ROLE OF LACTOSMART AS A NOVEL THERAPEUTIC AGENT IN ANTIMICROBIAL DEFENSE

Abstract

The emergence of multi – drug resistance (MDR ) in microorganisms against antibiotics has become a global problem [1,2,3]. Various conventional drugs with promised efficacy and specificity are unable to withstand the threat of antibiotic drug resistance [4,5,6]. The rising crisis of MDR bacteria has led to the channelization of relevant research in the direction of antimicrobial molecules from natural sources as potential novel antibiotics. The spectrum of innate immune proteins and their potent fragments herald a promising approach to fight the problem of drug resistance. Among the natural antimicrobial proteins, Lactoferrin (LF) has been identified as a potent host defense system based on its wide spectrum bactericidal and bacteriostatic activities [7,8,9,10,11,12,13] . In the past , several studies have demonstrated the antibacterial and antifungal effects of LF and its derivative peptides, for instance, lactoferricin B [14,15,16,17,18,19] and lactoferrampin [20,21]. Structurally, LF consists of two iron bound lobes, N -lobe (1-333) and C -lobe (345-692) [22,23,24,25]. Amongst the two lobes , the highly cationic properties of N- lobe are responsible for membrane disruption by interacting with anionic components present on bacterial surface [26,27]. It has been established that the lipid A component of the LPS is a known drug target for antimicrobial therapeutics [ 28,29]. One of the mechanisms by which Lf acts as an antimicrobial agent is through binding to pathogen associated molecular patterns (PAMP) such as Lipopolysaccharide (LPS), thereby disrupting the bacterial membrane integrity and activating the chemical signaling pathway[30- 32]. This leads to the secretion of pro- inflammatory responses which down regulates the release of cytokine production [33,34]. In the past, it had been reported that LF binds to LPS with its hexameric sequence present in the 18 - loop region of the lactoferricin [35-37] . In the present study , we have performed the partial digestion of LF with trypsin which generates a potent antimicrobial molecule of the size of about 21kDa (85-281). We have proposed its name as Lactosmart due to its higher potency against pathogens when compared to native LF as a whole protein . The lactosmart has been tested for antibacterial and antifungal properties along with its inhibitory potential of biofilm formation by Pseudomonas aeruginosa through established assays [41]. Our primary focus was on the comparison of LPS binding properties of lactosmart with native LF using surface plasmon resonance technique . The docking and molecular dynamics simulations (MD) studies with LPS have also been performed to further substantiate our claims. Through our studies , we have demonstrated that LF sequesters LPS through two binding sites which are situated on the N- lobe.