Altering the Composition of Caseicins A and B as a Means of Determining the Contribution of Specific Residues to Antimicrobial Activity

Abstract

Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR) are antimicrobial peptides generated through the bacterial fermentation of sodium caseinate, and on the basis of this and previous studies, they are active against many Gram-negative pathogens (Cronobacter sakazakii, Cronobacter muytjensii, Salmonella enterica serovar Typhimurium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas fluorescens) as well as the Gram-positive organism Staphylococcus aureus. Here we describe further studies with the aim of establishing the importance of specific (charged and nonpolar aliphatic) residues within the caseicin peptides and the effects that they have on the bacteria listed above. In order to achieve our objective, we created four derivatives of each caseicin (A1 to A4 and B1 to B4) in which specific residues were altered, and results obtained with these derivatives were compared to wild-type caseicin activity. Although conversion of cationic residues to alanine in caseicins B1 (R8A change), A1 (K2A), A2 (H3A), and A3 (K2A-H3A) generally resulted in their activity against microbial targets being reduced or unaltered, C. sakazakii DPC6440 was unusual in that it displayed enhanced sensitivity to three peptides (caseicins A1, A3, and B2) in which positively charged residues had been eliminated. While the replacement of leucine with alanine in selected variants (B3 and B4) resulted in reduced activity against a number of strains of Cronobacter and, in some cases, S. Typhimurium, these changes enhanced the activities of these peptides against DPC6440 and a number of S. aureus strains. It is thus apparent that the importance of specific residues within the caseicin peptides is dependent on the strain being targeted.In recent years, the milk protein casein has been identified as a rich source of antimicrobial peptides, many of which have been characterized extensively. The casein-derived caseicin A and B peptides, which are the subjects of this study, were originally identified following a bacterial fermentation of sodium caseinate and were previously shown to exhibit antibacterial activity against strains of Cronobacter sakazakii and Escherichia coli (15). These and a number of other casein-derived antimicrobials are primarily cationic in nature, and thus it was plausible that these functioned in a manner similar to that for classical cationic antimicrobial peptides (cAMPs). As well as being cationic, cAMPs are amphipathic and hydrophobic ␣-helical peptides which are active against a range of microbial targets as a consequence of permeabilization of the cytoplasmic membrane leading to loss of the proton gradient (6, 21, 25, 37). The importance of the charged residues within cAMPs is apparent from studies on peptide derivatives with reduced cationic charges, which were shown to exhibit reductions in antimicrobial activity (19). These observations prompted the initiation of corresponding investigations, described here, to assess the importance of a number of residues, including cationic amino acids, in caseicins A and B.The an...

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide α s2 -Casein f(183–207)

“…Template-based studies of peptide derivatives obtained through manipulation of the amino acid sequence have often been performed in order to identify properties that are important for AMP activity (12). Such studies have shed light on the importance of specific amino acids and residue positions in the activities of different peptides (13)(14)(15). However, it appears that the effect of particular manipulations is context dependent and varies according to the template sequence, in that analogous substitutions may have substantially different effects on different peptides or at different positions in the primary sequence (16).…”

Extensive Manipulation of Caseicins A and B Highlights the Tolerance of These Antimicrobial Peptides to Change

“…That study revealed that the replacement of the C-terminal arginine of caseicin B with alanine resulted in a generally greatly reduced activity against Gram-negative targets, whereas activity against S. aureus was not affected (22). Indeed, the results indicated that the anti-Gram-positive activity of the peptides was less sensitive to change than their antiGram-negative properties (22). Notably, the previous study focused on only a selection of the residues within these peptides, and these variants differed from the wild-type peptides by no more than one amino acid.…”

“…Milk-derived AMPs are of particular interest and have been investigated since the 1930s (11), resulting in the identification of a number of peptides which demonstrate activity against Gram-positive (14,19,29) or Gram-negative (9,17) pathogens. Two of the most noteworthy of the milk-derived AMPs are caseicins A (IKHQGLPQE) and B (VLNENLLR), which were identified following a bacterial fermentation of sodium caseinate (9) and have previously been found to be active against a range of pathogenic bacteria, such as Cronobacter sakazakii (9), Cronobacter muytjensii, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium (22). The precise mechanism by which caseicins A and B act remains unclear, and thus, to gain more insight into the importance of specific residues within these peptides, eight synthetic variants were generated which differed subtly from their natural equivalents (22).…”

“…Two of the most noteworthy of the milk-derived AMPs are caseicins A (IKHQGLPQE) and B (VLNENLLR), which were identified following a bacterial fermentation of sodium caseinate (9) and have previously been found to be active against a range of pathogenic bacteria, such as Cronobacter sakazakii (9), Cronobacter muytjensii, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium (22). The precise mechanism by which caseicins A and B act remains unclear, and thus, to gain more insight into the importance of specific residues within these peptides, eight synthetic variants were generated which differed subtly from their natural equivalents (22). That study revealed that the replacement of the C-terminal arginine of caseicin B with alanine resulted in a generally greatly reduced activity against Gram-negative targets, whereas activity against S. aureus was not affected (22).…”

“…The precise mechanism by which caseicins A and B act remains unclear, and thus, to gain more insight into the importance of specific residues within these peptides, eight synthetic variants were generated which differed subtly from their natural equivalents (22). That study revealed that the replacement of the C-terminal arginine of caseicin B with alanine resulted in a generally greatly reduced activity against Gram-negative targets, whereas activity against S. aureus was not affected (22). Indeed, the results indicated that the anti-Gram-positive activity of the peptides was less sensitive to change than their antiGram-negative properties (22).…”