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A Protein Secretion System Linked to Bacteroidete Gliding Motility and Pathogenesis

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A Protein Secretion System Linked to Bacteroidete Gliding Motility and Pathogenesis A protein secretion system linked to bacteroidete gliding motility and pathogenesis Keiko Satoa, Mariko Naitoa, Hideharu Yukitakea, Hideki Hirakawab, Mikio Shojia, Mark J. McBridec, Ryan G. Rhodesc, and Koji Nakayamaa,d,1 aDivision of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan; bLaboratory of Plant Genome Informatics, Department of Plant Genome Research, Kazusa DNA Research Institute, Chiba 292-0818, Japan; cDepartment of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201; and dGlobal COE Program at Nagasaki University, Nagasaki 852-8588, Japan Communicated by Roy Curtiss III, Arizona State University, Tempe, AZ, November 5, 2009 (received for review June 30, 2009) Porphyromonas gingivalis secretes strong proteases called gingi- such as Prevotella intermedia. porT orthologs have not been de- pains that are implicated in periodontal pathogenesis. Protein se- tected outside of the Bacteroidetes phylum, and they are also lacking cretion systems common to other Gram-negative bacteria are from some members of the Bacteroidetes, such as the intestinal lacking in P. gingivalis, but several proteins, including PorT, have anaerobes Bacteroides thetaiotaomicron and Bacteroides fragilis.In been linked to gingipain secretion. Comparative genome analysis this study, 11 additional genes that appear to be involved in gingi- and genetic experiments revealed 11 additional proteins involved pain secretion and activation were identified by comparative ge- in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, nome analysis and genetic experiments. and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were Results putative two-component system regulatory proteins. Real-time Identification of Genes Involved in Gingipain Secretion by RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, Comparative Genome Analysis and Genetic Experiments. Bacterial and sov were down-regulated in P. gingivalis porX and porY mu- protein secretion systems typically require multiple proteins that tants. Disruption of the F. johnsoniae porT ortholog resulted in form a complex in the cell envelope. PorT is required for P. defects in motility, chitinase secretion, and translocation of a glid- gingivalis gingipain secretion, and may interact with other pro- ing motility protein, SprB adhesin, to the cell surface, providing a teins to form the translocation machinery. porT homologs are link between a unique protein translocation system and a motility found in some other members of the phylum Bacteroidetes, such apparatus in members of the Bacteroidetes phylum. as C. hutchinsonii ATCC 33406, but not in others, such as B. thetaiotaomicron VPI-5482 (13–15). To identify possible addi- gingipain | Porphyromonas gingivalis | Flavobacterium | chitinase tional components of the gingipain secretion system, we identi- fied 55 genes in addition to porT that were present in P. gingivalis eriodontal disease, the major cause of tooth loss in industrial and C. hutchinsonii but absent in B. thetaiotaomicron (Fig. S1 and Pnations (1, 2), is one of the most frequently occurring infectious Table S1). P. gingivalis strains with deletion mutations in 46 of diseases in humans (3), and a chronic inflammatory disease that these genes were constructed, and gingipain activities were de- results in destruction of periodontal tissue and alveolar bone (4). The termined. Mutation of sov (PGN_0832), which was recently reported Gram-negative anaerobic bacterium Porphyromonas gingivalis is a to be involved in gingipain secretion (16), or of any of nine other major periodontal pathogen (5). P. gingivalis secretes extracellular genes, which we designated porK (PGN_1676), porL (PGN_1675), and cell-surface gingipain proteases that are major virulence factors porM (PGN_1674), porP (PGN_1677), porQ (PGN_0645), porU involved in periodontal pathogenesis (6, 7). Gingipains consist of (PGN_0022), porW (PGN_1877), porX (PGN_1019), and porY Arg-specific cysteine proteinases (Rgp) encoded by rgpA and rgpB, (PGN_2001), resulted in decreased Rgp or Kgp activity in cells and and the Lys-specific cysteine proteinase (Kgp) encoded by kgp (8– culture supernatants (Fig. 1A). The mutants displayed cell-associated 10). Gingipains have signal peptides to allow transit of the cyto- gingipain proproteins with high molecular masses as revealed by plasmic membrane via the Sec machinery, but the mechanism of immunoblot analysis with anti-Hgp44 and anti-Kgp antibodies (Fig. secretion across the outer membrane is not known. Studies of Gram- 1B). These proproteins were inactive, as previously described (12). negative bacteria belonging to the phylum Proteobacteria have Introduction of wild-type copies of por genes on plasmids into the fi appropriate mutants resulted in complementation of the ex- identi ed at least eight different protein secretion systems (11). Four fi of these (type I, III, IV, and VI secretion systems) transport proteins tracellular and cell-surface gingipain defects, con rming the roles of across the entire Gram-negative cell envelope and thus do not typ- the Por proteins in secretion (Fig. 1C). ically transport proteins with N-terminal signal peptides. Other se- Five of the genes described above, porK, porL, porM, sov,and porW F. johnsoniae gldK gldL cretion systems (type II and type V machineries, the two-partner , are similar to gliding motility genes , , gldM, sprA,andsprE, respectively (17, 18). In F. johnsoniae, gldN, secretion system, and the chaperone/usher system) mediate only the which is also involved in gliding motility, is located immediately final step (transit across the outer membrane) and rely on the Sec or downstream of gldK, gldL,andgldM. The P. gingivalis gldN ortholog twin arginine transport systems to escort proteins across the cyto- (PGN_1673), which we refer to as porN, lies immediately down- plasmic membrane. P. gingivalis is a member of the Bacteroidetes stream of porK, porL,andporM.AporN defective mutant of P. gin- phylum, and is thus not closely related to the Proteobacteria.Analysis givalis was constructed and found to exhibit almost no extracellular or of the P. gingivalis genome suggested that critical components of known bacterial protein secretion systems were lacking in P. gingi- valis, suggesting that some other machinery may be involved in Author contributions: M.J.M. and K.N. designed research; K.S., M.N., H.Y., H.H., M.S., and R. gingipain secretion. G.R. performed research; K.S., M.N., H.Y., H.H., M.S., and R.G.R. analyzed data; and M.J.M. Genetic and biochemical analyses indicate that the membrane and K.N. wrote the paper. protein PorT is involved in gingipain secretion (12). Genes related The authors declare no conflict of interest. to porT are found in many members of the large and diverse Bac- 1To whom correspondence should be addressed. E-mail: [email protected]. teroidetes phylum, including gliding bacteria such as Flavobacterium This article contains supporting information online at www.pnas.org/cgi/content/full/ johnsoniae and Cytophaga hutchinsonii, and nonmotile anaerobes 0912010107/DCSupplemental. 276–281 | PNAS | January 5, 2010 | vol. 107 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.0912010107 Downloaded by guest on October 1, 2021 A B 120 100 WT WT WT porU porX Kgp (whole cell) porY porQ WT WT kgp rgpA rgpB porL porK porM porP porN porW sov WT porL porP porK porM porN porW sov kgp rgpA rgpB KDa 80 KDa kgp rgpA rgpB KDa KDa 60 210 210 210 40 140 140 210 140 140 20 95 95 95 70 95 0 70 70 55 70 55 100 43 55 55 Kgp (culture supernatant) 43 43 43 80 36 36 36 36 60 28 28 28 28 40 19 19 19 19 20 0 -Kgp -HGP44 -Kgp -Kgp 120 Rgp (whole cell) 100 Relative activity (%) 80 C 120 60 Kgp/ cell 40 Kgp/ sup 20 100 0 Rgp/ cell 80 Rgp/ sup 100 Rgp (culture supernatant) 80 60 60 40 40 20 0 Relative activity (%) 20 ) ) ) ) ) ) ) ) ) WT ) 0 porU porL porK porM porQ porX) porN porY porP porW sov kgp rgpA rgpB PGN_0022 ( PGN_1676 ( PGN_1675 ( PGN_1674 ( PGN_0645 ( PGN_1019 ( PGN_1673 ( PGN_2001 ( PGN_1677 ( PGN_1877 ( PGN_0832 ( MICROBIOLOGY Fig. 1. Kgp and Rgp proteins and activities in wild-type and mutant P. gingivalis.(A) Rgp and Kgp activities in intact cells and in culture supernatants. P. gingivalis cells were grown anaerobically in enriched brain heart infusion medium at 37 °C for 24 h. All cultures had similar cell densities of OD600 of ∼1.0. Kgp and Rgp activities were measured at units per milliliter of cell suspensions or culture supernatants and are indicated as percent of activity relative to that of the wild type. (B) Accumulation of gingipain proproteins with high molecular masses in mutant cells. P. gingivalis cell lysates were subjected to SDS/PAGE and immunoblot analysis with anti-Kgp and anti-Hgp44. (C) Plasmid-mediated complementation. Rgp and Kgp activities in intact cells and in culture supernatants were measured for wild-type, mutant, and complemented cells. cell-surface gingipain activities (Fig. 1A). Gingipain activity was re- not appear to be encoded by nearby genes in either case. Given stored by complementation with plasmid-encoded wild-type PorN the similar phenotypes of the mutants it is possible that the two (Fig. 1C). porN mutant cells accumulated unprocessed gingipain proteins function together as a two-component signal trans- proproteins intracellularly (Fig. 1B), further confirming a role for duction system. To
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