歯周病原性細菌Porphyromonas gingivalis由来HtrAのプロテアーゼ活性および局在性について

HtrA (high-temperature requirement A) was initially described in Escherichia coli as a housekeeping protease in 1983. It is highly conserved in evolution, that is present in bacteria, yeast, plants and humans. In several microorganisms, HtrA is considered an important virulence factor that plays a regulatory role in high temperature and oxidative stress. HtrA also possesses chaperon activity at lower temperature. To investigate the role of HtrA in Porphyromonas gingivalis, a putative periodontopathogen, the gene of htrA was cloned from P. gingivalis W83 chromosomal DNA by polymerase chain reaction (PCR). The expression vector pGEX-htrA was constructed, and was transferred into E. coli BL21. A fusion protein, Glutathione S-transferase (GST)-HtrA was expressed under an atmosphere of isopropyl-thio-β-D-galactopyranoside (IPTG), and then purified by Glutathione Sepharose 4B affinity chromatography. Recombinant HtrA (rHtrA), was then obtained by using PreScission^<TM> protease to remove the GST region. The purified rHtrA was recognized by an anti-HtrA antiserum. Partial amino acid sequencing of rHtrA showed Ser-Gly-Ala-Ser-Ala-Ala-Leu, which was identical to a part of the reported deduced amino acid sequence of P. gingivalis HtrA. Proteolytic activity of rHtrA was examined, using α-casein and β-casein as the substrates. Purified rHtrA degraded β-casein, however, was unable to degrade α-casein and the other proteins tested. Proteolytic activity was inhibited by serine protease inhibitors, such as Nα-tosyl-L-lysine chloromethyl ketone (TLCK) and phenylmethylsulfonyl fluoride (PMSF). Therefore, P. gingivalis HtrA is considered to belong to the family of serine proteases. The localization of HtrA in bacterial cells was investigated by immunoblot analysis with an anti-HtrA antiserum, and it was suggested that HtrA existed in the outer membrane vesicles of P. gingivalis, which is involved in the virulency. The results thus suggest the possibility that HtrA contributes to the pathogenicity of P. gingivalis.

公開日:2010年1月24日で登録したコンテンツは、国立情報学研究所において電子化したものです。