The Characterization of The Mitogenic Factor Isolated from Extracellular Products of Aquatic Streptococcaceae
Date Issued
2009
Date
2009
Author(s)
Lin, Ming-Tzuo
Abstract
Bacteria secreted substances into environment are named extracellular products ( ECPs ). They are important virulence factors of many fish pathogens including Streptococcus spp. Most pathogenic bacteria and their products are tend to cause cytotoxic effect of cell, but some may have the mitogenic effect to induce cell proliferation, such as Pasteurella multocida, Bartonella hanselae and Helicobacter pylori etc. It was found that the ECP of streptococcus isolated from fish have mitogenic effect causing cell proliferation. Therefore, the this study focuses on the biology activity of the ECP of Lactococcus garvieae, including the mitogenic activity on fish cell line and chemotaxis effect on fish macrophage, to realize the relationship between ECP and pathogenic mechanism. First, ECP was produced by culturing L. garvieae on dialysis-membrane overlays, and purified using fast protein liquid chromatography (FPLC) technique purify the crude ECP, and then collecting the fractionated ECP. The fractions shown significant cell proliferation effect were further analyzed by using capillary electrophoresis (CE) and liquid chromatography-mass spectrometry (LC/MS). Mitogenic activity of ECP on fish cell line ( TO-2 ) was evaluated by MTT colorimetric assay. To demonstrate macrophage chemotaxis effect, we count the number of migrated macrophage with chemotaxis assay. The results showed that the ECP of L. garvieae significantly promoted TO-2 cell proliferation and caused macrophage migration. The biological activity with mitogenic and chemotaxis effect was found to be a group of low molecular weight peptides. The electrical neutral peptides analyzed with CE and LC/MS have been shown to be similar to the fragment of plantaricin SA6 secreted by Lactobacillus plantarum. Two peptides (ECP-P1, ECP-P2) were chemically synthesized demostrated TO-2 cell mitogenic activity and macrophage chemotaxis effect. Cell proliferation effect shows no dose dependency with the peptide concentration. However, low concentration (1 ppm) showed better mitogenic effect than high concentration. On the other hand, the chemotaxis effect was dose dependent on concentration. High concentration can cause more macrophage migration than low one.
Subjects
Lactococcus
ECP
Type
thesis
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