CLASSIFICATION AND MECHANISM OF BACTERIOCIN INDUCED CELL DEATH: A REVIEW

Multidrug resistance and toxicity associated with antimicrobial agents among pathogenic bacteria leading to a surge in morbidity and mortality in humans need bold proclamation in the area of research and development of new biological agents. The maximum propitious possibility we can see in the area of bacteriocins. Bacteriocins are ribosomally synthesized peptides produced by gram-positive and gram-negative bacteria which evince wide and narrow antimicrobial activity spectrum. They can survive in a highly competitive microbial environment. Bacteriocins attack their targeted bacterial cells through different mechanisms. Understanding different mechanisms that induced cell death will enable researchers to develop methodologies to limit this life-threatening problem. Therefore, in this study, we provide the updated information on the number of bacteriocins produced, their potential producers and different mode of action against relevant pathogenic bacteria.


INTRODUCTION
Today, the world suffers from a number of infectious diseases, which are mainly caused by pathogenic organisms. Pathogenic organisms inhibit the production of antimicrobial peptides inside the body and caused several life-threatening diseases Singh et al., 2021). The important part of natural immunity in human is the production of antimicrobial peptides which protects various disease-causing organisms like bacteria, fungi, yeast viruses and cancer cells (Reddy et al., 2004;. Bacteria itself release some antimicrobial peptides which are the biologically extra-cellular product of ribosomal synthesis (Klaenhammer, 1993;Pirzada et al., 2004). They are produced by both gram-positive and gram-negative bacteria including some archaea (Zheng et al., 2015). A large portion of bacteriocins from gram-negative bacteria resembles defensins which are the eukaryotic antimicrobial peptides (Baindara et al., 2018). Many bacteria are known for producing bacteriocins in humans, plants and various food products where they have a valuable place e.g. E. coli, Lactic acid bacteria (LAB), Weissellaconfusa, Streptococcus mutans, Streptococcus salivarius, Bacillus subtilis etc. Out of which LAB described as GRAS (generally regarded as safe) for human consumption (Balciunas et al., 2013;Indumathi et al. 2015;Sing, 2021). The bacteriocins show inhibitory action on food deterioration and foodborne pathogenic microorganisms, additionally, the bacteriocins from lactic acid microorganisms widely known for both food preservative and therapeutic potentials (Kumari et al., 2018;Mittal et al., 2020. Bacteriocins from different species of bacteria, in contrast to all other antibiotics, show killing action on the same or closely related species (Peter R, 1965). Each species of bacteria produces tens or even hundreds of different kinds of bacteriocins (Bindiya et al., 2016). Bacteriocins are a heterogeneous group of particles with different morphological and biochemical entities. They range from simple and low molecular weight protein to complex and high molecular weight protein. Moreover, the bacteriocins are non-immunogenic, biodegradable substances and possess cancer-cell specific toxicity (Kaur et al., 2015). They also act as the competitive agents between the microbial communities (Chao et al., 1981, Majeed et al., 2013, Riley et al., 1999. Researchers had conducted a deep study on various aspects of bacteriocins like the methods for their detection, characterization, purification and identification of genetic determinants from gram-positive and gram-negative micro-organisms (Catherine et al., 1993).

BACKGROUND OF THE REVIEW
Colicin is the very first bacteriocin discovered by Belgian scientist Gratia, (1925) a heat-liable product where he observed that Escherichia coli V inhibits Escherichia coli S during his search for the ways to kill the bacteria. The inhibition of one bacterial strain by another had been observed many times by Gratia. But the importance of bacteriocin can't explore much at that time due to the lack of knowledge about its structure and production which led to the dominance of chemically synthesized broad-spectrum antibiotics (Syngulon.com). Fredericq, (1946) revealed the proteinaceous nature of colicin and demonstrated that the inhibitory activity of bacteriocin was due to the presence of specific surface receptors of sensitive cells. After a long period, it is verified that a large number of bacteria produced some common molecules which inhibit the growth of other strains or species, these molecules were named bacteriocins (Jacob et al., 1953). Bacteriocins have been detected in all major lineages of eubacteria and some members of Archaebacteria and recently it becomes a viable alternative to conventional antibiotics (Torrebranca et al., 1995;Gillor et al., 2008;Cotter et al., 2013).
bacteriocin produced from LAB (gram-positive bacteria) into two main classes, lantibiotics (class I), not containing lanthionine lantibiotics (class II) whereas class III was individually designated as bacteriolysins. It was also suggested by the authors that class IV should be extinguished (Tumbarski et al., 2018). So, recently authors have altered the classification of gram-positive bacteria from four classes to three, while different authors have used a somewhat different description of subclasses (Mokoena, 2017). Yang et al. (2014) mentioned that microcin E492 derived from gram-negative bacterial sp. Klebsiella pneumonia, so class II should be categorized under microcins of gram-negative bacteria. Moreover, the bacteriocins are currently used in agro-food as a food preservative however it may be considered as potential candidates for further development and used in health contexts. The different classification and applications of bacteriocins are enlisted in Figure 1 and 2.

PROPERTIES OF BACTERIOCIN FOR INHIBITION
Bacteriocins have some special features which make them lethal towards pathogenic organisms. They must have a cationic (mostly at pH 7.0) and highly hydrophobic nature to be lethal as observed for the most bacteriocins belonged to Class I and II. They must be active at a wide range of pH, as found in the case of numerous small size bacteriocins where they show antibacterial activity at different pH ranging from 3.0 to 9.0. Their high isoelectric point promotes the interaction at physiological pH with the anionic surface of bacterial membranes which cause the insertion of hydrophobic moiety into the bacterial membrane which finally build up a trans-membrane pore that led to cell death due to gradient dissipation (Jack et al., 1995). They are diffusible toxins that do not require contact between bacteria like type six secretion system (T6SS) and contact-dependent inhibition (CDI) (Sharp et al., 2017). Bacteriocins are potent even at the pico to nanomolar concentration as compared to eukaryotic AMPs which acts at a micromolar concentration (Hassan et al., 2012). Low molecular protein must be heat stable to show the killing action on related pathogenic strains. The stabilization of secondary structures accompanies by the complex pattern of monosulfide and disulfide intramolecular bonds which acts to reduce the number of possible unfolded structures

MECHANISMS OF BACTERIOCINS
Bacteriocins kill the pathogenic bacteria in several ways, like pore-forming inhibition of cell wall, nucleic acid and protein synthesis ( Figure 3). Usually, they have a narrow killing spectrum as they are limited to the inhibition of closely related species and simultaneously they may have broad-spectrum activity against distantly related bacterial species (Singh et al., 2013;Klaenhammer, 1993; Adams and Moss, 2008; Kumariya et al., 2019) and plays a defensive role by inhibiting the invasion of other strains or by limiting the growth of neighbouring cells (Riley and Wertz, 2002b). The production of bacteriocins seems to be a hereditary feature associated with cytoplasmic genes i.e. bacteriocinogenic factors. Their mode of action varies greatly from one species to another (Daw and Falkiner, 1996

ANIMALS
• Used in preparation of veterinary medicines • Helpful in poultry and swine production • Also used in animal feed • Effective against the pathogens of animal origin Figure 3 Schematic diagram of mechanisms of bacteriocin induced cell death

INHIBITION BY PORE FORMATION
Pore formation is the well-known mechanism in which these antibacterial proteins binds to the specific receptors on cells and forms pores in the membrane which is also called as cell permeability and thus cause the death of pathogenic microorganism (Preciado et al., 2016). These antibacterial proteins are also called c PFTs are one of the wide categories of virulence factors as they constitute 25-30% of cytotoxic bacterial proteins (Alouf, 2003;Gonzalez et al., 2008). The diameter of the pore formed by these proteins varies from one species to another, ranging between 1-50 nm consisting of 6-50 or more units of PFPs (Peraro and van der Goot, 2016). The largest pores found in cholesterol-dependent cytolysins (CDCs) whose diameter ranges from 25-40 nm (Twetenet al., 2015). Generally, PFPs are genetically encoded large proteins (α-toxin) or small cationic peptides which are delivered to the targeted cell for production and insertion into the membrane (Panchal et al., 2002). Based on the secondary structure of the region that allows the formation of the pore by penetrating the host cell, PFPs are divided into two main classes: α-PFPs and β-PFPs which forms pores by bundles of αhelicals or by trans-membrane β-barrels respectively (Anderluhet al., 2008, Ostolazaet al., 2019). These antibacterial proteins are water-soluble monomers that bind to the lipid membrane of the cell and oligomerize to form structural assemblies called pre-pores. These pre-pores exposed the hydrophobic surface of the cell by undergoing some conformational changes that lead to the insertion into the lipid bilayer which forms a pore that causes the permeabilization of the cell membrane (Omersa et al., 2019). This mechanism is followed by β-PFPs while in the case of α-PFPs the insertion into the membrane is associated with a sequential oligomerization which then forms a partial or complete pore and the pore remains active in both cases. The β-pores are structurally more stable in comparison to αpores due to the inter-chain interactions between the hydrogen bonds (Ostolazaet al., 2019). The formation of oligomers is a common characteristic of PFPs that pierce the cell membrane of the pathogen (Cosentino et al., 2016). Pore-forming proteins disrupt the maintenance of the osmotic balance of the cell which leads to the cytolysis (Alouf, 2003). They make the path for the passage of ions, proteins or other constitutes through the targeted membrane. The loss of potassium and magnesium ion has been implicated as the primary cause of cell death (Konisky, 1982). Pore formation also causes rapid dissipation of transmembrane electrostatic potential which lead to the rapid death of bacterial cells (Prince et al., 2016). Nisin belongs to the lantibiotic family, an amphiphilic and cationic bacteriocin (3.4kDa) isolated from the different strains of Lactococcus lactis subsp. lactis, is one of the widely studied bacteriocins. It is an FDA approved and GRAS peptide with recognized potential for clinical use (Shin et al., 2016). It acts on the targeted cells through pore formation by the use of "Docking Molecule" mediated by cell wall precursor lipid II which forms stable pores of around 2-2.5 nm diameter (Wiedemann et al., 2004). Nisin binds to lipid II with the two lanthionine rings at the N-terminus, forming a pyrophosphate cage around the head group of lipid II and flexible hinge region cause the insertion of C-terminus in a transmembrane orientation which led to the formation of a stable pore (Prince et al., 2016). Kraaij et al., (1998) demonstrated the importance of translocation of the C-terminal region in pore formation. However, the C-terminus of NisI (immunity protein of Lactococcus lactis) found to inhibiting the nisin mediated pore formation by protecting the lipid II (Alkhatib et al., 2014). Further, nisin use all lipid II molecules to form the pore complex which uniformly consists of 8 nisin and 4 lipid-II molecules. These pores were able to resist the solubilization of the membrane environment by mild detergents (Hasperet al., 2004). The micromolar concentrations are necessary in the absence of lipid II while nanomolar concentrations are sufficient to form a pore in the presence of lipid II (Christ, 2007). Nisin also acts as an anionic selective carrier during the absence of anionic membrane phospholipids and forms nonselective, wedge-like, multistate, waterfilled pores in the presence of anionic phospholipids which results from the bending of lipid surface due to co-insertion of the surface-bound aggregate to it . The bacteriocins that kill the pathogens by pore formation are enlist in Table 1.

CELL WALL BIOSYNTHESIS INHIBITION
The antimicrobial peptides involved in the inhibition of biosynthesis of cell wall either by inhibiting peptidoglycan synthesis or by binding to the lipid II or may impair the cell wall functions are called as cell-wall active or membrane-active bacteriocins. This mechanism may involve a concerted action with pore formation as observed in nisin, a well-known bacteriocin widely used in food preservation. This mechanism is followed by both gram-negative and gram-positive bacteria. It comprises a wide variety of structures like lipid II-binding bacteriocins, two peptide lantibiotics and non-modified bacteriocins (Roceset al., 2012). In eukaryotic cells, cell membrane acts as the main target of bacteriocins where they enhance the expression of negatively charged cell surface molecules on the cancer cells makes them prone to the cytotoxic activity of bacteriocins (Kaur et al., 2015). Nisin is the first example of a membrane-targeted lantibiotics (Breukinket al., 2003). However, Tol et al. (2015) suggested that nisin variants that cluster lipid II kill L-form bacteria without involving the delocalization of peptidoglycan synthesis which is the primary killing mechanism of these lantibiotics. Lactococcin 972 (Lcn972) is the first unmodified, bacteriocin that binds to the cell wall precursor lipid II to inhibit the septum biosynthesis in Lactococcus lactis (Martínez et al., 2008). Scherer et al., (2015) revealed that an increase in the size of the nisin-lipid-II complex also plays a role in the inhibition of cell wall synthesis and also induce vesicle budding in the targeted cell membrane. However in some cases, the destabilization of the cell wall or outer membrane is brought by stress condition such as treatment of targeted cell with chemicals or by inducing some physical stress conditions like pH, heating, freezing etc., which may increase the sensitivity of targeted cell as observed for gram-negative bacteria (Costa et al., 2019). Besides all this, plantaricin NC8, a two-peptide non-lantibiotic class IIb bacteriocin composed of PLNC8α and PLNC8β and derived from Lactobacillus plantarum ZJ316 has been found to show antimicrobial activity against Micrococcus luteus 1.193 by following the mechanism of cell membrane disruption without targeting lipid II (Jiang et al., 2018). The bacteriocins that follow the cell wall inhibition mechanism for killing of pathogens are listed in Table 2.

Nuclease activity inhibition/ protein inhibition
Generally, the nuclease activity involves the breakdown of macromolecules like the disruption of bonds between nucleotides in nucleic acids such as DNA and RNA. Table 3. showed the list of bacteriocins that inhibits protein or nuclease activity of the targeted cell. The bacteriocins which follow this mechanism are also known as nuclease bacteriocins (NBs). Different nuclease bacteriocins are involved in the inhibition of DNA, RNA and protein synthesis together with permease function and show the primary effect on the deployment of energy by the bacterium (Reeves, 1972). They usually have a broad range of size, ranges from 178 to777 amino acid (Bindiyaet al., 2016). The colicins, plasmid encoded bacteriocin from Escherichia coli also shows nuclease activity. Even the colicin E1 and K inhibits all macromolecule synthesis without the arrest of respiration while others may act by cleaving the precise site of particular nucleic acid (Cascaleset al., 2007). They contain an N-terminal translocation domain, a central receptor binding domain and a C-terminal cytotoxic domain that binds a cognate immunity protein however the location of the translocation and receptor-binding domains in pyocins (bacteriocins from Pseudomonas aeruginosa) appears to be reverse (Atanaskovic et al., 2019). Translocations of nuclease colicins across the outer and inner membrane must be necessary to achieve their target in the cytoplasm (Cascales et al., 2007;de Zamaroczy et al., 2011). During translocation, the immunity proteins of nuclease colicins may be dissociated at the cell surface in a pmf-dependent step (Sharp et al., 2017). The nuclease bacteriocin delivered to the cytoplasm of a targeted cell which involves the DNA chromosomal cleavage randomly led to the cell death. Many nuclease colicins like colicin E2, E7, E8 and E9 found to exhibit their antimicrobial activity by the action of DNase which involves the non-specific cleavage of genomic DNA (Schaller et al., 1976;Chaket al., 1991;Cooper et al., 1984).HNH/ββα-Me motif acts as the catalytic centre of many colicins and pyocins DNases by hydrolyzing the phosphodiester bond through chelation with a single divalent metal ion (Klein et al., 2016). Walker et al., (2007) showed that the toxic action of nuclease colicins depends upon functional FtsH, an inner membrane AAA + ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm of a targeted cell as to cause cell death. LepB which is an important inner membrane enzyme of E. coliand a key membrane component of cellular secretion machinery offered a chaperonlike function for the penetration of several nuclease bacteriocins into a target cell in addition to this it was also reported as the necessary component of machinery hijacked by the tRNase colicin D for its import (Mora et al., 2015). Colicin like E3, E4, E6 exhibit RNase activity, out of which Colicin E3 is most widely studied, which is known to cleaves the 3' region of 16-S rRNA between A1493 and G1494 (E. coli numbering) in the decoding A-site and decreases the acceptance of cognate aminoacyl-tRNAs (aa-tRNAs) and thus slow down the protein synthesis and finally cause the death of the targeted cell (Ogawa et al., 2016).

ATP SYNTHESIS INHIBITION
Many bacteriocins also show their antimicrobial activity by inhibiting the ATP synthesis or by the release of ATP out of the cell. The bacteriocin that showed the ATP inhibition accompanied by other mechanisms is shown in Table 4. The ATP synthesis inhibition accompanied by either cell wall synthesis inhibition or by pore formation which allows the secretion or reduction of ATP along with other ionic molecules as stated by many researchers. There are many examples of bacteriocins that involved in ATP synthesis inhibition like mesentericin Y105 produced by Leuconostoc mesenteroides strain which is a pore-forming bacteriocin, had been found to show the effects on cell organelle, where it uncouples the mitochondria by increasing state 4 respiration and decreasing state 3 respiration. It also inhibits the ATP synthase and adenine nucleotide translocase of the organelle (Maftah et al., 1993). Similarly, microcin J25 also showed inhibition of ATP along with concomitant enhancement of ATP degradation. It was also observed for altering the membrane permeability and inhibiting the enzymatic activity of cytochrome C reductase (complex III) of the respiratory chain (Chirou et al., 2004). The increased ATPase activity found to be responsible for acid sensitivity of nisinresistant Listeria monocytogenes which cause cell death on the addition of an acid like hydrochloric acid or lactic acid (McEntire et al., 2004). Sometimes, as a consequence of a shift in the ATP equilibrium, the ATP is hydrolysed into ADP and AMP due to the efflux of phosphate through the channels (Guihard et al., 1993). Here, we represent the list of some bacteriocins that involves in the inhibition of ATP synthesis either as a primary or as a secondary action of these antimicrobial proteins.

CONCLUSION
As described above, we can recapitulate that how these bacteriocins are inhibiting the growth of bacteria replacing the hazardous chemical preservatives in agro-food industries and become prominence for society as they involve in the killing of pathogens by following mechanisms. Due to their diversity in various aspects like mode of action, uses and their habitat they may provide new and more advanced pathways for researchers in the area of medical, pharma, agriculture and food biotechnology for the sake of humanity. To overcome, antibiotic-resistant related issue in the medical sector this can warrant an alternative and provide the researchers to remove insurmountable difficulties.  (2005). Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832.