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

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.

Oxygen deprivation affects the antimicrobial action of LL-37 as determined by microplate real-time kinetic measurements under anaerobic conditions.

Some agents, including Escherichia coli and group A Streptococcus pyogenes cause infections in oxygen depleted sites. LL-37 is a human host defence peptide shown previously to play an important role in controlling infections caused by these bacteria. However, the effect of oxygen levels on the antimicrobial activity of LL-37 remains obscure. In order to test the effect of oxygen (or lack thereof) on LL-37's activity against E. coli and S. pyogenes, a method for adapting commonly used microtiter plates for real-time growth-kinetic (and growth-inhibition) measurements under anaerobic conditions was developed. Using the proposed method, anaerobic conditions were attained in the microplate within 30 min and were maintained for at least five days. Anaerobiosis was further confirmed by comparing the growth of two anaerobic oral species (Porphyromonas gingivalis and Fusobacterium nucleatum) in anaerobic compartments of microtiter plates versus aerobic ones. Both species grew only in the anaerobic compartments of the plates as indicated by the growth curves generated. The sensitivities of E. coli and S. pyogenes to LL-37 were tested under anaerobic conditions and compared to those in aerobic ones. The oxygen facultative E. coli grew to a higher density under aerobic conditions and its sensitivity to LL-37 was increased under anaerobiosis. The microaerophilic pathogen S. pyogenes grew faster and to a higher density under anaerobic conditions and was much more resistant to LL-37 under oxygen deprivation. Our results suggest that resistance to antimicrobial agents of microbes infecting anaerobic-microaerophilic sites should be tested under oxygen-restricted conditions.

Do dental impression materials play a role in cross contamination?

OBJECTIVE: Dentists are required to institute infectious control procedures. Dental impression materials possessing antimicrobial properties may aid in reducing the risk of cross contamination since impression materials might play a role as carriers. The purpose of this study was to evaluate the antimicrobial properties of four impression materials. METHOD AND MATERIALS: Four impression materials--Orthoprint, Impregum Penta, Aquasil Ultra Monophase, and Permlastic--were evaluated by the direct contact test. The materials were tested in contact with Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. The materials were mixed; allowed to set; and examined immediately and after aging for 24, 48, and 72 hours and 1 week. Two-way ANOVA, one-way ANOVA, and Tukey multiple comparison procedures were applied to the results. RESULTS: Impregum Penta presented the broadest antibacterial spectrum of all the materials tested. There was a complete growth inhibition of S aureus and S epidermidis, and it sustained this ability for at least 7 days. It also showed an antifungal effect by partially inhibiting the growth of C albicans, a quality that was seen only immediately after setting. Aquasil Ultra showed an antifungal effect only immediately after setting. Permlastic showed a complete growth inhibition when in contact with C albicans and sustained this ability for at least 7 days. No significant antimicrobial properties were recorded for Orthoprint. When in contact with E faecalis, no significant antibacterial properties were recorded for any of the materials. CONCLUSION: None of the tested materials exhibited a long-lasting or complete antibacterial and antifungal property. Therefore, disinfection of impressions is essential.