Cationic Antimicrobial Peptides Inactivate Shiga Toxin-Encoding Bacteriophages

Abstract

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Keywords: Escherichia coli O157; anti-infective agents; antimicrobial peptides; bacteriophages (phages); hemolytic uremic syndrome (HUS).

Figure 1

Bacteriophage inactivation by cationic peptides. (A) Peptide P.2. (B) Peptide 6.2. After pre-incubation of bacteriophages with different concentrations of peptides the inactivation was measured by titration of E. coli, strain (ATCC 37197). Prism 5.0 software (GraphPad Software) was used to determine statistical significance between different samples. Peptide 2: ^*p < 0.05, ^***p < 0.0001. Peptide 6.2: ^**p < 0.05, ^***p < 0.0001.

Figure 2

Bacteriophage inactivation by cationic peptides. (A) Peptide 5 (B) Peptide 8 (C) Peptide 8.1. After pre-incubation of bacteriophages with different concentrations of peptides the inactivation was measured by titration of E. coli, strain (ATCC 37197). Prism 5.0 software (GraphPad Software) was used to determine statistical significance between different samples using one-way analysis of variance (ANOVA). Peptide 5: ^*p = 0.0313. Peptide 8: ^**p < 0.005, ^***p < 0.0001. Peptide 8.1: ^*p < 0.05, ^**p = 0.008.

Figure 3

Bacteriophage inactivation by control peptides. (A) Omiganan, a commercial cationic beta sheet peptide (B) Random sequence, a cationic non alpha helical peptide. Prism 5.0 software (GraphPad Software) was used to determine statistical significance between different samples using one-way analysis of variance (ANOVA). Omiganan: ns. Random: ns.

Figure 4

Zeta potential measurements of bacteriophage incubated with peptides P5 or Omiganan (OMI), or in buffer solution (control). Each point represents the mean of 2 independent batches ± SEM. ^**P < 0.01; ^***P < 0.001, one-way ANNOVA followed by a Dunnett post-test for multiple comparisons vs. the control.

Figure 5

Average hydrodynamic diameter of bacteriophage incubated with peptides 5 or in buffer solution (control). Ns, Not significant, t-test (unpaired).

Figure 6

Schematic representation of bacteriophage-peptide interaction and its role in bacterial infection.