Features and applications of Ent35-MccV hybrid bacteriocin: current state and perspectives.

Bacteriocins are peptides of ribosomal synthesis that are active against bacteria related to the producing strain. They have been widely used in the food industry as biopreservatives. The generation of hybrid peptides by combining the genes that encode two different bacteriocins has made it possible to study the mechanisms of action of the bacteriocins that compose them and also develop new peptides with improved biotechnological applications. Hybrid bacteriocins may be obtained in several ways. In our laboratory, by combining enterocin CRL35 and microcin V (Ent35-MccV), we obtained a broad-spectrum peptide that is active against both Gram-positive and Gram-negative bacteria. Ent35-MccV is sensitive to the action of intestinal proteases and is heat resistant, which makes it a good candidate for use as a biopreservative. For this reason, the peptide was tested in skim milk and beef burgers as food models. We also obtained more potent variants of the hybrid by modifying the central amino acid of the hinge region that connects the two bacteriocins. This review also discusses future applications and perspectives regarding the Ent35-MccV and other hybrid peptides.Key Points• Ent35-MccV is a new broad-spectrum bacteriocin.• The mechanism of action of bacteriocins can be studied using hybrid peptides.• Genetic engineering allows obtaining improved bacteriocin derivatives.• Hybrid peptides can be used in the food, pharmaceutical, and veterinary applications.

Obtaining an Ent35-MccV derivative with mutated hinge region that exhibits increased activity against Listeria monocytogenes and Escherichia coli.

The present paper describes the generation of derivatives from the hybrid peptide called Ent35-MccV, active against Gram-positive and Gram-negative bacteria. This peptide has a triple glycine hinge region between enterocin CRL35 and microcin V. In order to obtain variants of Ent35-MccV with greater biotechnological potential, a saturation mutagenesis was carried out in the hinge region. As a result, we obtained a bank of E. coli strains expressing different mutated hybrid bacteriocins in the central position of the hinge region. From all these variants, we found that the one bearing a tyrosine in the central region of the hinge (Ent35-GYG-MccV) is 2-fold more active against E. coli and 4-fold more active against Listeria than the original peptide Ent35-MccV. This derivative was purified and characterized. The development and evaluation of alternative hinges for Ent35-MccV represents a step forward in the bioengineering of antimicrobial peptides. This approach fosters the rational design of peptides with enhanced antimicrobial activity.

The case for class II bacteriocins: A biophysical approach using "suicide probes" in receptor-free hosts to study their mechanism of action.

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called "suicide probes". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.

Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragacea.

Sea anemones produce water-soluble toxins that have the ability to interact with cell membranes and form pores within them. The mechanism of pore formation is based on an initial binding step followed by oligomerization and membrane insertion. Although the final structure of the pore remains unclear, biochemical studies indicate that it consists of a tetramer with a functional radius of approximately 1.1 nm. Since four monomers seem to be insufficient to build a pore of this size, the currently accepted model suggests that lipids might also participate in its structure. In this work, the crystallization and preliminary crystallographic analysis of two crystal forms of fragaceatoxin C (FraC), a newly characterized actinoporin from Actinia fragacea, are described. The crystals diffracted up to 1.8 A resolution and the preliminary molecular-replacement solution supports an oligomeric structure of about 120 A in diameter.