS but lose A-LPS 13,14,17 . We thus hypothesized that mutation of one or more of these genes was responsible for the A-LPS deficient phenotype in strain HG66. Among the above genes, we found that wbpB from strain ATCC 33277 conferred the pigmentation phenotype on strain HG66 ( Figure 1A). HG66 cells expressing the wbpB gene from ATCC 33277 showed greater hemagglutination and gingipain activities on the cell surface compared with strain HG66 ( Figure 1B, C). We have recently shown that cell lysates from strain HG66 exhibit no diffuse HBP35 and Rgp bands on a gel due to a lack of A-LPS 13 . Immunoblot analyses revealed that HG66 cell lysates expressing wbpB from ATCC 33277 contained A-LPS when probed with an anti-A-LPS antibody. The lysates also exhibited the diffuse HBP35 and Rgp bands that are also observed in the wild-type ATCC 33277 or W83 strains ( Figure 2A). Interestingly, an anti-O-LPS antibody recognized higher molecular mass immunoreactive products in the HG66 cells expressing wbpB from ATCC 33277 compared with strain HG66 (Figure 2A). We next confirmed that purified LPS from HG66 cells expressing wbpB from ATCC 33277 contained both A-LPS and O-LPS by immunoblot analyses ( Figure 2B). These results suggested that strain HG66 might encode a non-functional wbpB gene. Therefore, the coding sequence of the wbpB gene from strain HG66 was amplified by PCR and sequenced. We found that a nonsense mutation occurred at amino acid Q 240 of the WbpB protein ( Figure 2C, Supplemental Text 2). WbpB is an oxidoreductase that forms UDP-GlcNAc(3 keto)A from UDP-GlcNAcA ( Figure 3). Additionally, WbpB is a component of the Wbp pathway, which synthesizes UDP-ManNAc(3NAc)A, a precursor of the ManNAc(3NAc)A residue in the B-band Oantigen of P. aeruginosa 33,34 . The P. gingivalis WbpB protein had high similarity to P. aeruginosa WbpB and Bordetella pertussis WlbA (Supplemental Figure 1). A truncated WbpB protein lacking C-terminal 83 amino acids is likely to be non-functional, although we did not confirm expression at the mRNA or protein levels.
Genes in the Wbp pathway are involved in the biosynthesis of A-LPS. As WbpB belongs to the Wbp pathway, we next hypothesized that the nucleotide-activated sugar formed by the Wbp pathway in P. gingivalis was recognized by an anti-A-LPS antibody. Genomic analysis revealed that PGN_1243 and PGN_0631 (ugdA) were WbpA homologs, PGN_0168 (wbpB) was a WbpB homolog, PGN_1236 (porR) was a WbpE homolog, and PGN_0002 was a WbpD homolog; however, no WbpI homologs were found (Supplemental Figures 1, 2, 3, 4). It has been shown that the ugdA, wbpB and porR gene products are involved in the biosynthesis of A-LPS 4,16,17 . We focused on the PGN_0002 protein because it may be the last enzyme of the Wbp pathway in P. gingivalis, and it has not yet been characterized. PGN_0002 showed high similarity to P. aeruginosa WbpD and Bordetella pertussis WlbB, and both of these proteins possessed acetyltransferase activity, allowing the formation of UDP-GlcNAc(3NAc)A from UDP-GlcNAc(3NH 2 )A (Supplemental Figure 4). We constructed full PGN_1243 and PGN_0002 gene deletions; we were able to generate a PGN_0002 mutant, but not a PGN_1243 mutant. The PGN_0002 mutant exhibited a pigmentless phenotype and reduced hemagglutination and gingipain activities on the cell surface compared with wild-type cells ( Figure 4A, B, C). Complementation of the PGN_0002 gene or expression of P. aeruginosa wbpD restored the pigmentation phenotype and, hemagglutination and gingipain activities on the cell surface ( Figure 4A, B, C). Immunoblot analyses revealed that the wbpB, porR and PGN_0002 mutants were not immunoreactive to anti-A-LPS, and no HBP35 and Rgp diffuse bands were observed ( Figure 5A). Interestingly, anti-O-LPS immunoreactive bands in the wbpB, porR and PGN_0002 mutants exhibited a lower molecular mass than those of the wild-type cells. The ugdA mutant showed decreased immunoreactivity to anti-A-LPS and reduced HBP35 and Rgp diffuse bands. The PGN_1243 gene products may compensate for the WbpA-like activity in the ugdA mutant. We also confirmed that purified LPSs from various P. gingivalis mutants in the Wbp pathway possessed O-LPS, but lacked A-LPS ( Figure 5B).

Discussion
Our previous study revealed that CTD  via the type IX secretion system 6 . P. gingivalis has approximately 30 CTD proteins. Of these, 19 have been shown to be LPS-modified, suggesting that they bind to the A-LPS 35 . Specifically, RgpB, TapA and HBP35 have been experimentally shown to be anchored to the cell surface by binding to A-LPS 25 10 has demonstrated that the structure of APS is relatively similar to that of yeast mannan and is predicted to be synthesized by a1R6 and a1R2 mannosyltransferases. However, the genes encoding the mannosyltransferases have not been identified. Recently, the vimF gene product, which is one of proteins necessary for A-LPS biosynthesis, has been demonstrated to possess galactosyltransferase activity 22 . In addition, not all of the mannosidases are involved in A-LPS synthesis 37 . Therefore, the postulated structure of APS may not yet be fully understood.
In this study, we found that a wbpB gene mutation was responsible for the non-pigmented phenotype of strain HG66, and we further demonstrated that genes in the Wbp pathway were involved in the biosynthesis of A-LPS. The existence of ManNAc(3NAc)A was first reported in P. aeruginosa LPS more than 30 years ago 38 . It has been recently shown that the ManNAc(3NAc)A in P. aeruginosa is enzymatically synthesized by WbpA, WbpB, WbpE, WbpD and WbpI, proteins that together have been designated the Wbp pathway 33,34,39 . Bordetella pertussis has a similar Wbp pathway and possesses two wbpA gene homologs. These homologs are present separately and apart from the other gene homologs located in the wlb gene cluster 40 .
Almost all of the genes in the Wbp pathway are located within a gene cluster in P. aeruginosa and B. pertussis. Gene homologs of wbpA, wbpB, wbpE and wbpD were located separately in the P. gingivalis genome, and no wbpI gene homologs were found in the genome ( Figure 6). ManNAc(3NAc)A, the C2-epimer of GlcNAc(3NAc)A, is present not only in the LPS of P. aeruginosa and B. pertussis, but also in the cell wall polysaccharide of the Gram-positive thermophile, Bacillus stearothermophilus 41 . GlcNAc(3NAc)A has been reported to be present in the LPS of a number of P. aeruginosa strains, including P1-III and P14, the N-linked glycans of the methanogenic archaea, Methanococcus voltae and Methanococcus maripalidus, and the cell surface polysaccharide of the Gram-negative spirochete, Treponema medium [42][43][44][45] . A partial genome research survey revealed that Thermus thermophiles (Deinococcus-Thermus), Zobellia galactanivorans (Bacteroidetes), Wolinella succinogenes (Epsilonproteobacteria) and Photorhabdus asymbiotica (Gammaproteobacteria) possess 5 genes in the Wbp pathway. Conversely, P. gingivalis and Odoribacter splanchnicus (Bacteroidetes), Sulfurihydrogenibium azorense (Aquificae) and Xenorhabdus bovienii (Gammaproteobacteria) possess 4 genes in the Wbp pathway and lack the wbpI gene  Figure 6). The presence of 5-Wbp pathway gene homologs suggests that a particular species may utilize ManNAc(3NAc)A as a structural sugar, whereas the presence of 4-Wbp pathway gene homologs (lacking wbpI) suggests that a particular species may utilize GlcNAc(3NAc)A as a structural sugar. As ManNAc(3NAc)A and GlcNAc(3NAc)A have C2-and, C3-acetylated and C6-uronic acid forms, they are called di-acetylated mannuronic acid and glucuronic acid, respectively. Considering the conservation and the distribution of these molecules from archaea to Gram-positive and Gram-negative bacteria, di-acetylated uronic acid(s) may be structurally advantageous to endure a variety of environmental stresses.
It has been shown that O-glycosylated proteins are widely distributed in bacteria within the Bacteroidetes phylum 46 . Coyne et al. 46 generated a specific antibody against the sugar portion of the Oglycosylated proteins in Bacteroides fragilis. Although this antibody mainly recognized protein bands from Bacteorides species, it also recognized a few P. gingivalis protein bands. To date, OMP85, Mfa1, PGN_0743 (a probable FKBP PPIase), PGN_0876 (a TPR domain protein), PGN_1513 (a hypothetical protein) and PGN_0729 (an outer membrane protein 41 precursor) have been reported to be glycoproteins in P. gingivalis [47][48][49] . As these glycoproteins each exhibit discrete bands in SDS-PAGE gels, they may be glycosylated by commonly known O-linked or N-linked glycosylation systems. Conversely, A-LPS-modified CTD proteins are expressed via the T9SS and are A-LPS dependent. However, the exact binding mechanism between CTD proteins and A-LPS is not known.
Based on the previously known molecules involved in the biosynthesis of A-LPS, we identified the gene mutation responsible for the non-pigmented phenotype of strain HG66 and found that genes in the Wbp pathway were involved in the biosynthesis of A-LPS. To date, the presence of di-acetylated glucuronic acid in P. gingivalis LPS molecules has not been documented. The analysis of di-acetylated glucuronic acid will shed light on novel features of the T9SS-dependent glycosylation system.

Methods
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Supplemental Tables 1 and 2, respectively [50][51][52] .
Chemicals. The proteinase inhibitors Na-p-tosyl-L -lysine chloromethyl ketone (TLCK) and iodoacetamide were purchased from Wako (Japan), and leupeptin was obtained from the Peptide Institute (Japan).
Construction of P. gingivalis strains. The oligonucleotides used in this study are listed in Supplemental Table 3. The general manipulation of DNA, restriction and mapping of plasmids and transformation of E. coli and P. gingivalis have been described in detail elsewhere 26 . The chromosomal DNA from P. gingivalis ATCC 33277 was used as the template for cloning purposes. The construction of various mutants from P. gingivalis ATCC 33277 or complemented strains from the mutants is described in Supplemental Text 1.
Sequencing of the wbpB gene of P. gingivalis HG66. The coding region of the wbpB gene from strain HG66 was amplified using PGN_0168upFw/PGN_0168upRev primers and cloned into the pGEM-T Easy vector. Sequencing was performed by direct sequencing using the purified PCR product or by the common method using the cloned vector with SP6/T7 primers.  Japan). In brief, appropriate amounts of the bacterial cell as well as the bacterial culture supernatant, were added to the reaction mixture (0.25 ml) containing 5 mM cysteine, 20 mM sodium phosphate buffer, pH 7.5, and 10 mM each fluorogenic substrate. After 10 min incubation at 40uC, the reaction was terminated by adding 100 mM sodium acetate buffer, pH 5.0, containing 10 mM iodoacetic acid (0.25 ml). The released 7-amino-4-methyl-coumarine was measured at 460 nm (excitation at 380 nm) by fluorescence spectrophotometer Beckman Coulter DTX 800 (Brea, CA).
Hemagglutination activity. Overnight cultures of P. gingivalis strains in enriched BHI medium were centrifuged, washed once with phosphate-buffered saline (PBS), and suspended in PBS at an optical density of 1.0 at 595 nm. The bacterial suspensions were then diluted in a two-fold series with PBS. A 100-ml aliquot of each suspension was mixed with an equal volume of defibrinated sheep erythrocyte suspension (1% in PBS) and incubated in a round-bottom microtiter plate at room temperature for 3 h.
Preparation of P. gingivalis LPS. A fully grown 200-ml culture was centrifuged, washed once with distilled water and suspended with 5 ml of 10 mM Tris-HCl (pH8.0) containing 2% SDS. Next, 20 mg/ml of DNase and RNase (Sigma, St. Louis, MO. USA) was added at 37uC for 1 h, and 20 mg/ml of proteinase K (Takara, Japan) was added at 37uC overnight. After the overnight incubation, the samples were mixed with preheated 90% phenol at 68uC and stirred at 68uC for 20 min. The samples were www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 5056 | DOI: 10.1038/srep05056 centrifuged at 7,000 rpm for 20 min. The aqueous solutions were dialyzed against milliQ water to remove residual phenol. The dialyzed solutions were centrifuged at 100,000 3 g for 3 h, and the precipitated samples were dissolved in milliQ water as the LPS fraction. LPS was visualized by silver staining.
Gel electrophoresis and immunoblot analysis. SDS-PAGE and immunoblot analysis were performed as described previously 13,19,26 . For visualization of LPS, modified SDS-PAGE containing 4 M urea in the separating gel or Tris-Tricine SDS-PAGE was used.
Preparation of antiserum. An anti-HBP35 rabbit polyclonal antibody 53 was used to detect HBP35, mAb 1B5 was used to detect A-LPS 12 , mAb TDC-5-2-1 was used to detect O-LPS and mouse polyclonal antiserum against the amino acid region (E 361 -L 375 ) within the catalytic domain of RgpB (PGN_1416) was used to detect Rgp 13 . To prepare rabbit antiserum against the amino acid region (E 361 -L 375 ) within the catalytic domain of RgpB (PGN_1416), a rabbit was immunized by EveBioscience Co., Ltd. (Wakayama, Japan), and the antiserum was named anti-Rgp (rabbit) to distinguish it from anti-Rgp (mouse) described above.
Statistical analysis. The significance of all described comparisons was established using two-tailed unpaired t tests on triplicate samples with a significance level of 0.01.