Joon Lee

Extracellular Gelatinase of Enterococcus faecalis Destroys a Defense System in Insect Hemolymph and Human Serum

ABSTRACT We isolated Enterococcus faecalis from the body fluids of dead larvae of the greater wax moth, Galleria mellonella. Extracellular gelatinase (GelE) and serine protease (SprE), both of which are considered putative virulence factors of E. faecalis, were purified from the culture supernatant of E. faecalis. In an attempt to elucidate their virulence mechanisms, purified GelE and SprE were injected into hemolymph of G. mellonella and evaluated with regard to their effects on the immune system of insect hemolymph. As a result, it was determined that E. faecalis GelE degraded an inducible antimicrobial peptide (Gm cecropin) which is known to perform a critical role in host defense during the early phase of microbial infection. The results obtained from the G. mellonella-E. faecalis infection model compelled us to assess the virulence activity of GelE against the complement system in human serum. E. faecalis GelE hydrolyzed C3a and also mediated the degradation of the alpha chain of C3b, thereby inhibiting opsonization and the formation of the membrane attack complex resultant from the activation of the complement cascade triggered by C3 activation. In contrast, E. faecalis SprE exhibited no virulence effect against the immune system of insect hemolymph or human serum tested in this study.

Comparative Study on Characteristics of Lysozymes from the Hemolymph of Three Lepidopteran Larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli

Lysozymes were purified from the hemolymph of three immunized Lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli to compare their physico-chemical properties and antibacterial activities with those of chicken lysozyme. Four lysozymes including the one from chicken had similar molecular masses and chromatographic behavior on reverse phase-high pressure liquid chromatography. Western blotting analysis using an antibody raised against G. mellonella revealed that lysozyme cross-reacted with two other insect lysozymes but not with commercial chicken lysozyme. Antibacterial activities of lysozymes were measured in two types of tests: radial diffusion assay and colony count assay. Our antibacterial tests revealed that all lysozymes have strong activities against Gram-positive bacteria and three insect lysozymes still retain a little potency against Gram-negative bacteria, while chicken lysozyme has no activity against Gram-negative bacteria. Taken together, we conclude three Lepidopteran lysozymes have a common distinct structure and have an antibacterial activity, which is absent in chicken lysozyme, against Gram-negative bacteria.

Biological Activities of Synthetic Analogs of Halocidin, an Antimicrobial Peptide from the Tunicate Halocynthia aurantium

ABSTRACT Halocidin is a heterodimer antimicrobial peptide previously isolated from the tunicate Halocynthia aurantium. Based on the larger monomer (18Hc) of halocidin, nine halocidin congeners, including a series of 6 peptides truncated successively from the carboxyl-terminal end of 18Hc and 3 analogs (18HcKK, K19Hc, and K19HcKK), which have lysine residues in place of two internal histidines or have a lysine added to the amino terminus of the 18Hc molecule, were prepared. Each peptide was also converted into a homodimeric version. The antimicrobial activities of halocidin congeners truncated from the C terminus were dramatically decreased, suggesting that the full length of 18Hc is required for maintaining its maximum antimicrobial activity. Dimer forms of halocidin congeners exhibited stronger antimicrobial activities than the monomer of the corresponding peptide. Four dimer peptides (di-18Hc, di-18HcKK, di-K19Hc, and di-K19HcKK) were analyzed for antimicrobial activities against 10 clinically isolated antibiotic-resistant bacteria in elevated concentrations of NaCl or MgCl2. Of the peptides studied here, di-K19Hc retained invariably strong activity against all bacteria in diverse conditions and also showed much reduced hemolytic activity against human erythrocytes.

Bombyx mori cell line as a model of immune‐system organs

We tested 11 Bombyx mori cell lines for induction of cecropin B gene (CecB) expression. After the immune challenge, CecB expression was induced in seven cell lines. A mixture of the cell‐free supernatant from the immune‐responsive cell lines and lipopolysaccharide activated a promoter of CecB in the non‐immune‐responsive cell line, indicating that secreted factor(s) is involved in CecB activation. The expressed sequence tags of one of the immune‐responsive cell lines, NISES‐BoMo‐Cam1, contained genes encoding proteins similar to Relish, Cactus, clip‐domain serine protease, serpin, lectin, peptidoglycan recognition protein, 6tox and gloverin, in addition to seven known B. mori immune‐inducible genes. These results show that NISES‐BoMo‐Cam1 cells can be used as an in vitro model of the immune system organs of B. mori.

Characterization of Kunitz-type protease inhibitor purified from hemolymph of Galleria mellonella larvae.

We characterized a Kunitz-type protease inhibitor (Gm KTPI) obtained from the hemolymph of Galleria mellonella larvae immunized with Escherichia coli. The structural analysis of the cloned cDNA showed that it consists of 56 residues derived from the precursor of 75 amino acids. The peptide was constitutively produced in the fat bodies, but not in the midgut nor the integument of larvae. In our analysis of stage-dependent expression, its transcript was detected within the midgut, the fat bodies and the integument of the prepupae, which undergo tissue remodeling. The inhibition assays showed that Gm KTPI was capable of inhibiting only the trypsin-like activity of the larval midgut extracts. Furthermore, it was determined that Gm KTPI induced the activation of extracellular signal-regulated kinase (ERK) in the fat bodies and integument cells, and this kinase is known to perform a central role in cell proliferation signaling. Its effect on ERK activation was also verified in a control experiment using a human endothelial cell culture. Collectively, it was suggested that Gm KTPI might be responsible for the protection of other tissues against proteolytic attack by trypsin-like protease(s) from larval midgut during metamorphosis, and might play a role in the proliferation of cells in the fat body and integument.