A REVIEW : LANTIBIOTICS , A PROMISING ANTIMICROBIAL AGENT

Rise in multi drug resistant bacteria has been a public health problem. This has necessitated for the exploration of novel antimicrobials. Bacteriocins, probiotic bacteria, and bacteriophages are considered as alternatives to antibiotics. To this context, lantibiotics could be the future candidate for antimicrobial agent. Lantibiotics are synthesized ribosomally and after posttranslational modification active peptide is produced. Lantibiotics are lanthionine and methyllanthionine containing peptides exhibiting activity against multi drug resistant pathogens. Besides its application as alternatives to old antibiotics, they can be used as food preservatives, additives, probiotics, and prophylactics.


INTRODUCTION
Lantibiotics are antimicrobial peptides produced by most of the Grampositive bacteria.These peptides are effective against most of the Grampositive bacteria and few Gram-negative bacteria, for example, nisin Z at higher concentrations can affect Escherichia coli and other Gram-negatives like Neisseria or Helicobacter pylori.The first lantibiotic nisin was discovered in 1920s (Rogers & Whittier 1928) which is used as a safe food preservative.However, the ribosomal origin of such modified peptides was revealed only after 60 years, when epidermin biosynthetic gene cluster from Staphylococcus epidermidis Tü 3298 was sequenced (Schnell et al. 1988).Research on lantibiotics has gained renewed interest due to the emergence of antibiotic resistant strains such as, methicillin resistant Staphylococcus aureus (Rubin et al. 1999), vancomycin resistant Enterococcus faecium, multi drug resistant Gram-negative pathogens -Pseudomonas aeruginosa, Acinetobacter baumanii and Enterobacteriaceae (Livermore 2004) etc.Since then, hundreds of lantibiotics have been described, and most of them are produced by Gram-positive bacteria (Emma et al. 2013).Likewise, many more lanthipeptide gene cluster have been characterized from actinomycetes viz-cinnamycin from Streptomyces cinnamoneus DSM 40005 (Widdick et al. 2003), SapB from Streptomyces coelicolor (Kodani et al. 2004), SapT from Streptomyces tendae (Kodani et al. 2005), actagardine from Actinoplanes garbadinensis (Boakes et al. 2009), deoxyactagardine B (DAB) from Actinoplanes liguriae (Boakes et al. 2010), venezuelin from Streptomyces venezuelae (Goto et al. 2010), and microbisporicin from Microbispora corallina (Foulston et al. 2010).Furthermore, lanthipeptide gene clusters are also found in proteobacteria, chlamydiae, bacteroidetes and cyanobacteria (Li et al. 2010, Marsh et al. 2010).

Biosynthetic gene cluster
Lantibiotics are synthesized ribosomally and post translationally modified (Schnell et al. 1988, Arnison et al. 2012).The genes required for the biosynthesis of lantibiotics are arranged in cluster.The genes are designated by generic locus lan (eg.nis for nisin (Gross & Morell 1971), gdm for gallidermin (Kellner et al. 1988), pse for pseudomycoicidin (Basi-Chipalu et al. 2015) etc. Gene clusters can be found on conjugative transposable elements (e.g., nisin), on the chromosome of the host (e.g., subtilin), or on the plasmids (e.g., epidermin, lacticin 481) (Chatterjee et al. 2005).The lantibiotics are characterized by Lanthionine (Lan), methyllanthionine (MeLan) and other modified amino acids such as didehydroalanine (Dha) and didehydrobutyrine (Dhb) (Schnell et al. 1988).The representative gene clusters for biosynthesis of specific lantibiotics are shown in fig 1 (see appendix).The gene cluster consist of structural gene lanA that encodes prepeptide, modification gene lanB,C,M, L, labKC encoding enzymes that introduce thioether rings, transporter (lanT) gene that exports modified peptide as well as cleaves leader peptide, and / or extracellular protease (lanP) that removes the leader, and immunity gene [lanI (H) and/or lanFEG] that protect the producer bacteria from its own product.Normally, parts of gene cluster are organized in the form of operon.The structural gene is often clustered as first ORF.Besides this, there is no uniform gene order in the individual gene clusters (Siezen et al. 1996, Willey & van der Donk 2007).Other genes that are necessary for modification, transport, processing, immunity, and regulation are arranged close to the structural genes.The regulation of lantibiotic biosynthesis is done either by a quorum-sensing or by growth phase dependent mechanisms (Chatterjee et al. 2005).Quorum sensing system consists of a receptorhistidine kinase (lanK) and its cognate transcriptional response regulator (lanR) (McAuliffe et al. 2001, Bierbaum & Sahl 2009).The active lantibiotic peptide acts as a triggering molecule which leads to a signal cascade initiated by autophosphorylation of the lanK histidine residue (Kuipers et al. 1995, Schmitzet al. 2006).Subsequently, the phosphate group is transferred to the LanR that often functions as a transcriptional activator that ultimately regulates transcription of the target genes (van Kraaij et al. 1999, Yonezawa & Kuramitsu 2005, Willey & van der Donk 2007).

Classification of Lantibiotics
Recently, lantibiotics are classified on the basis of their biosynthetic pathway (Willey & van der Donk 2007, Knerr & van der Donk 2012).In Class I lantibiotics (eg.nisin), lanB dehydratase catalyses dehydration of Ser or Thr to didehydroalanine (Dha) and Z-didehydrobutyrine (Dhb), respectively.lanC cyclases catalyses the formation of the characteristic lanthionine (Lan from Ser) and methyllanthionine (MeLan, from Thr) thioether crosslinks by intramolecular addition of Cys thiols to Dha/Dhb.Then ABC transporter lanT transports the mature prepeptide and protease lanP cleaves the leader peptide (van der Meer et al. 1993, Meyer et al. 1995).In class II lantibiotic (eg.mersacidin), both dehydration and cyclization steps are catalysed by a single enzyme lanM (Siezen et al. 1996).In class III lantibiotics (Sap B and Sap T), the peptides are modified by RamC.These peptides lack antimicrobial activity; instead they perform morphogenic and signaling functions for the producer cells.Likewise, the labyrinthopeptins are also categorized under the class III lantibiotics, because they are also posttranslationally processed by the RamC-like kinase-cyclase Lab KC enzyme (Meindl et al. 2010).The gene clusters of the labyrinthopeptins also possess two lanT like transporters without a dedicated protease (Müller et al. 2011, Knerr & van der Donk 2012).In class IV lanthipeptides (eg.venezuelin), the peptides are modified by the lanthionine synthetase lanL (Goto et al. 2010).Similar to the enzymes of the class III lantipeptides, it also has a lanT-like transporter, but no dedicated protease or protease domain (Goto et al. 2010, Knerr & van der Donk 2012).

Post-translational modification
Initially lantibiotics are synthesized as inactive prepeptide which is later converted to active peptide by extensive post translational modifications.The post translational modifications include the dehydration of serine and threonine residues to form the didehydro amino acids Dha and Dhb (Fig. 2), respectively (see appendix).This step is catalyzed by modification enzyme (lan B/M/ L/Lab KC).The Dha and Dhb residues later react with the nearby C-terminally located Cys residues to form thioether rings -lan and MeLan, respectively which is catalyzed by lanC/lanM (Tang & van der Donk 2012, 2013).Final step is catalyzed by lanT/lanP, which exports the modified peptide and cleaves its leader peptide to produce an active form.

Mode of action Lantibiotics kill the target cells by different mechanisms (Asaduzzaman & Sonomoto 2009).
Lantibiotics show activity against Gram-positive bacteria while not effective against Gram-negative bacteria (Castiglione et al. 2008).Generally, Gramnegatives are not affected by lantibiotics due to the presence of protective outer membrane which restrict the entry of lantiobiotics into the cytoplasmic membrane.However some lantibiotics like nisin Z can affect Gram negatives -Escherichia coli, Neisseria or Helicobacter pylori at higher concentration.The activity of this lantibiotic might be due two mechanisms: i. the self promotion uptake, ii. the destabilization of the outer membrane by binding of lantibiotics to the lipopolysachharides (Nagao et al. 2009).Similarly, the lantibiotic produced by Bifidobacterium longum DJO10A has a prospect to inhibit members of the Enterobacteriaceae.In addition, microbisporicin from Acinetobacter also exhibited the activity against Gram-negative bacteria such as Moraxella catarrhalis, Neisseria sp., and Haemophilus influenza (O'Sullivan & Lee 2011).The mode of action of lantibiotics is primarily based on two mechanisms: pore formation and inhibition of peptidoglycan synthesis (Brötz et al. 1998b).

Pore formation
Pore formation is one of the important mechanisms of lantibiotics for its antimicrobial activity.The pores formed by the lantibiotics may have the diameter of 2 nm and the pores' half life is about few to several hundred milliseconds (Brötz et al. 1998).Many lantibiotics make a lipid II mediated pores in the target bacteria (Brötz et al. 1998).In this context, lipid II seems to enable the peptides to integrate into the membrane (van Heusden et al. 2002) as well as stabilizes the resulting pores (Breukink et al. 1999, Wiedemann et al. 2004).Nisin is the one in which there has been an extensive study on the mechanism of pore formation.Ramseier in 1960 showed the first evidence on pore formation.He observed a leakage of intracellular compounds from clostridial cells treated with nisin.In addition, Brötz et al. (1998b) and Breukink et al. (2003) also showed that nisin binds to lipid II, using it as a docking molecule to form a stable and efficient pore.Later, Hsu and colleagues described that, nisin binds to the pyrophosphate moiety of the lipid II by five hydrogen bonds (Hsu et al. 2003).This interaction results in the insertion of the elongated C-terminus of nisin into the membrane (van Heusden et al. 2002;Hasper et al. 2004).This insertion causes an efflux of ions and small cytoplasmic compounds that leads to the dissipation of the membrane potential.Nisin is said to cause both mechanisms (pore formation and inhibition of cell wall biosynthesis) for its antimicrobial activity, however, pore formation is the primary cause.This could be explained by the rapid killing of cells by pore formation before inhibition of peptidoglycan synthesis could take place (Brötz et al. 1998b, Wiedemann et al. 2001).Mechanism of pore formation by binding to lipid II is also shown by two peptide lantibiotics viz haloduracin (Oman et al. 2011) and lacticin 3147 (Morgan et al. 2005).In case of lacticin 3147, first A1 peptide binds to the lipid II and forms a complex.Later, the A2 peptide binds to the complex of peptide A1 and lipid II and forms a trivalent complex.This trivalent complex leads to the deeper insertion of the complex into the membrane (Deegan et al. 2006, Wiedemann et al. 2006Oman & van der Donk 2009).This complex arrangement allows the A2 peptide to adopt a transbilayer orientation, thereby resulting in the pore formation.These pores are comparatively smaller (0.6 nm) than those formed by the nisin (Bonelli et al. 2006).Haloduracin also forms pores by similar mechanism as shown by lacticin 3147 (Oman et al. 2011).Streptococcin A-FF22 and Pep5 are example of lantibiotics that can form unstable pores.These pores dissipate the membrane potential but do not release the large molecules from the cell (Kordel et al. 1988, Jack et al. 1994).Likewise, small peptides such as gallidermin and epidermin can also form pores that depend on the thickness of the bacterial membrane (Bonelli et al. 2006).Thus, the antibacterial activity of epidermin and gallidermin may not always be due to the pore formation but could also be due to inhibition of peptidoglycan synthesis (Xiulan et al. 2012).

Inhibition of peptidoglycan biosynthesis
The inhibition of peptidoglycan biosynthesis is another important mode of action of lantibiotics.Lipid I and lipid II are the essential precursors for cell wall biosynthesis (Linett & Strominger 1973).Lantibiotics bind to lipid I and lipid II, the essential precursors for cell wall biosynthesis.This binding makes the transpeptidase and transglycosylase unable to utilize lipid II, which ultimately inhibits the peptidoglycan biosynthesis.Nisin is able to accumulate the lipid II thus inhibits the synthesis of peptidoglycan (Reisinger et al. 1980).The ability of the nisin and epidermin to inhibit the conversion of lipid I to lipid II was described by Brötz et al. in 1998.Likewise, in the nisin-like lantibiotics having conserved two Nterminal lanthionine rings dissipate the lipid II from its functional location in the cell wall biosynthesis complex (Hasper et al. 2006).This dissipation of lipid II results in the accumulation of UDP-linked peptidoglycan precursors in the cytoplasm, thus inhibiting peptidoglycan synthesis (Castiglione et al. 2007(Castiglione et al. , 2008)).Mersacidin, actagardine, and cinnamycin are example of other lantibiotics that also inhibits cell wall biosynthesis by binding to lipid II, specifically bind to transglycosylases (Brötz et al. 1997, Hsu et al. 2003).The lantibiotics of the mersacidin group target the acetylglucosamine moiety and most probably the sugar and phosphate residues of the lipid II.All mersacidinlike lantibiotics possess a conserved TxS/TxEC motif (Hsu et al. 2006, Böttiger et al. 2009).Especially, the Glutamate residue in the motif is essential for the antibacterial action of mersacidin (Szekat et al. 2003).Moreover, the bioactivity of mersacidin is ion dependent, since, the presence of calcium ions increases its activity in vivo.Since lipid II is negatively charged, calcium ions can transfer a net positive charge to the neutral peptides like mersacidin, which might result in enhanced membrane interaction and consequently in deeper membrane insertion (Böttiger et al. 2009).Plantaricin C, a positively charged peptide in which calcium might stabilize the conformation that promotes the membrane insertion.An ion independent mode of action was observed for an uncharged lacticin 481.It contains a positively charged amino acid on its N-terminus that might be sufficient for membrane interaction and formation of lipid II complexes (Böttiger et al. 2009).

Other functions
Many lantibiotics have other biological functions than pore formation and hindrance on peptidoglycan biosynthesis.Lantibiotics like SapT and SapB exhibit a morphogenetic rather than an antibacterial effect.Because of their amphiphilic nature, these peptides serve as the biosurfactants, facilitating the emergence of aerial hyphae (Kodani et al. 2004(Kodani et al. , 2005)).Nisin and Pep5 induce autolysis of certain staphylococcal strains, leading to a break down of the cell wall at the septa of dividing cells (Bierbaum & Sahl, 1987).Likewise, nisin, subtilin and sublancin inhibit the spore outgrowth from Bacillus and Clostridium species (Hurst, 1981, Paik et al. 1998).The lantibiotics of cinnamycin subgroup (duramycin, duramycin B, duramycin C, cinnamycin and ancovenin) shows antibactericidal activity only against a few bacterial strains and Bacillus sp.Specifically, duramycin impairs ATP dependent protein translocation (Chen & Tai 1987) and interferes with calcium uptake (Navarro et al. 1985).In addition, duramycin also blocks the transport of chloride, sodium and potassium (Xie et al. 1983, Stone et al. 1984) and inhibits the proton pump of clathrin coated vesicles (Nakamura et al. 1984).Likewise, nukacin ISK-1, also a lantibiotic, exhibit a bacteriostatic mode of action rather than a bactericidal.It does not affect the membrane potential or form pores, but it reduces the width of the cell wall, causing incomplete formation of the septum, and thus preventing active growth (Assaduzzaman & Sonomoto, 2009).

Application of lantibiotics
Lantibiotics have vast array of application is areas like, food industy, medicine, health care, etc.The characteristics like low molecular weight , a broad range of antimicrobial activity, lack of toxicity and low immunogenicity (Asaduzzaman & Sonomoto, 2009, Dischinger et al. 2014) make them applicable in multiple areas.Nevertheless, lantibiotics need to overcome some obstacles before they can be used in the medical applications.For example, Nisin has few drawbacks such as a low stability at the physiological pH of the gastrointestinal tract, a low solubility, and has the tendency to interact with the blood components (Dischinger et al. 2014).At present there is only one lantibiotic, nisin, which has been used commercially.It has been used as a powerful and safe food preservative in processed dairy products, canned fruits and vegetables since it has no known toxicity to humans (Delves-Broughton 1990).Nisin in addition exhibits an antimicrobial activity against food spoilage bacteria like Listeria monocytogenes (Cotter et al. 2005).It is applied in veterinary medicine as well for the treatment of bovine mastitis (Broadbent et al. 1989).Furthermore, as nisin is effective against clinically relevant human pathogens, like Helicobacter pylori, it might be an effective drug in peptic ulcer treatment too.Moreover, it is also used in treatment of oral decay, enterococcal infections and treatment of enterocolitis (Ryan et al. 2002, Nascimento et al. 2006).Other applications of nisin include the inhibition of experimental vascular graft infections caused by methicillin-resistant Staphylococcus epidermidis (Ghiselli et al. 2004).In addition to its antibiotic effect, nisin also inhibits sperm motility, showing its potential as a contraceptive agent (Aranha et al. 2004).Apart from nisin, many other lantibiotics have also been investigated for their possible applications as antimicrobials.Mersacidin and actagardine show a notable activity against methicillin resistant Staphylococcus aureus (MRSA) infection, bacterial mastitis, oral decay, acne, etc (Limbert et al. 1991).Likewise, gallidermin and epidermin are affective against acne, eczema, follicultis, and impetigo, thus, they might be used for personal care products.Cinnamycin might be used against inflammation and viral infections, and for blood pressure regulation (Ryan et al. 2002).Similarly, Pep5 and epidermin prevent the adhesion of coagulasenegative staphylococci, specifically S. epidermidis, to siliconisated catheters (Fontana et al. 2006).Mutacin 1140 may prevent dental cavities.Duramycin and ancovenin can be used for inflammation and blood pressure regulation respectively.
Lacticin 3147 prevents Propionibacterium acnes from causing acne, thus these substance may also be used as additives in cosmetics and personal care products (Kellner et al. 1988, Lawton et al. 2007).Salivaricins are effective against Streptococcus pyogenes strains.Therefore, the salivaricin A producer was supplemented as a probiotic to milk drinks.Moreover, chewing gums and lozenges containing salivaricin-producing strains have been developed (BLIS Technologies).There are several actinomycete lanthipeptides described having clinical applications.NVB302 (an actagardine derivative) and Moli1901 (also known as lancovutide or duramycin, a structural analogue of cinnamycin) successfully completed Phase I and Phase II clinical trials for the treatment of Clostridium difficle infections (Crowther et al. 2013) and cystic fibrosis (Grasemann et al. 2007, Oliynyk et al. 2010).Likewise, NAI-107 (also known as microbisporin) is in a late stage of preclinical development for the treatment of multi drug resistant Gram-positive pathogens (Jabes et al. 2011).Furthermore, lantibiotics could be used in food products.McAuliffe et al. (1999) and Rodriguez et al. (2001) had described the possible use of starter cultures of Lacticin 3147 or lacticin 481 producing Lactococcus lactis to inhibit Listeria monocytogenes in cheese production.

CONCLUSION
Lantibiotics have a huge potential as alternatives for antimicrobial drugs.Till date, only nisin is used commercially as a food preservative.Although many lantibiotics are under clinical and preclinical trials to explore them as therapeutics, none is yet known to have approved for use as pharmaceuticals.Apart from its use as an agent against multi drug resistant bacteria, it could also be used for personal care products, veterinary medicine, and other biotechnological purpose.Currently, there is a very limited knowledge on mode of action of lantibiotics which yet need to be explored thoroughly.Bioinformatics data with subsequent protein engineering could help discover more lantibiotics.