Anin vivostudy of novel bioactive peptides that inhibit the growth ofEscherichia coli

Abstract

We have created a system in which synthetically produced novel bioactive peptides can be expressed in vivo in Escherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth of E. coli on minimal media. The inhibitors could be placed into one of two groups, 1-day inhibitors, which were partially inhibitory, and 2-day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide-protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C-terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth of E. coli and what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

“…It showed a very wide spectrum of activity against gram-positive and -negative bacteria (Table 3). By comparing some structural characteristics of this peptide with those of isracidin and lactoferricin (23,46), the ␤-CN f184-210 has a very similar length (26 amino acids), a lower positive charge, a higher content of nonpolar hydrophobic residues (15 of the 26 amino acids), and some proline residues very near the C-terminal end of the peptide which could act to make its degradation by peptidases more difficult (44). ␤-CN f184-210 inhibited the indicator culture, E. coli F19, at a MIC of ca.…”

“…Antimicrobial milk proteins, such as lactoferrin, its pepsin-derived peptide fragments (lactoferricins), casocidin-I, and isracidin were described in the early literature (5,23,46). More recently, antimicrobial and antifungal peptides were designed by using combinational libraries (4), and novel antibacterial peptides were expressed in vivo in Escherichia coli (44) or purified from a pepsin digest of human milk which corresponds to -casein (CN) f63-117 (24). Compared to the other potential functions of bioactive peptides, this remains to be further studied due to the interesting features of antimicrobial peptides.…”

Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria

“…Specific inhibition of ProRS in the cell was demonstrated by a decrease in the intracellular pool of charged tRNA Pro . Walker et al (47) expressed 20,000 peptides in E. coli and found that 21 of them were growth inhibitory; however, the targets of these peptides were unknown. This study showed the importance of using peptides fused to proteins or amino acid motifs to improve stability.…”

Screening and cloning of antimicrobial DNA sequences using a vital staining method

“…This capacity is particularly useful in isolating antimicrobial genes whose products occur at low abundance levels, or in organisms that are not easily available, thus difficult to be discovered by traditional protein chemistry methods. Typically, on a membrane filter of 100 mm in diameter, thousands of colonies can be cultured, stained and examined, without need of replication and individual colony transfer as reported early (Walker et al, 2001) thereby drastically improving the screening capacity and reducing operational costs.…”