Acidocin A

Pediocin-like bacteriocins are among the natural antimicrobial agents attracting attention as scaffolds for the development of a new generation of antibiotics. Acidocin A has significant structural differences from most other members of this subclass. We studied its antibacterial and cytotoxic activity, as well as effects on the permeability of E. coli membranes in comparison with avicin A, the typical pediocin-like bacteriocin. Acidocin A had a more marked tendency to form an alpha-helical structure upon contact with detergent micelles, as was shown by CD spectroscopy, and demonstrated considerably less specific mode of action: it inhibited growth of Gram-positive and Gram-negative strains, which were unsusceptible to avicin A, and disrupted the integrity of outer and inner membranes of E. coli. However, the peptide retained a low toxicity towards normal and tumor human cells. The effect of mutations in the pediocin box of acidocin A (on average, a 2-4-fold decrease in activity) was less pronounced than is usually observed for such peptides. Using multiplex analysis, we showed that acidocin A and avicin A modulated the expression level of a number of cytokines and growth factors in primary human monocytes. Acidocin A induced the production of a number of inflammatory mediators (IL-6, TNFα, MIG/CXCL9, MCP-1/CCL2, MCP-3/CCL7, and MIP-1β) and inhibited the production of some anti-inflammatory factors (IL-1RA, MDC/CCL22). We assumed that the activity of acidocin A and similar peptides produced by lactic acid bacteria might affect the functional state of the human intestinal tract, not only through direct inhibition of various groups of symbiotic and pathogenic bacteria, but also via immunomodulatory effects.

Acidocin A, a bacteriocin produced by Lactobacillus acidophilus TK9201, is active against closely related lactic acid bacteria and food-borne pathogens including Listeria monocytogenes. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential ion-exchange and reversed-phase chromatographies. The molecular mass was determined by high-performance liquid chromatography gel filtration to be 6,500 Da. The sequence of the first 16 amino acids of the N terminus was determined, and oligonucleotide probes based on this sequence were constructed to detect the acidocin A structural gene acdA. The probes hybridized to the 4.5-kb EcoRI fragment of a 45-kb plasmid, pLA9201, present in L. acidophilus TK9201, and the hybridizing region was further localized to the 0.9-kb KpnI-XbaI fragment. Analysis of the nucleotide sequence of this fragment revealed that acidocin A was synthesized as an 81-amino-acid precursor including a 23-amino-acid N-terminal extension. An additional open reading frame (ORF2) encoding a 55-amino-acid polypeptide was found downstream of and in the same operon as acdA. Transformants containing this ORF2 became resistant to acidocin A, suggesting that ORF2 encodes an immunity function for acidocin A. The 7.2-kb SacI-XbaI fragment containing the upstream region of acdA of pLA9201 was necessary for acidocin A expression in the acidocin A-deficient mutant, L. acidophilus TK9201-1, and other Lactobacillus strains.

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ~7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.