Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus.
The mechanism of the enhanced bactericidal action to Escherichia coli of the lysozyme having a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) at its C terminus was investigated. The modified lysozyme, hydrophobic pentapeptide-fused lysozyme (HLz), was secreted in the culture medium from yeast harboring the expression plasmid, in which a synthetic DNA fragment encoding a hydrophobic pentapeptide was introduced to the 3'-end of the coding region of the lysozyme cDNA. Although CD analysis showed that HLZ was considerably different from wild-type lysozyme (WLz) in the secondary and tertiary structures, it retained 76% of the lytic activity of WLz. When E. coli cells were exposed to the WLz or HLz, the survival cells were significantly reduced only in the case of HLz. Periplasmic proteins from the HLz-treated cells were released to an extent similar to that from the WLz-treated cells, indicating that HLz has nearly the same action as WLz with respect to the disruption of the outer membrane and peptidoglycan. Experiments with E. coli phospholipid liposomes revealed that HLz dissipated the valinomycin-induced transmembrane electrochemical potential, but WLz did not. These results suggest that the enhanced bactericidal action of HLz to E. coli is due to disruption of the electrochemical potential of the inner membrane in cooperation with the inherent function of the lysozyme to the outer membrane and peptidoglycan.
Ibrahim
H R
HR
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Yamada
M
M
Matsushita
K
K
Kobayashi
K
K
Kato
A
A
D000469:Alkaline Phosphatase / Q000032:analysis; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000737:chemistry / Q000633:toxicity*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity; D008081:Liposomes; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity*; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity; D012441:Saccharomyces cerevisiae; D001616:beta-Galactosidase / Q000032:analysis
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D017931:DNA Primers; D008070:Lipopolysaccharides; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D009113:Muramidase; D001616:beta-Galactosidase