Kiyoshi Konishi

Identification of a Porphyromonas gingivalis novel protein sov required for the secretion of gingipains.

Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in‐frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP‐7, and PtpA were not affected. Immunoblot analysis using anti‐RgpB antiserum showed that Arg‐gingipains were poorly secreted in an outer membrane or into an extracellular portion but accumulated within the cells of 83K3, suggesting the secretion of gingipains is defected in 83K3. Taken together, our findings indicated that Sov is a novel protein required for the secretion of gingipains and suggested that the secretion system for gingipains is different from the conserved secretion systems.

PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system.

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.

Hsa, an adhesin of Streptococcus gordonii DL1, binds to α2-3-linked sialic acid on glycophorin A of the erythrocyte membrane

Bacterial recognition of host sialic acid‐containing receptors plays an important role in microbial colonization of the human oral cavity. The aggregation of human platelets by Streptococcus gordonii DL1 is implicated in the pathogenesis of infective endocarditis. In addition, we consider that hemagglutination of this organism may act as an additive factor to increase the severity of this disease. We previously reported that this interaction requires the bacterial expression of a 203‐kDa protein (Hsa), which has sialic acid‐binding activity. In the present study, we confirmed that erythrocyte surface sialoglycoproteins are the receptors for Hsa. We examined the effects of proteinase K, chymotrypsin, phospholipase C, and α(2‐3) or α(2‐3, 6, 8) neuraminidase on hemagglutination activity and found that the interaction occurs between Hsa and α2‐3‐linked sialic acid‐containing proteins of erythrocytes. We expressed recombinant NR2, which is the putative binding domain of Hsa, fused with GST in Escherichia coli BL21. Dot‐blot analysis demonstrated that GST‐HsaNR2 binds both glycophorin A (GPA) and band 3. Moreover, GPA and a small amount of band 3 were detected by GST pull‐down assays. These findings indicate that S. gordonii Hsa specifically binds to GPA and band 3, α2‐3‐linked sialic acid membrane glycoproteins.

The role of Sov protein in the secretion of gingipain protease virulence factors of Porphyromonas gingivalis

Porphyromonas gingivalis transports Arg-gingipains and Lys-gingipain across the outer membrane via an unknown pathway. Recently, we found that the sov gene of P. gingivalis W83 was required for this step. In the present study, we characterized the Sov protein. We constructed a P. gingivalis strain that expresses histidine-tagged Sov instead of Sov. Subcellular fractionations and a histidine-tag pulldown experiment showed that histidine-tagged Sov was present in an outer membrane fraction. Furthermore, antiserum raised against the terminal regions of Sov obstructed the secretion of Arg-gingipains from wild-type W83 cells. A deletion study showed that the region from Phe2495 to the C-terminus Gln2499 of Sov is essential for gingipain secretion. Anti-histidine-tag immunoglobulins interfered with the secretion of Arg-gingipains by P. gingivalis cells that expressed histidine-tagged Sov. In conclusion, we found that Sov is an outer membrane protein participating in the secretion of gingipains and that the C-terminal region of Sov is exposed to the extracellular milieu and involved in the modulation of Sov function.

Binding of the Streptococcus gordonii DL1 Surface Protein Hsa to the Host Cell Membrane Glycoproteins CD11b, CD43, and CD50

ABSTRACT Infective endocarditis is frequently attributed to oral streptococci. The mechanisms of pathogenesis, however, are not well understood, although interaction between streptococci and phagocytes are thought to be very important. A highly expressed surface component of Streptococcus gordonii, Hsa, which has sialic acid-binding activity, contributes to infective endocarditis in vivo. In the present study, we found that S. gordonii DL1 binds to HL-60 cells differentiated into monocytes, granulocytes, and macrophages. Using a glutathione S-transferase (GST) fusion to the NR2 domain, which is the sialic acid-binding region of Hsa, we confirmed that the Hsa NR2 domain also binds to differentiated HL-60 cells. To identify which sialoglycoproteins on the surface of differentiated HL-60 cells are receptors for Hsa, intrinsic membrane proteins were assessed by bacterial overlay and far-Western blotting. S. gordonii DL1 adhered to 100- to 150-kDa proteins, a reaction that was abolished by neuraminidase treatment. These sialoglycoproteins were identified as CD11b, CD43, and CD50 by GST pull-down assay and immunoprecipitation with each specific monoclonal antibody. These data suggest that S. gordonii DL1 Hsa specifically binds to three glycoproteins as receptors and that this interaction may be the initial bacterial binding step in infective endocarditis by oral streptococci.

Identification of a novel Porphyromonas gingivalis outer membrane protein, PG534, required for the production of active gingipains

Gingipains are secreted endopeptidases important for the virulence and proliferation of Porphyromonas gingivalis; however, their secretion and biogenesis process is not yet fully elucidated. The PG0534 gene of P. gingivalis W83 encodes a novel protein, PG534, of unknown function. In a PG0534 deletion mutant 83K25, the activities of Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) were reduced to 4-22% of those of the wild-type W83, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. This indicates that PG534 is required for the gingipain activity. Immunoblot analysis using anti-Rgp or anti-Kgp antiserum showed that abnormal forms of gingipains were detected in the extracellular fraction from 83K25, suggesting that 83K25 exhibits dysfunctional gingipain secretory activity. Normal carbohydrate biogenesis of lipopolysaccharide is required for production of the active gingipains; however, lipopolysaccharide was not deficient in 83K25. Subcellular fractionation and immunoblot analysis using anti-PG534 antiserum localized PG534 to the outer membrane. In conclusion, we identified PG534 as a novel outer membrane protein required for the biogenesis of gingipains.

Porphyromonas gingivalis C‐terminal signal peptidase PG0026 and HagA interact with outer membrane protein PG27/LptO

Outer membrane protein PG27 is essential for secretion/maturation of conserved C-terminal domain (CTD) proteins such as gingipains, HagA, and PG0026. To determine the binding partner(s) of PG27, we used a Porphyromonas gingivalis mutant strain, 83K48, which expressed functional histidine-tagged PG27. Purification of histidine-tagged PG27 from 83K48 found that 136-kDa and 264-kDa proteins accompanied histidine-tagged PG27. Mass spectrometry revealed the 136-kDa protein and 264-kDa protein to be PG0026 and PG1837 (HagA), respectively. PG0026 is a C-terminal signal peptidase which cleaves the CTDs of other CTD proteins. A cross-linking and a native electrophoresis studies suggested the interaction between histidine-tagged PG27 and HagA and the interaction between histidine-tagged PG27 and PG0026. We constructed Porphyromonas gingivalis gene disrupting mutants, and characterized them. PG0026 was required for the full activities of gingipains, whereas HagA was not. A mutation disrupting PG0026 affected localization of PG27, but a mutation disrupting PG1837 showed little effect on the expression and localizations of PG27 and PG0026. To determine the functional role of HagA, we used PG1837-disruptant 83K54 which expressed functional histidine-tagged PG27. Histidine-tagged PG27 in 83K54 was co-purified with not only PG0026 but also several contaminated proteins. These results suggest that PG0026 interacts with PG27 in the absence of HagA, and that the binding state of a PG27-PG0026 complex was affected and stabilized by HagA. Taken together, these data suggest that secreted PG0026 anchors to the cell by interacting with PG27, is stabilized by HagA, and functions in processing of other CTD proteins such as gingipains.

Characterization of a quinol peroxidase mutant in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.

Analysis of Porphyromonas gingivalis PG27 by deletion and intragenic suppressor mutation analyses.

PG27 is required for secretion of virulence factor gingipains, and has recently been proposed as LptO, which is involved in O-deacylation of lipopolysaccharide. In the present study, a predicted 14 anti-parallel β-strand structure of PG27 was ascertained. Deletion study showed that the region from Asp382 to the C-terminal His391 of PG27 is dispensable for the function of PG27. Analysis of C-terminal deletion mutants revealed that the region in strand S14 (Asn369-Gly385) is important for activity. Of the gingipain-defective mutants, ΔThr378-His391 and ΔPhe377-His391 produced amounts of PG27 comparable to those produced by wild-type cells, suggesting that Thr378-Phe381 contains essential residues for the function of PG27. In contrast, ΔPhe381-His391, ΔAla380-His391, ΔLeu379-His391 and ΔArg376-His391 produced no detectable PG27. The defects of the ΔAla380-His391 mutant were suppressed by changing either Ala346 or Ala359 of PG27 to valine. Importantly, Ala346 and Ala359 are located close to Leu379 in the structural model of PG27. A359V compensated for the instability of PG27, but not the gingipain-defective phenotypes, of other deletion mutants tested, suggesting that Ala380 and Phe381 of PG27 are important for the stability of PG27. Lastly, we found that the C-terminal region of PG27 may be located in the periplasm. Taken together, these findings fit well with a predicted β-barrel structure model for PG27, and show that strand S14 is important for its function.

Streptococcus gordonii promotes rapid differentiation of monocytes into dendritic cells through interaction with the sialic acid-binding adhesin

Infective endocarditis is frequently attributed to oral streptococci. Although the pathogenetic mechanisms are not well understood, interaction between streptococci and phagocytes is thought to be important for infective endocarditis. In this study, HL-60 cell-derived monocytes were characterized following interaction with Streptococcus gordonii DL1. Exposure of monocytes to S. gordonii DL1 induced up-regulation of the dendritic cell (DC) markers CD83, CD1a, CD86, and interleukin-12, while monocyte markers PU.1 and MafB, which are typically present at low levels in mature DCs, were down-regulated. Interaction of HL-60-derived monocytes with S. gordonii DL1 was instructive for DC differentiation in the absence of released cytokines. Furthermore, neither the filtered culture medium of S. gordonii nor the hsa mutant, deficient in sialic acid-binding activity, was able to induce the differentiation of HL-60 cells. Taken together, these data suggest that monocytes stimulated with S. gordonii DL1 rapidly undergo monocyte-to-DC differentiation through interaction with the bacterial surface receptor Hsa and that this response may be the initial step in infective endocarditis by oral streptococci.