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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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Self-restriction of Fsp25586 is not efficient.Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).
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pone-0111329-g005: Self-restriction of Fsp25586 is not efficient.Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).

Mentions: Autocatalytic processing is common in type Va secretion systems [55], [56], [57] and in subtilisins [58]. Although F. nucleatum ATCC 25586 remains refractory to plasmid transformation, others and us were previously successful with plasmid expression in F. nucleatum ATCC 23726 [43], [59]. As can be seen in figures 1E and 5A, the serine protease detected in the growth medium of F. nucleatum ATCC 23726 (Fsp23726) is approximately 56 kDa. Zymogram analysis of culture supernatants prepared from F. nucleatum ATCC 23726 expressing Fsp25586 of strain ATCC 25586 revealed the presence of the 99 kDa Fsp25586 protease in addition to the typical 56 kDa protease of ATCC 23726 (Fig. 5B). The fact that Fsp25586 was not cleaved when expressed in F. nucleatum ATCC 23726 suggests that the processing of Fsp25586 is not efficient compared to that in Fsp23726 (and the orthologs in the other tested F. nucleatum strains, Table 1). It is possible that Fsp25586 lacks the restriction site that is cleaved to release the catalytic domain from the autotransporter domain, or that this cleavage site is not exposed for cleavage.


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)

Self-restriction of Fsp25586 is not efficient.Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111329-g005: Self-restriction of Fsp25586 is not efficient.Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).
Mentions: Autocatalytic processing is common in type Va secretion systems [55], [56], [57] and in subtilisins [58]. Although F. nucleatum ATCC 25586 remains refractory to plasmid transformation, others and us were previously successful with plasmid expression in F. nucleatum ATCC 23726 [43], [59]. As can be seen in figures 1E and 5A, the serine protease detected in the growth medium of F. nucleatum ATCC 23726 (Fsp23726) is approximately 56 kDa. Zymogram analysis of culture supernatants prepared from F. nucleatum ATCC 23726 expressing Fsp25586 of strain ATCC 25586 revealed the presence of the 99 kDa Fsp25586 protease in addition to the typical 56 kDa protease of ATCC 23726 (Fig. 5B). The fact that Fsp25586 was not cleaved when expressed in F. nucleatum ATCC 23726 suggests that the processing of Fsp25586 is not efficient compared to that in Fsp23726 (and the orthologs in the other tested F. nucleatum strains, Table 1). It is possible that Fsp25586 lacks the restriction site that is cleaved to release the catalytic domain from the autotransporter domain, or that this cleavage site is not exposed for cleavage.

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