Antibacterial Action of Protein Fraction Isolated from Rapana venosa Hemolymph against Escherichia coli NBIMCC 8785

Abstract

Natural products and especially those from marine organisms are being intensively explored as an alternative to synthetic antibiotics. However, the exact mechanisms of their action are not yet well understood. The molecular masses of components in the hemolymph fraction with MW 50-100 kDa from Rapana venosa were determined using ImageQuant™ TL v8.2.0 software based on electrophoretic analysis. Mainly, three types of compounds with antibacterial potential were identified, namely proteins with MW at 50.230 kDa, 62.100 kDa and 93.088 kDa that were homologous to peroxidase-like protein, aplicyanin A and L-amino acid oxidase and functional units with MW 50 kDa from R. venous hemocyanin. Data for their antibacterial effect on Escherichia coli NBIMCC 8785 were obtained by CTC/DAPI-based fluorescent analysis (analysis based on the use of a functional fluorescence probe). The fluorescent analyses demonstrated that a 50% concentration of the fraction with MW 50-100 kDa was able to eliminate 99% of the live bacteria. The antimicrobial effect was detectable even at a 1% concentration of the active compounds. The bacteria in this case had reduced metabolic activity and a 24% decreased size. The fraction had superior action compared with another mollusc product-snail slime-which killed 60% of the E. coli NBIMCC 8785 cells at a 50% concentration and had no effect at a 1% concentration. The obtained results demonstrate the high potential of the fraction with MW 50-100 kDa from R. venosa to eliminate and suppress the development of Escherichia coli NBIMCC 8785 bacteria and could be applied as an appropriate component of therapeutics with the potential to replace antibiotics to avoid the development of antibiotic resistance.

Keywords: Escherichia coli NBIMCC 8785; Rapana venosa hemolymph; antimicrobial effect.

Figure 2

MS/MS spectrum of peptide [M + H]⁺ at m/z 1274.68 from the protein band at 61.106 kDa of the fraction MW 50–100 kDa of R. venosa hemolymph. The peptide amino acid sequence was determined manually following a series of b and y-fragment ions. After alignment of the identified amino acid sequence (LDWPVLFNDR) for peptide [M + H]⁺ at m/z 1274.68 with a database for extracellular proteins from gastropods by the Basic Local Alignment Search Tool (BLAST), we established a high homology (83%) with Aplysianin-A of Aplysia kurodai. In this way were determined the other proteins presented in Table 1 [31].

Figure 1

(A) Depiction of 12.0% SDS-PAGE analysis, visualized by staining with Coomassie G-250. Positions: (1) protein marker in the range 6.5–200 kDa (SigmaMarker^TM, Sigma-Aldrich, Saint Louis, MO, USA); (2) fraction from R. venosa hemolymph with MW 50–100 kDa. (B) Electrophoretic profile of a standard protein molecular marker (electrophoretic Lane 1) analyzed by ImageQuant^TM TL. (C) Analysis of the electrophoretic profile of fraction Rv 50–100, electrophoretic Lane 1, using ImageQuant™ TL v8.2.0 software.

Figure 3

The well diffusion method for determination of the antibacterial activity of the fraction with MW 50–100 kDa. Deep inoculation of E. coli was applied.

Figure 4

Fluorescence analysis for living and dead cells in E. coli bacteria after addition of 1%, 5%, 10% and 50% of the protein fraction with MW 50–100 kDa isolated from the hemolymph of R. venosa (all cells are colored blue with DAPI; only metabolically active cells are colored red with CTC). The images are with 1000× magnification.

Figure 5

Digital analysis of images obtained from the fluorescent staining of E. coli samples with 1%, 5%, 10% and 50% of the protein fraction with MW 50–100 kDa from the hemolymph of R. venosa: (A) percentages of alive cells in the samples; (B) fluorescence intensity; (C) average areas of cells in the samples.

Figure 6

Digital analysis of images obtained from the fluorescent staining of E. coli samples with 1%, 5%, 10% and 50% of the fraction with MW 50–100 kDa from R. venosa and the fraction with MW below 10 kDa from C. aspersum: (A) percentages of living cells in the samples; (B) fluorescence intensity; (C) average areas of the cells in the sample.

Figure 7

Illustration of the antibacterial damaging effect of a 10% solution of hemolymph from Rapana venosa against E. coli NBIMCC 8785. (A) control 18 h culture of E. coli NBIMCC 8785. (B) E. coli NBIMCC 8785 after 6 h exposure to rapana hemolymph—10% solution. The damage to the surface layers of the bacterial cells is clearly visible with SEM. (C) E. coli NBIMCC 8785 after 6 h exposure to rapana hemolymph—10% solution. The damage to the surface layers of the bacterial cells is clearly visible with AFM-2D. (D) E. coli NBIMCC 8785 after 6 h exposure to rapana hemolymph—10% solution. The damage to the surface layers of the bacterial cells is clearly visible with AFM-3D.