Team:SDU-Denmark/Bacteriocins

Learn more about Bacteriocins

Overview

Bacteriocins are antimicrobial peptide compounds that are ribosomally produced by Gram-positive or Gram-negative bacteria as inactive or active pre-peptides ZENDO, T. (2013). Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria. Bioscience, Biotechnology, and Biochemistry, 77(5), 893–899. . Bacteriocins were first described in 1925 by Gratia, and were reported as highly specific antibiotics produced by E. coli Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. The bacteriocins show bacteriostatic and/or bactericidal activity against other related bacteria, and can be broad- or narrow-spectrum Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. The bacteriocins effect is through adhesion to specific receptors present on the external surface of the target bacteria, or by other unknown mechanism Zendo, T., Yoneyama, F., & Sonomoto, K. (2010). Lactococcal membrane-permeabilizing antimicrobial peptides.. Hereby the bacteriocins induce metabolic changes by forming an open pore where nutrients diffuse out of the cell and therefore also induces morphological changes, which might result in the death of the bacteria or inhibition of its virulence factors. The bacteriocins are found to play a fundamental role in controlling pathogenic and non-wanted flora, as well as establish beneficial bacterial populations and thus serve as an alternative and/or supplement to traditional antibiotics used today. Thereby facing the problem of antimicrobial resistance (AMR).

Gene localization and regulation

The bacteriocins are located in operon clusters. These clusters contain genes encoding the bacteriocin, corresponding immunity and also genes that encodes peptides responsible for post translational modification Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. . The operon can be located either on plasmids, chromosomes or transposons of plasmids or chromosomes, which gives the complexity to the bacteriocins. However most bacteriocin operons are located on plasmids, as it has been suggested to help the phylogenetic dissemination of bacteriocins among lactic acid bacteria (LAB) Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. .

The immunity systems makes the bacteriocin-producing bacteria resistant towards its own bacteriocin Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. For the bacteriocins produced by Gram positive LAB there are two types of immunity systems known today. The first being the Lan I protein, which provides immunity to the producing cell by being attached to the outside of the cytoplasmic membrane. The localization prevents pore formation by the bacteriocin molecule as the Lan I protein is continuously transporting the bacteriocin molecule, that has been inserted into the membrane, back to the surrounding medium. The second immunity system depends on separate dedicated multicomponent ABC transporter called LAN EFG Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. . However the mechanism of immunity systems are similar in their way of functioning, as they, in general, work by removing the bacteriocin or by competitive inhibition of the bacteriocin receptor Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142..

The regulation of the bacteriocin expression is a subject for external induction factors (IF). The regulating system is composed of three components; an inducing peptide - either IF or pheromone-activating factors, a transmembrane histidine kinase or a pheromone receptor and a response regulator Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. IF are small peptides that are ribosomally synthesized at low levels as a pre-peptide and are cleaved before secretion. IF factors are either secreted by the bacteriocin producer-strain itself or concurrent of the bacteriocin producing strain Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. The possibility of the production of IF factors by other strains makes, the regulatory and controllable aspect of bacteriocin production of a specific strain, a target for quorum sensing. IF binds to the correspondent histidine protein kinase and thus activate it to phosphorylate a response regulator, which then stimulates the transcription of target genes by binding to specific repeats that are present in many bacteriocin operons. The bacteriocin production is thereby autoregulated by the inducer peptide (IF), making it possible for the respective strain to possess positive or negative feedback or to be regulated, or even to regulate, related or competing strains. The production of IF are dependent of environmental conditions such as temperature and pH.

Bacteriocin secretion

The proteinaceous bacteriocins are secreted out of the bacteria cell in their exponential growth phase by guidance of their respective N-terminal leader sequence Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Through the export process the leader sequence is proteolytically processed and some of the peptides are modified post-translationally. The export results in a mature and active peptide through enzymatically modification Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. , Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Bacteriocins produced by gram-positive lactic acid bacteria (LAB) are defined as GRAS (Generally Recognized As Safe), and/or probiotics as their presence serve to provide an advantage in the human gastrointestinal tract Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142..

Classification of bacteriocins

The bacteriocins, in general, are cationic and amphipathic molecules, that can be characterized and classified according to their biochemical characteristics, structural appearance, genetics and their producing strain. Bacteriocins from Gram-positive bacteria can be divided into 4 major groups: Class I, lantibiotics, which are post-translational modified. Class II, small-unmodified peptides. Class III, large-unmodified peptides, and Class IV, complex bacteriocins. In our project most of the bacteriocins are class II, because they are small and typically not modified post-translationally.

Class I Include lantibiotics, which are denominated by the presence and unusual amount of the modified amino acid lanthionine in the peptide structure. The lantibiotics are small, have a low molecular weight (<5 kDa), heat-stable peptide inhibitors that contain several post-translational modified amino acids. The amino acids are modified in a two-step process that includes enzymatic dehydration by specific amino acids followed by thiol attack from neighboring cysteine amino acids, which results in a condensation between two neighboring residues and thus the formation of a fully structured and functional lantibiotic. The lantibiotics can be divided into subclasses according to the overall structure, as they form more complex operons when needing additional genes encoding enzymes for posttranslational modification Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Class Ia compose screw-shaped linear and positively charged peptides that are membrane targeting peptides as they act through pore formation by depolarizing the cytoplasmic membrane of their target Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. . Class Ib composes globular, negatively or “neutral” peptides that act by interference with cellular enzymatic reactions.

Class II includes non-lantibiotic peptides. These are small (<10 kDa), heat stable and possess an amphiphilic helical structure, that allows them to be membrane active peptide inhibitors through depolarization Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. The genes encoding Class II bacteriocins, have a more widespread localization on the chromosome compared to the class I bacteriocins. Class II bacteriocins require accessory proteins to facilitate the membrane translocation and the enzymatic cleavage of the N-terminus Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Class II bacteriocins are also divided into subclasses: The Class IIa non-lantibiotics contain a consensus sequence YGNGV in the N-terminal part Zacharof, M. P., & Lovitt, R. W. (2012). Bacteriocins Produced by Lactic Acid Bacteria A Review Article. APCBEE Procedia, 2(2), 50–56. . The peptides are synthesized with a leader sequence that is proteolytically processed. The C-terminal is responsible for the specific cell wall permeabilization through pore formation, which results in dissipation of the proton motive force and thus accelerating the consumption of ATP, which cause death of the bacteria Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Class IIa is listeria active peptides. Class IIb requires two different peptides for activity and shows a synergistic action Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. The majority has GxxxG motifs, which interacts with corresponding motifs in the bacterial cell membrane, thus causing lysis by dissipation of the membrane potential. Class IIc include other bacteriocins, which have a covalent bond between the C-terminus and the N-terminus resulting in a cyclic structure Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142..

Class III includes large (>30 kDa), heat stable peptides that possess a complex activity and protein structure but remains unmodified. The C-terminal contain a recognition site for the target cell while the N-terminus has homology to endopeptidases involved in cell wall synthesis. The Class III bacteriocins are thus different is their function by promoting cell wall lysis Balciunas, E. M., Castillo Martinez, F. A., Todorov, S. D., Gombossy De Melo Franco, B. D., Converti, A., & Pinheiro De Souza Oliveira, R. (2013). Novel biotechnological applications of bacteriocins: A review. Food Control, 32, 134–142.. Class IV are complex bacteriocins that possesses a carbohydrate or lipid.

Chosen Bacteriocins

Figure 1 Graphically shows the adherence of a bacteriocin to specific receptors present on the external surface of the target bacteria. The bacteriocin form an open pore where nutrients diffuse out of the cell, thus inducing morphological changes in the bacterial cell membrane. ZENDO, T. (2013). Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria. Bioscience, Biotechnology, and Biochemistry, 77(5), 893–899. .

There exist a lot of different types of bacteriocins, and they are produced from several bacterial strains. Many of the bacteriocin producing LAB, shows high activity against bacteria which initiate spoilage in food, but some of them can probably also be used as a treatment against infection Zendo, T., Yoneyama, F., & Sonomoto, K. (2010). Lactococcal membrane-permeabilizing antimicrobial peptides..

LacticinQ (BioBrick: K2018006) is a bacteriocin produced by Lactococcus lactis QU5 and has shown bactericidal activity against Staphylococcus aureus Ma, Q., Yu, Z., Han, B., Wang, Q., & Zhang, R. (2012). Expression and Purification of LacticinQ by Small Ubiquitin-Related Modifier Fusion in E.coli. The Journal of Microbiology, 50(2), 326–331.. LacticinQ is resistant to heat treatment and pH variations, which makes it more stable than other bacteriocins. LacticinQ does not require a specific receptor, but acts when it meets its target bacteria Ma, Q., Yu, Z., Han, B., Wang, Q., & Zhang, R. (2012). Expression and Purification of LacticinQ by Small Ubiquitin-Related Modifier Fusion in E. coli. The Journal of Microbiology, 50(2), 326–331.. LacticinQ is a class IId bacteriocin. It functions by forming large toroidal pores by disrupting and redistributing membrane lipid organization. The event causes ATP leakage of protein molecules from the target cell, and thus elicits important metabolites needed for bacterial survival. The pore forming event thereby introduce LacticinQ as a bactericidal agent. LacticinQ has an N-terminal formylated methionine, and gene sequencing indicates that it is synthesized without a leader peptide. The peptide sequence consist of 53 amino acid, and is cationic. It forms two amphiphilic alfa-helix with a hinge region between ZENDO, T. (2013). Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria. Bioscience, Biotechnology, and Biochemistry, 77(5), 893–899. , Zendo, T., Yoneyama, F., & Sonomoto, K. (2010). Lactococcal membrane-permeabilizing antimicrobial peptides..

ThuricinS (BioBrick: K2018003) is a cationic peptide bacteriocin, classified in Class IId. ThuricinS is produced by Bacillus thuringiensis, which produce a large number of antibiotics including bacteriocins. ThuricinS targets a broad spectrum of pathogens, including Pseudonoas aeruginosa and Enterobactoer cloacae, these are often found in correlation to burn and wound infection. ThuricinS exhibit a strong bactericidal effect by forming pores in the cytoplasmic membrane of sensitive cells, thus inducing membrane permeability Chehimi, S., Pons, A.-M., Sablé, S., Hajlaoui, M.-R., & Limam, F. (2010). Mode of action of ThuricinS, a new class IId bacteriocin from Bacillus thuringiensis. Canadian Journal of Microbiology, 56(2), 162–167...

Laterosporulin (BioBrick: K2018002) is also a cationic bacteriocin and belongs, as well, to the class IId Singh, P. K., Solanki, V., Sharma, S., Thakur, K. G., Krishnan, B., & Korpole, S. (2015). The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module. FEBS Journal, 282(2), 203–214. . It is produced by Brevibacillus spp., and exhibit a broad spectrum of antibacterial activity against bacterias like: B. subtilis , S. aureus, E. coli, P. aeruginosa, and L. monocytogenes. However B. subtilis and S. aureus are more sensitive to the bacteriocin than other strains Singh, P. K., Chittpurna, A., Sharma, V., Patil, P. B., & Korpole, S. (2012). Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by Brevibacillus sp. Strain GI-9.. Laterosprulin inhibits growth of bacteria by increasing the permeability of important substances for bacterial survival, such as ions, amino acids, ATP, etc.Singh, P. K., Solanki, V., Sharma, S., Thakur, K. G., Krishnan, B., & Korpole, S. (2015). The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module. FEBS Journal, 282(2), 203–214. . Laterosporulin contains many polar amino acids, where the most important is cysteine. The polar characteristic of the amino acids gives it an amino acid profile that is comparable with human beta-defensin-like proteins, which are antimicrobial peptides implicated in the resistance of epithelial surfaces to microbial colonization.

PyocinS5 (BioBrick: K2018018) is produced by P. aeruginosa and elicit its effect against other strains of P. aeruginosa. S type Pycins is composed of an effector and an immunity component. The effector component is exerting a killing activity, which includes DNase activity. The effector component, which ranges from 498-776 amino acids, consists of three domains: a receptor binding domain, a translocation domain, which translocates it across the membrane, and a killing domain. The immunity component ranges from 87–153 amino acids and gives the pyocinogenic strains immunity to their produced bacteriocins by production of immunity proteins, thus making the pyocinogenic strains insensitive to their own pyocins. Pyocin causes the cell membrane of target cells to be permeable and thereby causing leakage of intracellular materials, which cause cell death Ling, H., Saeidi, N., Haji Rasouliha, B., & Wook Chang, M. (2010). A predicted S-type pyocin shows a bactericidal activity against clinical P. aeruginosa isolates through membrane damage. FEBS Letters, 584, 3354–3358..

Laterosporulin-ThuricinS (BioBrick: K2018008). From the bacteriocins Laterosporulin and ThuricinS we have designed another hybrid Laterosporulin-ThuricinS. No study have yet reported the effect of these bacteriocins as a hybrid and it is therefore still unknown whether these bacteriocins corporate and have synergistic effect or not.

LacticinQ-LacticinZ (BioBrick: K2018009). In our project we have designed a combination of bacteriocins, which is a hybrid made of LacticinQ and LacticinZ. LacticinZ has only three different amino acids from LacticinQ, and is similar in structure and activity. In our project we have found synergistic effects of the hybrids. Please check out Demonstrate and Results.