Streptococcus parasanguis pepO encodes an endopeptidase with structure and activity similar to those of enzymes that modulate peptide receptor signaling in eukaryotic cells

Abstract

Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguis FW213. In this study, we isolated and sequenced DNA fragments linked to fimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from the fimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.

FIG. 1

Physical map of pepO and the fimA operon region of S. parasanguis. The diagram shows a simplified restriction map of a 5.1-kb DNA fragment containing pepO, three genes (fimC, fimB, and fimC) of the fimA operon, and tpx. Arrows indicate the locations and orientations of the ORFs. The lines below the map indicate the sizes and locations of probes and plasmid inserts. The Km^r marker, shown by an open arrowhead, was inserted at the KpnI restriction site of pepO. Restriction sites were as follows: B, BamHI; E, EcoRI; G, BglII; K, KpnI; P, PstI; S, SalI; and T, SstI.

FIG. 2

Sequence of the deduced S. parasanguis PepO protein and alignment with related enzymes. Amino acid residues, indicated by the standard one-letter code, are numbered on the left. Highlighted residues were identical to those of the S. parasanguis PepO protein. Gaps are indicated by dashes. Arrowheads indicate the HEXXH motif and residues in the mammalian enzymes known to be critical for enzyme activity. The sequences shown are as follows: PepO(Sp), S. parasanguis PepO (GenBank accession no. AF029677); PepO(Ll), L. lactis PepO (53); ECE(Hs), H. sapiens ECE isoform 1 (41); and NEP(Hs), H. sapiens NEP (32).

FIG. 3

Northern analysis. RNA (8 μg) was fractionated on a 0.44 M formaldehyde–1.2% agarose gel, blotted, and hybridized to an α-³²P-labeled 1,062-bp internal PCR product from pepO. Molecular size markers (in kilobases) are shown at the left. The 16S rRNA runs at approximately 1.6 kb.

FIG. 4

Southern hybridization analysis of the pepO region of wild-type S. parasanguis FW213 and two putative pepO mutant derivatives. Chromosomal DNA from each strain was digested with EcoRI and fractionated on a 0.75% agarose gel. DNA fragments were transferred to Hybond-N⁺ membranes and hybridized to either pVT1327 (A) or the Km^r determinant aphA3 (B) that had been α-³²P labeled. Lane 1, FW213 DNA; lanes 2 and 3, DNAs from two putative pepO mutant derivatives. Sizes in kilobases are noted at the left.

FIG. 5

TLC of metenkephalin hydrolysis by extracts from S. parasanguis FW213 and pepO mutant VT1346. Protein (15 μg) from FW213 (A) or VT1346 (B) was incubated with 1.25 mM metenkephalin for 0 to 10 min (corresponding to lanes) before termination of the reaction. Endopeptidase activity was detected by TLC. Abbreviations: F-M, phenylalanylmethionine; F, phenylalanine; M, methionine; Y, tyrosine; Y-G-G, tyrosylglycylglycine; Y-G-G-F-M, metenkephalin.

FIG. 6

Phylogenetic tree based on bacterial and eukaryotic PepO sequence homologues. The phylogenetic tree was constructed based on CLUSTAL W alignments of the amino acid sequences listed in Table 2. Numbers on the branches indicate the frequency (percentage) with which the corresponding cluster occurred during bootstrap analysis. ECE-1 and ECE-2, ECE isoforms 1 and 2, respectively; NEP, NEP 24.11; PEX, zinc metallopeptidase homologue (52).