Anti-influenza Activity of a Bacillus subtilis Probiotic Strain

Abstract

Among Bacillus bacteria, B. subtilis is the species that produces the most antimicrobial compounds. In this study, we analyzed the activity of probiotic strain B. subtilis 3 against the influenza virus. The antiviral effect of this strain has been demonstrated in vitro and in vivo A new peptide, P18, produced by the probiotic strain was isolated, purified, chemically synthesized, and characterized. Cytotoxicity studies demonstrated no toxic effect of P18 on Madin-Darby canine kidney (MDCK) cells, even at the highest concentration tested (100 μg/ml). Complete inhibition of the influenza virus in vitro was observed at concentrations of 12.5 to 100 μg/ml. The protective effect of P18 in mice was comparable to that of oseltamivir phosphate (Tamiflu). Further study will assess the potential of peptide P18 as an antiviral compound and as a promising candidate for the development of new antiviral vaccines.

Keywords: Bacillus subtilis; antiviral peptide; influenza virus; probiotics.

Copyright © 2017 American Society for Microbiology.

Figures

FIG 1

Antiviral activity of B. subtilisin vitro and in vivo. (A) MDCK cells were inoculated with the influenza virus following treatment with a B. subtilis strain (106 CFU per well). Viral titers were analyzed by titration in MDCK cells. *, P < 0.05. (B) Mice (10 per group) received a single dose of B. subtilis (107 CFU per mouse) or PBS by oral gavage. After 24 h, both groups of animals were infected intranasally with influenza virus. Survival was monitored for up 14 days postinfection.

FIG 2

Isolation and characterization of B. subtilis peptides. (A) HPLC separation of the protein fractions from B. subtilis 3. Peptides (2 mg/ml) were applied to a TSKgel DEAE-5PW column and the fractions were collected by elution with NaCl solutions of increasing ionic strength (0.01 and 1 M) with 0.01 M Tris-HCl (pH 7.4). (B) Protein fraction interactions with antibodies to peptides from B. subtilis 3. Each collected peptide fraction was dried and analyzed by ELISA using antibodies against B. subtilis peptides. Fraction 11 showed the highest activity of interaction with antibodies. (C) Gel electrophoresis analysis of fraction 11. Homogeneity of the fraction was analyzed in 12% polyacrylamide together with molecular mass standards and stained with Coomassie blue.

FIG 3

MALDI-TOF mass spectrum of fraction 3 (from Fig. 2C). Spectrum was acquired using the instrument in reflectron mode and calibrated using a standard peptide mixture.

FIG 4

MALDI-TOF mass spectrum of the chemically synthesized peptide P18. Peptide TVAAPSVFIFPPSDEQLK (P18) was synthesized at the highest available purity (90%).

FIG 5

Characterization of peptide P18. (A) Cytotoxicity of P18 peptide was analyzed by MTT assay in MDCK cells. Viability of cells in the control wells (no peptide added) was scored as 100%. Other samples were normalized to this value. (B) Monolayers of MDCK cells were infected with the influenza virus. After 1 h of incubation, serial dilutions of peptide P18 were added to the wells and no peptide was added to the control wells. Viral titers were analyzed 3 days postinfection by titration in MDCK cells.

FIG 6

Efficacy of peptide P18 in vivo. Mice were treated with PBS, P18, or oseltamivir phosphate before infection with influenza virus (A) or after infection (B). On day 4 postinfection, the lungs from three mice in each group before infection (solid bars) and postinfection (open bars) were removed, and viral titers were evaluated in each supernatant by TCID50 analysis in MDCK cells (C). *, P < 0.05.

FIG 7

Experimental design. Animals were allocated to six groups (13 mice in each group): (i) control, PBS pretreatment; (ii) control, oseltamivir phosphate pretreatment; (iii) P18 pretreatment; (iv) control, PBS posttreatment; (v) control, oseltamivir phosphate posttreatment; (vi) P18 posttreatment. One day before the infection with virus, PBS pretreatment control mice received 0.2 ml PBS per os; oseltamivir phosphate pretreatment control mice received oseltamivir phosphate (1 mg/kg) by gavage. P18 pretreatment animals received P18 (0.1 mg/kg) by oral gavage. Mice from the posttreatment groups were infected with influenza virus and, after 24 h, were treated with PBS, oseltamivir phosphate, or P18.