Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes

Abstract

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, "Treponema vincentii," and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5' hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes.

FIG. 1.

Neighbor-joining phylogenetic tree for treponeme strains examined in this study and named human oral species as determined from 16S rRNA sequence comparisons. GenBank accession numbers are given in braces. Numbers in brackets refer to treponeme phylogenetic groups defined previously (5).

FIG. 2.

Phylogenetic tree of treponeme prtP genes. Tree based on amino acid comparisons with PAM250. GenBank accession numbers are given in braces.

FIG. 3.

Phylogenetic tree of treponeme prcA genes. Tree based on amino acid comparisons with PAM250. GenBank accession numbers are given in braces.

FIG. 4.

Operon map of sequenced genes. Solid lines with arrowheads indicate sequenced portions of each ORF, drawn to scale. The amino acid length of each ORF is indicated, without parentheses for completed sequences and with parentheses for incomplete sequences. The positions of key degenerate primers used to amplify the ORFs are indicated by vertical lines below small, labeled arrowheads. Paralog names and group numbers are indicated above the appropriate ORFs. Genus, species, and strain designations for each ORF are indicated at the right. GenBank accession numbers are given in braces.

FIG. 5.

Potential cleavage sites of PrcA. The aligned amino acid sequences for six PrcA genes are shown above. The two potential cleavage sites in PrcA proposed by Lee et al. (18) are indicated with arrows. The positions of the amino acids at the cleavage sites are numbered from the initial methionine with subscripts. The T. socranskii subsp. 04 is a partial sequence, and the numbering of amino acids refers to the start of the partial sequence. The second site is more conserved in the PrcA sequences. While it is not known if PrcA-II is cleaved, there is a conserved PH amino acid sequence at the corresponding position.

FIG. 6.

Putative lipoboxes at the amino termini of PrcA and PrtP. The cysteine in the aligned sequences occurs 19 to 34 amino acids from the initial methionine.

FIG. 7.

Comparison of PrtP deduced amino acid sequences around the active-site residues. The active-site aspartate (D), histidine (H), and serine (S) are boldfaced and underlined.