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Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Doron L, Coppenhagen-Glazer S, Ibrahim Y, Eini A, Naor R, Rosen G, Bachrach G - PLoS ONE (2014)

Bottom Line: Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa.In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease.The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position.

View Article: PubMed Central - PubMed

Affiliation: Institute of Dental Sciences, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

ABSTRACT
Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

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Sequence alignment of fusolisin.ClustalW alignment of Fsp25586, the available partial sequence of the homologues serine protease Fsp23726, Fsp10953 and Fsp49256. The predicted catalytic triad Asp, His and Ser are highlighted.
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pone-0111329-g004: Sequence alignment of fusolisin.ClustalW alignment of Fsp25586, the available partial sequence of the homologues serine protease Fsp23726, Fsp10953 and Fsp49256. The predicted catalytic triad Asp, His and Ser are highlighted.

Mentions: Amino acid sequence alignment (Fig. 4) of Fsp25586 revealed a high homology (71% similarity and 61% identity) with that of Fsp49256, 71% similarity and 60% identity with that of Fsp10953 and 63% similarity and 57% identity with the available partial sequence of the homologous serine protease Fsp23726. Previous annotation of the FN1426 (Fsp25586) and the FNV0835 (Fsp49256) open reading frames revealed a signal peptide and three other functional domains [49]. The N-terminal, peptidase domain [amino acids 131–471 in Fsp25586 and 1–406 in Fsp49256] were found to belong to the peptidase S8 domain family. The C-terminal domain (amino acids 788–1047 in Fsp25586 and 690–995 in Fsp49256) belong to the autotransporter superfamily. While the C-terminal autotransporter domain of Fsp25586 and Fsp49256 were highly conserved (93% identity and 98% similarity), a higher divergence was found between the catalytic domains of the proteases of the two species (37% identity, 47% similarity). As a member of the S8 family of subtilisins, the amino acid sequence analysis of fusolisin revealed that the arrangement of the active site catalytic triad is Asp-His-Ser [52], [53] that was identified using NCBI's conserved domain database (CDD) [54] in the amino acid sequences of Fsp49256, Fsp10953, Fsp23726 and Fsp25586, and can be seen in Fig. 4.


Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Doron L, Coppenhagen-Glazer S, Ibrahim Y, Eini A, Naor R, Rosen G, Bachrach G - PLoS ONE (2014)

Sequence alignment of fusolisin.ClustalW alignment of Fsp25586, the available partial sequence of the homologues serine protease Fsp23726, Fsp10953 and Fsp49256. The predicted catalytic triad Asp, His and Ser are highlighted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214739&req=5

pone-0111329-g004: Sequence alignment of fusolisin.ClustalW alignment of Fsp25586, the available partial sequence of the homologues serine protease Fsp23726, Fsp10953 and Fsp49256. The predicted catalytic triad Asp, His and Ser are highlighted.
Mentions: Amino acid sequence alignment (Fig. 4) of Fsp25586 revealed a high homology (71% similarity and 61% identity) with that of Fsp49256, 71% similarity and 60% identity with that of Fsp10953 and 63% similarity and 57% identity with the available partial sequence of the homologous serine protease Fsp23726. Previous annotation of the FN1426 (Fsp25586) and the FNV0835 (Fsp49256) open reading frames revealed a signal peptide and three other functional domains [49]. The N-terminal, peptidase domain [amino acids 131–471 in Fsp25586 and 1–406 in Fsp49256] were found to belong to the peptidase S8 domain family. The C-terminal domain (amino acids 788–1047 in Fsp25586 and 690–995 in Fsp49256) belong to the autotransporter superfamily. While the C-terminal autotransporter domain of Fsp25586 and Fsp49256 were highly conserved (93% identity and 98% similarity), a higher divergence was found between the catalytic domains of the proteases of the two species (37% identity, 47% similarity). As a member of the S8 family of subtilisins, the amino acid sequence analysis of fusolisin revealed that the arrangement of the active site catalytic triad is Asp-His-Ser [52], [53] that was identified using NCBI's conserved domain database (CDD) [54] in the amino acid sequences of Fsp49256, Fsp10953, Fsp23726 and Fsp25586, and can be seen in Fig. 4.

Bottom Line: Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa.In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease.The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position.

View Article: PubMed Central - PubMed

Affiliation: Institute of Dental Sciences, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

ABSTRACT
Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

Show MeSH
Related in: MedlinePlus