Novel Protein Encoded in the Treponema denticola PrtP Protease Operon

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary proteins (PrcA1 and PrcA2), is believed to contribute to periodontal disease by disrupting or modulating intercellular host signaling pathways.

Objectives: Transcription analysis showed that a gene immediately upstream of prcA-prtP is part of the protease complex operon. In the present study we first determined that this gene, designated prcB, encodes a T. denticola protein and then initiated characterization of its role in expression of the protease complex.

Methods: PrcB was cloned in E. coli and modified with a C-terminal 6xHis tag. This construct was introduced into T. denticola to generate allelic replacement mutant strains. Expression of PrcB and protease proteins was assayed both in E. coli and in T. denticola by immunoassay. Potential interaction between recombinant PrtP and PrcB-6xHis was assayed by nickel affinity chromatography and immunoassay.

Results: PrcB-6xHis was expressed both in E. coli and in two distinct T. denticola mutants. One mutant contains the entire prcB(6xHis)-prcA-prtP operon expressed from its native promoter, while the other expresses prcB(6xHis) but is disrupted in prcA-prtP. PrcA and PrtP proteins were expressed and cell extracts showed wild-type levels of gelatinase activity in the mutant carrying the entire operon, but not in the mutant with disrupted prcA-prtP. PrtP associated with PrcB-6xHis when E. coli extracts containing PrcB-6xHis and PrtP were subjected to nickel affinity chromatography.

Conclusion: The PrcB polypeptide is expressed in T. denticola at much lower levels than PrcA and PrtP, consistent with the lack of a consensus ribosome binding site upstream of the non-standard GTG start codon. The 6xHis tag on PrcB does not affect protease activity. Continuing studies will further characterize the function of PrcB in expression and assembly of the protease complex.

Supported by NIH DE013565.