Review Article

Microbial Lipopeptides and their Medical Applications

Khem Raj Meena, Abhishek Sharma and Shamsher S. Kanwar*
Department of Biotechnology, Himachal Pradesh University, India


*Corresponding author: Shamsher S. Kanwar, Department of Biotechnology, Himachal Pradesh University, Shimla, India


Published: 08 Nov, 2017
Cite this article as: Meena KR, Sharma A, Kanwar SS. Microbial Lipopeptides and their Medical Applications. Ann Pharmacol Pharm. 2017; 2(21): 1111.

Abstracts

Many bacterial species are capable of producing small peptides linked to lipidic moiety and interestingly these lipopeptides (LPs) possess antimicrobial activities against fungal and/ or bacterial pathogens. A number of novel bacterial lipopeptides (s) have been screened and identified predominantly from Bacillus spp. for their potential therapeutic applications for the human welfare. The researches on the bacterial LPs make them novel antibiotics to combat the diseases of animals, human as well as plants. Microbial lipopeptides are about ~ 1.0-1.2 kDa molecules which have some specific/unique physicochemical and biochemical properties that put them in the category of antibiotics. In the context of biological control, three families of LPs i.e. Surfactins, Iturins and Fengycins have been studied for their potent antagonistic activities against various human as well as plant diseases. Therefore, these lip opeptides are being considered as alternatives to the combat pathogens which are resistance to conventional antibiotics and hence cause life-threatening diseases. Besides antimicrobial properties, these LPs exhibit antiparasitic, antithromolytic, antiviral, haemolytic, antitumor and anticancer activities with ability to trigger apoptosis/ programmed cell death in the malignant cells. The bacterial LPs have lower toxicity for animals& humans, high biodegradability, low irritancy and good compatibility with human skin.
Keywords: Lipopeptides; Surfactin; Iturin; Fengycin; Antibacterial; Antifungal; Antitumor activities


Introduction

The members of the genus Bacillus are recognized as microbial factories for the extracellular production of lipopeptide-like low molecular mass bioactive molecules [1-4]. Microorganisms produce a wide variety of bioactive chemical compounds during their growth [5]. The demand of lipopeptides (LPs) is surging by leaps and bounds due to their utility for the human welfare [2]. Bacillus subtilisproduces mainly three types of LPs (Figure. 1) with broader biotechnological and biopharmaceutical applications [6]. Among the Bacillusspp. lipopeptide classes; Iturin and Surfactin remain attractive antibiotics for their antifungal and antibacterial applications [6]. The Mycosubtilin, Polymyxin etc are also examples of LPs [7,8]. Amongst various types of LPs (Table 1), Iturin containing an array of 7 amino acids has a molecular mass of ~1.04 kDa and possesses a strong antifungal activity. In contrast, the Surfactin (~1.03 kDa), a cyclic heptapeptide antibiotic also contains seven amino acids Glu-Leu-Leu-Val-Asp-Leu-Leu (ELLVDLL)and exhibits a strong bio surfactant activity. The seven amino acids of the Surfactin linked with β-hydroxy fatty acid chain involve C12 to C16 carbon atoms to form a cyclic lactone ring structure [9]. Surfactin shows an array of biological activities such as antiviral, haemolytic, antimycoplasma and antibacterial activities. These therapeutic properties of the Surfactin make it a prime drug to resolve a number of global issues for the human welfare, in the context of medicine [10,11], industry [12] and in the environmental protection [13]. Fengycins (Plipastatins A and B)comprise third family of the LPs. The Fengycins contain a peptide chain of 10 amino acids with a lactone ring and a β-hydroxyl fatty acid chain (C14 to C18), which may be saturated or unsaturated. The bacterial LPs have low irritancy, high biodegradability, lower toxicity and good compatibility with human skin [14].
Biomedical and therapeutic applications of LPs
Among several categories of bio surfactants, LPs are particularly interesting because of their high surface activities and antibiotic potential against an array of pathogens. The first lipopeptide Polymyxin A, was discovered and isolated in 1949 from a soil bacterium Bacillus polymyxa [15]. CubicinR (Daptomycin), the first cyclic lipopeptide antibiotic was approved in USA by Food and Drug Administration (FDA) for the treatment of serious blood and skin infections caused by certain Gram-positive microorganisms [16]. Members of theBacillus genusare considered dedicated microbial factories for the large scale production of such type of bioactive molecules [4, 17 and 18]. Surfactin and its derivatives also act as antiviral agents, antibiotics, antitumor agents, immunomodulators or specific toxin-inhibitors (Table 2). Conjugates of LPs and Th cell epitopes also constitute effective adjuvants for in vitro immunization of either human mononuclear cells or mouse B cells and result in an increased yield of antibody secreting hybridoma. Actinobacteriaspecies of the genus Streptomyces are also reported to produce diverse antimicrobial lipopeptides (s) with their applications in pharmaceutical industries [19]. Another lipopeptide (Polymyxin) binds to LPS by electrostatic interaction through its N-terminal fatty acyl tail and targets for its anti-bacterial activity [20]. Synthetic lipopeptides are widely used as vaccine adjuvant to enhance immune response but bacterial-derived recombinant lipopeptides such as Lipo-Nter, is a novel adjuvant that can be used to induce superior anti-tumor effects as compared to a synthetic lipopeptides(s) [21].
Mycoplasma is a smallest free-living organism and parasite of eukaryotic cells and is also one of the major contaminants that affect mammalian tissue cultures. Mycoplasmas are serious causative agents of diseases of both humans and animals, such as acute respiratory inflammation (including pneumonia), diseases of the urogenital tract and in the pathogenesis of AIDS [22]. Treatment with antibiotics is a most effective procedure for eliminating or suppressing mycoplasma infection in the cell cultures. The principal representative of the lipopeptide family is Surfactin, which is produced by abacterium Bacillus subtilis. Surfactin shows remarkable membrane-active and surface-interface properties resulting in a number of excellent biological activities, which are of great relevance in healthcare and biotechnology-based processes. Surfactin is used commercially for curing of cell cultures and cleansing of biotechnological products of mycoplasma contamination [23].The general, antibiotic therapies are successful in long lasting decontamination and do not show undesirable side effects/ cytotoxic effects on eukaryotic cells. Surfactins have versatile bioactive properties with significant anti mycoplasma activity [24]. The disintegration of mycoplasma is obviously due to a physicochemical interaction of the membrane active Surfactin with the outer part of the lipid membrane bilayer, which causes permeability changes and at higher concentrations leads finally to disintegration of the mycoplasma membrane system by its detergent-like effect.
Cyclic LPs: Potent mosquito larvicidal agent(s)
Mosquitoes are blood feeding insects which serve as vectors for spreading human diseases such as malaria, yellow fever, encephalitis, West Nile fever, lymphatic filariasis etc. The culture cell-free broth of a Surfactin producing B. subtilis strain was found to effectively kill the larval and pupal stages of mosquito species such as Culexquinquefasciatus, Anopheles stephensi and Aedesaegypti. As some biocontrol agents or insecticides are effective against mosquito pupae, this could be a good tool for application in malaria control programmes [32]. Further, growing public awareness about the environmental and human risk associated with chemical pesticides, emergence of pesticide-resistant insect populations as well as rising prices of chemical pesticides has invariably stimulated the search for new eco friendly vector-control biological tools [33]. In this context, several biological control agents have been tested in India and in many other parts of the world to evaluate their potential to control the mosquito vectors. Toxins from certain strains of bacteria, like Bacillus thuringenesis var. Israelensis(Bti) and B. sphaericues(Bs) have been shown to be highly effective against mosquito larvae at very low dosage and they are also safe to non-target organisms. However, the biolarvicide formulation from Bs strain is reported to be less effective against Anophelesculicifacies and appeared hardly effective against Aedesaegypti [34]. A potential key strategy for delaying resistance to mosquitocidal proteins is to use a mixture of toxins that acts at different targets within the insects [35].
Antiparasitic activity of Surfactin
Microsporidia are defined as highly specialized fungi. Nosemaceranae is one of the etiologic agents of nosemosis, a worldwide disease [35]. Surfactin is considered as a molecule capable of reducing parasitosis development, acting either by direct exposure on to spores or by its incorporation in the luminal of beemidgut [36]. Surfactin functions as a competitive inhibitor of NAD+ and an uncompetitive inhibitor of acetylated peptide(s). Surfactin was also found to be a potent inhibitor of intra-erythrocytic growth of P. falciparumin vitro [37]. Surfactin can also be used as alternative treatment for nosemosis. When exposed to Surfactin, the spores of Nosemaceranae, the causative agent of parasitic infection in Apismellifera, revealed a significant reduction in infectivity [36]. Moreover, when Surfactin is administered and is introduced into the digestive tract of a bee, it also leads to a reduction in parasitoids development [36].
Antiviral activity of Surfactin
Surfactin is also active against several viruses, including the Semliki Forest virus, Herpes simplex virus (HSV-1 and HSV 2), Vesicular stomatitis virus, Simian immunodeficiency virus, Feline calicivirus and the Murine encephalomyocarditis virus. The length of the carbon chain in cyclic Surfactin lipopeptide influences its capacity for viral inactivation [38]. The inactivation of enveloped viruses, especially herpes viruses and retroviruses by Surfactin is significantly more efficient than that of non-enveloped viruses. This suggests that the antiviral action of Surfactin is primarily due to the physicochemical interaction between the membrane active surfactant property of Surfactin and the virus lipid membrane. One important factor for virus inactivation is the number of carbon atoms in the acyl chain of Surfactin. The capacity for virus inactivation increases with rising fatty acid hydrophobicity. During the inactivation process of viruses, Surfactin permeates into the lipid bilayer thereby inducing complete disintegration of the envelope containing the viral proteins involved in virus adsorption and penetration of the target cells. Its absence accounts for the loss of viral infectivity. Thus Surfactins have demonstrated antiviral activities [29].It has also been observed that antimicrobial LPs containing Surfactin inactivate the Porcine parvovirus, Pseudo rabies virus, Bursal disease virus and Newcastle disease virus [39] in the cell-free state.
Antitumor activity and induction of apoptosis by ROS/JNK pathway by Surfactin
Surfactin is a potent lipopeptide considered as a versatile bioactive molecule with antitumor activity [29].Surfactin has been reported to show antitumor activity against Ehrlich’s ascites carcinoma cells. The effect of Surfactin as cytotoxic agent on the proliferation of a human colon carcinoma cell lines such as HCT-15 and HT29 [40] has also been reported. The inhibition of growth of transformed cells by Surfactin was due to the cell-cycle arrest and induction of apoptosis via the suppression of cell survival regulating signals such as ERK and PI3K/ Akt [41]. The percentage of viable cells decreased with increasing Surfactin concentrations and exposure time that indicated its cytostatic/ cytotoxic effect against breast cancer cell lines like T47D and MDA-MB-231 [42]. Another study revealed that Surfactin inhibits proliferation and also induces apoptosis of human breast MCF-7 cancer cells trough a ROS/ JNK-mediated mitochondrial/ caspase pathway (Figure. 2) in a dose-dependent manner [43]. Surfactin generates the reactive oxygen species (ROS), which activate the mediator of survival and JNK and ERK1/ 2, which are the key regulators in the apoptosis process. This showed that the action of Surfactin seems to be realized via two independent signalling mechanisms [11]. The induction of apoptotic cell death is an emerging strategy for the prevention as well as treatment of cancer.
LPs induced apoptotic pathway
Oxidative stress induced by the LPs leads to the production of ROS in LPs-treated cancer cells. This oxidative stress further results in induction of apoptosis in the cell, as evident by the fragmentation/ condensation of nuclei. Another marker of apoptotic cell death is DNA nicking, which is indicated by FACS- based TUNEL assay. Extensive nicking of DNA depends on the concentration of the LPs. However, surprisingly, caspase-3 band could not be detected using biotin-conjugated polyclonal rabbit anti active human caspase-3 antibody. Further, other important biomolecules reported to be involved in apoptosis such as PARP-1, Apoptosis-Inducing Factor (AIF) and cytochrome-C were also assayed. PARP-1 is a nuclear enzyme that regulates transcription under homeostatic conditions while during stress, it responds to DNA damage and facilitates DNA repair. In caspases independent death processes, some researchers have shown that PARP-1 plays an important role of initiator, activation of which is caused by DNA damage [44]. Many agents that cause DNA damage lead to PARP-1 activation. Some of these agents include H2O2, DNA-alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and a neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Most of these agents generate ROS that lead to direct DNA damage. It is reported that oxidative stress/ ROS lead to activation of both PARP-1 and AIF. In one of the pathways authors have shown that excessive activation of PARP-1 leads to an intrinsic caspase-independent cell death program, in which PAR polymer appears to be a pro-death signalling molecule that acts as a nuclear/ mitochondrial signal to release AIF from the mitochondria (Figure 3). Once released, AIF is translocated to the cytoplasm and nucleus, where it induces chromatin condensation [45]. Subsequent to its own expulsion from mitochondria, AIF also triggers release of mitochondrial cytochrome C and sometimes caspase activation [45]. In one of the experiments with cell-free systems, incubation of isolated HeLa nuclei with recombinant AIF resulted in peripheral chromatin condensation and DNA loss was associated with high molecular weight (50 kb) DNA fragmentation [46]. AIF released from mitochondria and subsequent cell death was shown to be triggered by excessive cellular calcium influx resulting in an over-activation of poly (ADP-ribose) polymerase-1 [47].
Thrombolytic activity of Surfactin
The plasminogen-plasmin system involves in the dissolution of blood clots in a variety of pathological and physiological processes requiring proteolysis. Zymogen plasminogen is proteolyticallyactivated by urokinase-type and tissue type plasminogen activator [38].Activation of plasminogen and prourokinase is an important mechanism in the initiation as well as propagation of fibrinolytic activity. Surfactin at concentrations of 3–20 μ moL/L enhanced activation of prourokinase and also led to conformational change in the plasminogen that further increased fibrinolysis in vitro and in vivo [48]. In a rat pulmonary embolism model, Surfactin C increased the lysis of plasma clot, when injected in the combination with prourokinase. Surfactin was also able to prevent platelet aggregation, led to inhibition of additional fibrin clot formation [22] and also enhanced fibrinolysis with the facilitated diffusion of fibrinolytic agents [49]. Detergent property of Surfactin has little role in the antiplatelet activity but it appeared to be caused by action on downstream signalling pathways [50]. Moreover, Surfactin has advantages over other thrombolytic agents because it has fewer side effects and therefore it has potential for long-term use as a clot-bursting agent.
Antiobesity activity of the LPs
Obesity is considered as an exceeding life style disorder especially in developing countries. It is prevailing in new world countries as a result of fast food intake, including high fructose corn syrup added products consumption and lack of physical activity [51]. Pancreatic lipase inhibitory activity has been largely used for the exploration of potentially effective natural products [52]. Lipopeptides as a unique class of bio-surfactants have recently emerged as promising molecules owing to their structural novelty, versatility and diverse properties that are useful for advanced therapeutic applications [53]. Bacillus subtilis SPB1 lipopeptide may be a major drug of future to treat the obesity-related metabolic disorders. B. subtilis lipopeptides can be administered orally, in order to achieve an effective control on body weight [54]. Bacillus subtilis SPB1 crude lipopeptide biosurfactant has both protective and curative action on obese persons and it reduced the body weight of obese rats and thus appeared to treat hyperlipidemia without apparent side effects. Bacillus subtilis LPs reduced the body weight of mouse by reducing the serum pancreatic lipase activity [54].
The LPs no doubt appears to a novel class of antibiotics which astonishingly exhibit a wide variety of surfactant, antibacterial, anti mycoplasma, antifungal, antiviral, antiparasitic, antilarval, antithrombocytic and antitumor/ anticancer activities. Among bacterial genera, the Bacillus spp. are prominent producers of extracellular LPs. However, to commercially employ LPs as potent antibacterial or antifungal molecules, they need to be produced in adequate quantities by cloning of appropriate gene(s) in the efficient expression vectors. Targeting of tumour/ cancer cells efficiently could be achieved by their conjugation with tumour specific surface exposed binding molecules. The low molecular mass, cell membrane compatibility, eco-friendliness and bio-degradability make them an interesting class of antibiotics.


Table 1

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Table 1
Distinctive properties of lipopeptides.s

Figure 1

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Figure 1
Three prominent types of lipopeptides.

Figure 2

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Figure 2
Proposed mechanisms involved in the antitumor activity of Surfactin. Abbreviations: AIF, apoptosis-inducing factor; Cyt c, cytochrome c; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase and ROS, reactive oxygen species.

Figure 3

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Figure 3
Lipopeptide(s)-induced apoptosis pathway.

Acknowledgements

This work has been funded by Department of Biotechnology, Ministry of Science and Technology, New Delhi under a DBT-JRF Fellowship grant awarded to one of the authors (KRM) vide a Letter No. DBT-JRF/2011-12/270. The authors are thankful to BTIS, Department of Biotechnology, Ministry of Science and Technology, New Delhi and Department of Biotechnology, Himachal Pradesh University, Shimla for the financial support for this work.

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