To identify proteins other than gingipains secreted by the PorSS,

To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel Bleomycin in vivo electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of

the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. The Gram-negative bacterium Porphyromonas gingivalis, a major pathogen of check details periodontal disease, possesses a number of virulence factors, including fimbriae, hemagglutinins, lipopolysaccharides

and proteinases. Extracellular and surface proteinases with high hydrolytic activities named gingipains are of particular importance as they have the ability to destroy periodontal tissue directly and/or indirectly (Potempa et al., 2000; Andrian et al., 2007). Gingipains are encoded by three separate genes, rgpA, rgpB and kgp, on the P. gingivalis chromosome (Curtis et al., 1999). The kgp and rgpA genes encode polyproteins comprising the signal peptide, propeptide, Lys-

and Arg-specific proteinase domains, adhesin domains and C-terminal Thiamine-diphosphate kinase domain (CTD). The rgpB gene encodes a protein comprising the signal peptide, propeptide, Arg-specific proteinase domain and CTD. These proteins are synthesized as polyproteins in the cytoplasm, are translocated across two membranes, inner and outer membranes, and secreted onto the bacterial cell surface. In our previous studies (Sato et al., 2010; Shoji et al., 2011) we found that gene products of rgpA, rgpB and kgp were translocated across the outer membrane by the Por secretion system (PorSS) in which porK, porL, porM, porN, porO, porP, porQ, porT, porU, porV (PG27, lptO), porW and sov genes were involved. Expression of some of these genes is regulated by a two-component system, the PorX response regulator and PorY histidine sensor kinase (Sato et al., 2010). Primary gene products of rgpA, rgpB and kgp have common motifs in their CTD regions. The P. gingivalis genome encodes a number of putative CTD-containing proteins (Seers et al., 2006). Nguyen et al. (2007) showed that CTD-containing proteins were also found in predicted proteins of other bacteria in the Bacteroidetes phylum, such as Prevotella intermedia and Tannerella forsythia. Among P.

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