Contribution of S-layer proteins to the mosquitocidal activity of Lysinibacillus sphaericus

Abstract

Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.

Figure 1. Detection of S-layers in spore-preparations and vegetative cultures.

Pellet (P) and supernatant (S) fractions of spore-preparations from 2362 and C7 strains, obtained from the alkaline treatment described in Materials and Methods, were subjected to SDS-PAGE 12.5% (A) and Western Blot analysis for detection with specific antibodies against BinA or BinB or S-layer proteins (B). Purified S-layers from 2362 and C7 strains were obtained from vegetative cultures as described in Materials and Methods. 6 µg of each preparation were subjected to SDS-PAGE 12.5% electrophoresis (C) and analyses by Western Blot with specific antibody against the 2362 strain’s S-layer (D).

Figure 2. Detection of S-layers in spores by immunofluorescence.

Spores preparations from C7 (A and B) were cleaned as described in Materials and Methods. A: Microscopic white light observation. B: Fluorescent (652 nm excitation and 668 nm emission) observation of the S-layer in the same preparation.

Figure 3. Binding assays of S-layer to chitin derivatives.

Insoluble preparations of chitosan, crab chitosan, chitin (10 µg) were vigorously mixed with 50 µg of the different proteins and allow standing for 16 h at 32°C: S-layers from either 2362 or C7 strains, Bovine serum albumin (BSA). Free protein was determined and % bound calculated. Six independent experiments with duplicate samples were performed. Bars show the mean ± SD. Mann Whitney-U test was used to determine statistically significant differences between S-layers proteins and BSA control protein. *, P<0.05. With colloidal chitin, N-acetylglucosamine (NAG) (25 mM) was also added to verify binding inhibition. Five independent experiments with duplicate samples were performed. Mann Whitney-U test was used to determine statistically significant differences with and without NAG. **, P<0.05.

Figure 4. Hemolytic activity from S-layer of L. sphaericus 2362 and C7.

Hemolysis was analyzed with 1% sheep red blood cells suspension in PBS. Bovine serum albumin (BSA) protein was used for unspecific effect. Six independent experiments with duplicate samples were performed. Bars show the mean ± SD. Mann Whitney-U test was used to determine statistically significant differences between S-layers proteins and BSA control protein. *, P<0.05.