Isolation, Characterization, Kinetics, and Enzymatic and Nonenzymatic Microbicidal Activities of a Novel c-Type Lysozyme from Plasma of Schistocerca gregaria (Orthoptera: Acrididae)

Abstract

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30–50°C, and 0.05 M Ca2+ or Mg2+; and has a Km of 0.5 μg/ml and a Vmax of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a Km of 0.93 mg/ml and a Vmax of 1.63.

Molecular characterization of a c-type lysozyme from the desert locust, Schistocerca gregaria (Orthoptera: Acrididae)

“…We have previously reported that a bacterial challenge with E. coli induced an increased lysozyme activity in the hemolymph of 5 th instars larvae of S. gregaria and a maximum of activity was found 6 h after the injection of the bacteria (Mohamed et al, 2013). In the present study, by western blot analysis and the use of a polyclonal antiserum directed towards SgLys (Elmogy et al, 2015 and Supplementary Figure S2), a single protein band with a molecular weight of about 15.7 kDa was stained in the hemolymph of control larvae (not injected with E. coli)( Figure 5) which size corresponds to the lysozyme with its signal peptide still attached. We suggest that this apparent As a matter of fact, Liu et al (2004) reported the presence of two forms of lysozymes, one of 14.4 kDa and the other of 16.4 kDa, in salivary gland homogenates of Helicoverpa zea.…”

“…In order to understand the antimicrobial mechanism of S. gregaria lysozyme, we recently purified and characterized this antimicrobial molecule from the plasma of this insect (Elmogy et al, 2015;Mohamed et al, 2013). In the present work, we report, for the first time, the isolation and cloning of a complete cDNA encoding S. gregaria lysozyme (SgLys) and demonstrate the protein's primary, secondary and three dimensional structures.…”

“…The Trp residues are responsible for inducing functionally active conformation of the channel due to their specific preference for the interface (Cross et al, 1999;Hu et al, 1993;Kelkar and Chattopadhyay, 2007). The time-dependent rate of passage of the cytoplasmic β-galactosidase into the incubation medium (Elmogy et al, 2015) indicates a proceeding process of the inner membrane permeabilization by the SgLys which is most probably due to the referred insertion ability and pore formation.…”

Insect immunology and hematopoiesis

“…Unlike AMPs, which are produced in response to an immune insult, lysozymes are usually present in low, constitutive levels and are transcriptionally upregulated following infection. Although lysozymes are classically active in the lytic antibacterial response (Elmogy et al, 2015; Kwon et al, 2014b), some also have anti- Plasmodium and anti-fungal activity (Kajla et al, 2011; Sowa-Jasilek et al, 2014), and can activate the phenoloxidase-based melanization pathway (Li and Paskewitz, 2006; Rao et al, 2010). In addition to being involved in immunity, some insect lysozymes are expressed in the gut and function in digestion (Ito et al, 1995; Ursic-Bedoya et al, 2008).…”

Newly identified C‐type lysozyme in Chinese soft‐shelled turtle (Pelodiscus sinensis) exhibits potent antimicrobial activity

“…The antibacterial activity of C‐type lysozyme has been widely reported, with a different spectrum of inhibition among different species (de‐la‐Re‐Vega, García‐Galaz, Díaz‐Cinco, & Sotelo‐Mundo, ; Mai & Hu, ; Yu, Sun, Li, & Sun, ). Some C‐type lysozyme only showed lytic activity against Gram‐positive bacteria (Fujimoto, Toshimori‐Tsuda, Kishimoto, Yamano, & Morishima, ), while other lysozymes displayed bacteriolytic activities against both Gram‐positive and Gram‐negative bacteria, and even fungi (Ding et al, ; Elmogy et al, ). In the present study, the r Ps lysC was able to inhibit Gram‐negative bacteria and Gram‐positive bacteria, but showed no inhibitory activity against fungi.…”