Proteases of Treponema denticola outer sheath and extracellular vesicles

Abstract

Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath-and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 ؋ 10 4 M ؊1 min ؊1. Inhibition of the enzyme by phenylboronic acid was rapid (<1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola

“…Chymotrypsin-like protease activity associated with T. denticola outer membranes was due to CTLP [17,18]. As expected, and as shown in Table 1, strain CKE had greatly reduced enzymatic activity against SAAPNA, con¢rming that CTLP accounted for most of the chymotrypsin-like protease activity of T. denticola.…”

“…3B, and data not shown). Earlier studies implied that the high molecular mass native Msp oligomeric complex might be structurally associated with the CTLP complex [5,18]. The lack of oligomeric Msp and hexagonal array in strain CKE suggested that CTLP expression might in£uence the production or assembly of the Msp hexagonal array.…”

The autolysin of Porphyromonas gingivalis is involved in outer membrane vesicle release

“…P. gingivalis vesicles were prepared according to the method of Rosen et al [24] with some modi¢cations. P. gingivalis cultures were grown in supplemented TSB and 20-ml volumes were used to prepare vesicles.…”

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola

“…In a dentilisin-de¢cient mutant, the amount of the oligomer in the outer sheath decreases [22], and the hexagonal array disappears [23]. It is possible that the protease participates in the construction of the outer sheath component, since this protease can be isolated from membrane associated fractions such as the outer sheath and the vesicles [24]. Further study is required to determine the location of the enzyme and to evaluate the physical role of this enzyme.…”

“…The machinery of the release of the surface material is important for its pathogenicity. Rosen et al [24] observed that the vesicles released from T. denticola were constituted of components similar to those of the outer sheath and they included dentilisin. On the other hand, Caimano et al [10] frequently observed vesicles which did not react with anti-Msp.…”