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 Ca(2+) or Mg(2+); 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.

Keywords: Schistocerca gregaria; antibacterial activity; kinetics; lysozyme c; muramidase.

Fig. 1.

Purification of lysozyme from plasma of fifth instar Schistocerca gregaria with cation-exchange chromatography on CM-Sepharose Fast Flow ion-exchange column. Proteins obtained with 60% (NH₄)₂SO₄ saturation of hemolymph fifth instars S. gregaria immunized with pricking by a thin needle previously dipped into a concentrated pellet (2 × 10⁸ cells/ml) of Escherichia coli were applied to column (20 × 2 cm) equilibrated with 0.5 M Na-acetate (pH 6.5) buffer. Elution was carried out with a gradient of 0.5–3.0 M of the same buffer at a flow rate of 1 ml/min (——, A₂₈₀;…….., Na-acetate concentration; ○- - - -○, anti-Micrococcus lysodeikticus activity). Five microliter of the fraction samples were used for the inhibition zone assay. Active fractions (anti-M. lysodeikticus fractions), tubes 31–39, were pooled, desalted, lyophilized, and stored at −20°C for further use.

Fig. 2.

Electrophoresis. (A) SDS/PAGE (12%), under reducing condition, of fractions obtained during purification of the Sgl. Samples were taken of each step during the purification and electrophoresed. The following samples were separated: (A-1) Lane 1, AMRESCO mid molecular weight standards; lane 2, crude hemolymph; lane 3, the salted out proteins from 60% (NH₄)₂SO₄ saturation. (A-2) Lane 1, mid molecular weight standards; Lanes 2–10, pooled active fractions from CM-Sepharose Fast Flow column. (B) SDS-PAGE, 12%, under reducing conditions. Lane 1, marker proteins; lane 2, the isolated Sgl. The molecular mass of Sgl under reducing conditions was estimated to be 15.7 kDa. (C) Native PAGE, 10% acrylamide, of the isolated Sgl. (D) Native IEF/PAGE patterns (pH range 3-10), 10%, of the isolated Sgl. The pH gradient was obtained with a mixture containing 4% (v/v) ampholytes 3-10 (Sigma Ampholyte high resolution). Lane 1, IEF Mix 3.6-9.3, Sigma; lane 2, the isolated Sgl. Proteins in the gels were stained with COBB R-250.

Fig. 3.

Estimation of Sgl molecular weight by gel filtration on Sephadex G-75 column. A column (2.6 × 60 cm, void volume 318 ml) was equilibrated with 0.5 M Na-acetate buffer, pH 6.5; then calibrated with a set of marker proteins at 25°C and a regression correlation between elution volume and the logarithm of the molecular weight of each marker protein was made. The Sgl and marker proteins were eluted at a flow rate of 2.6 ml/min. Marker proteins, inset of figure, (Biolabs) were used. Mr, relative molecular weight.

Fig. 4.

Ouchterlony double immunodiffusion showing cross-reactivity of Sgl antiserum with the purified Sgl. Center well (anti-Sgl) containing Sgl antiserum. Upper peripheral wells, Sgl, containing purified Sgl. Sgl and anti-Sgl were stained with COBB.

Fig. 5.

N-terminal amino acid sequence of the isolated S. gregaria lysozyme (Sgl) aligned with some c-, i-, and g- type lysozymes. Accession numbers are as follows: c-type: Locusta migratoria (AHE76131); Galleria mellonella (P82174); Manduca sexta (AAB31190); Triatoma brasiliensis (AAU04569), G. gallus (HEWL) (AAA48943); g-type: Azumapecten farreri (AGA95494); i-type: Nilaparvata lugens (AGK40910). Similar amino acids are marked with a dot (.).

Fig. 6.

(A) Effect of pH on the lytic activity of the Sgl. The buffers used were 0.2 M sodium acetate (pH 4.0 to 6.5), 0.2 M sodium phosphate (pH 7 to 8.5), and 0.1 M Tris-HCl (pH 9.0 to 10.0). The muramidase activity of lysozyme samples was tested using a turbidimetric standard assay against M. lysodeikticus. Lysozyme activity was shown as percentage of the highest activity. (B) Heat stability of the isolated Sgl. Lysozyme aliquots were adjusted to pH 6.2, placed at the indicated temperatures for 10 min, and tested for lytic activity using a turbidimetric standard assay against M. lysodeikticus. Aliquots were removed at the different temperatures and assayed for lysozyme activity which is shown as percentage of the highest activity. Data represent the mean of three replicates. (C) Effects of the divalent cations Ca²⁺ and Mg²⁺ on the lytic, muramidase, activity of the Sgl at ionic strength 0.1. The ionic strength was regulated with NaCl. Effects were observed by measuring the activity of lysozyme in 0.1 M sodium acetate buffer at pH 6.2 supplemented with CaCl₂ or MgCl₂. The 100% activity represents a specific lysozyme activity of U/mg protein in 0.1 M sodium acetate buffer at pH 6.2. Lysozyme activity was shown as percentage of the lowest activity.

Fig. 7.

(A) Determination of Michaelis-Menten constant (K_m) of the muramidase activity of the Sgl and HEWL (as a standard reference) by Lineweaver-Burk plot. Enzymatic assay (turbidemetric standard assay) was carried out in 0.2 M sodium acetate buffer, pH 6.2 at 37°C. M. lysodeikticus was used as substrate. S is expressed in mg/ml. Each point is the mean of three replicates. K_m was calculated from the reciprocal of the x intercept and measured as OD/mg protein/30 min at 450 nm. (B) Determination of Michaelis-Menten constant (K_m) of chitinase activity (at pH 6.0) of the isolated Sgl and HEWL. K_m was determined by Lineweaver-Burk plot, (S is expressed in mg/ml), using colloidal chitin as a substrate and measured as OD/mg protein/h at 585 nm.

Fig. 8.

Kinetics of permeabilization of the inner membrane (in parallel to antibacterial activity) by the Sgl against E. coli ML-35 p. The release of β-galactosidase from the lactose permease-deficient E. coli ML-35 p under the effect of the Sgl in an incubation medium and monitored by tracing the production of o-nitrophenol over time (1, 2, 3, and 4 h post incubation) from the specific substrate o-nitrophenyl-β-D-galactopyranoside. The absorbance (which is proportional to the β-galactosidase activity) was plotted against the incubation time. Values shown represent the mean of three experiments. In the control, an equivalent volume of sterile ddw replaced the Sgl solution. HEWL was used as a standard reference.

Fig. 9.

The effect of Sgl on activity of prophenoloxidase and phenoloxidase (PO) in plasma. Aliquots of preactivated plasma, using laminarin, were incubated with the Sgl purified from plasma, HWEL, BSA, or PBS. L-dopamine substrate was added to each sample and PO activity was monitored after certain intervals of time at 470 nm for 30 min. The bars represent the SE of mean of four replicates.