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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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PMSF inhibits growth of F. nucleatum but not of E. coli.(A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P<0.05 compared to PMSF-treated bacteria, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.
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pone-0111329-g008: PMSF inhibits growth of F. nucleatum but not of E. coli.(A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P<0.05 compared to PMSF-treated bacteria, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.

Mentions: The genes coding for Fsp25586 and Fsp49256 were located in genomic loci involved in metabolic functions suggesting a nutritional role for fusolisin. Indeed, growth of F. nucleatum in a complex medium was inhibited by the AEBSF (not shown) and PMSF serine protease inhibitors (Fig. 8). Both inhibitors did not affect growth of E. coli used as control (Fig. 8), ruling out that growth attenuation of F. nucleatum by both serine-protease inhibitors resulted from non specific toxicity. P. gingivalis is a proteolytic anaerobic periodontal pathogen frequently isolated together with F. nucleatum[3]. The gingipain proteases produced by P. gingivalis are cysteine proteases which are not inhibited by PMSF. Addition of filter-sterilized gingipain-containing supernatant collected from a P. gingivalis culture [60], relieved the PMSF inhibitory effect on F. nucleatum’s growth (Fig. 8). Addition of the P. gingivalis supernatant did not affect the pH of the reaction mixture and did not reduce the inhibitory activity of PMSF as tested on trypsin under similar conditions (data not shown). These results suggest that P. gingivalis can enable fusolisin-independent growth of F. nucleatum.


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)

PMSF inhibits growth of F. nucleatum but not of E. coli.(A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P<0.05 compared to PMSF-treated bacteria, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111329-g008: PMSF inhibits growth of F. nucleatum but not of E. coli.(A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P<0.05 compared to PMSF-treated bacteria, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.
Mentions: The genes coding for Fsp25586 and Fsp49256 were located in genomic loci involved in metabolic functions suggesting a nutritional role for fusolisin. Indeed, growth of F. nucleatum in a complex medium was inhibited by the AEBSF (not shown) and PMSF serine protease inhibitors (Fig. 8). Both inhibitors did not affect growth of E. coli used as control (Fig. 8), ruling out that growth attenuation of F. nucleatum by both serine-protease inhibitors resulted from non specific toxicity. P. gingivalis is a proteolytic anaerobic periodontal pathogen frequently isolated together with F. nucleatum[3]. The gingipain proteases produced by P. gingivalis are cysteine proteases which are not inhibited by PMSF. Addition of filter-sterilized gingipain-containing supernatant collected from a P. gingivalis culture [60], relieved the PMSF inhibitory effect on F. nucleatum’s growth (Fig. 8). Addition of the P. gingivalis supernatant did not affect the pH of the reaction mixture and did not reduce the inhibitory activity of PMSF as tested on trypsin under similar conditions (data not shown). These results suggest that P. gingivalis can enable fusolisin-independent growth of F. nucleatum.

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