Composition and Localization of Treponema denticola Outer Membrane Complexes

Abstract

The Treponema denticola outer membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated that prcB, located upstream of and cotranscribed with prcA and prtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of both prcA and prtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-highmolecular-weight outer membrane complex.

The Major Outer Sheath Protein (Msp) of Treponema denticola Has a Bipartite Domain Architecture and Exists as Periplasmic and Outer Membrane-Spanning Conformers

“…Herein, we show that Msp, like TprC, has a bipartite architecture; importantly, only the C-terminal domain is surface exposed in T. denticola and able to form a ␤-barrel that can insert into liposomes and form channels. Consistent with immunolabeling studies localizing Msp to both the OM and periplasm of T. denticola (19,45), we show that the native protein exists as OM-integrated and periplasmic trimers with distinct physical properties. Based upon our collective results, we propose a model whereby a newly exported, partially folded intermediate can be either processed for OM insertion by the ␤-barrel assembly machinery (BAM) or remain periplasmic, ultimately forming a stable, water-soluble trimer.…”

“…Figure 6A shows that native, full-length Msp in the membrane fraction partitioned exclusively into the detergent phase, while Msp in the soluble fraction was recovered only in the aqueous phase. Native Msp forms SDS-stable trimers that dissociate upon boiling (18,19). The SDS-PAGE gels in Fig.…”

“…Over the years, numerous activities have been described for Msp (3,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19); our studies provide a topologic framework for assessing these claims. As one example, binding and biological assays of Msp often have employed isolated, native protein (7,8,10,11,13,14,64).…”

“…In addition to forming large, depolarizing channels in artificial and HeLa cell membranes (6,7), Msp binds to extracellular matrix components and cell adhesion molecules (8)(9)(10)64), induces cytopathic effects in epithelial cells (11), perturbs cytoskeletal actin dynamics and intracellular calcium flux in fibroblasts (12,13), inhibits neutrophil chemotaxis (14,15), and activates neutrophils, inducing them to release matrix metalloproteases and other tissue-damaging enzymes (16,17). Msp also forms detergent-stable trimers that physically associate with dentilisin (11,18,19), a protease complex that degrades extracellular matrix and disrupts intercellular junctions (20,21). Notably, despite the considerable attention devoted to Msp over the years, very little is known about its structure and membrane topology.…”

Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities

“…PrcB is a lipoprotein that interacts with PrtP pre-proteinase and is required for enzymic activity. The CTLP complex on the surface of T. denticola comprises PrtP, PrcA1 and PrcA2 (processed forms of PrcA), PrcB and Msp (major outersheath protein) (Godovikova et al, 2011).…”