Abstract
Surface adhesins of pathogens and probiotics strains are implicated in mediating the binding of microbes to host. Mucus-binding protein (Mub) is unique to gut inhabiting lactic acid bacteria; however, the precise role of Mub proteins or its structural domains in host-microbial interaction is not well understood. Last two domains (Mubs5s6) of the six mucus-binding domains arranged in tandem at the C-terminus of the Lp_1643 protein of Lactobacillus plantarum was expressed in E. coli. Mubs5s6 showed binding with the rat intestinal mucus, pig gastric mucins and human intestinal tissues. Preincubation of Mubs5s6 with the Caco-2 and HT-29 cell lines inhibited the binding of pathogenic enterotoxigenic E. coli cells to the enterocytes by 68% and 81%, respectively. Pull-down assay suggested Mubs5s6 binding to the host mucosa components like cytokeratins, Hsp90 and Laminin. Mubs5s6 was predicted to possess calcium and glucose binding sites. Binding of Mubs5s6 with these ligands was also experimentally observed. These ligands are known to be associated with pathogenesis suggesting Mub might negotiate pathogens in multiple ways. To study the feasibility of Mubs5s6 delivery in the gut, it was encapsulated in chitosan-sodium tripolyphosphate microspheres with an efficiency of 65% and release up to 85% in near neutral pH zone over a period of 20 hours. Our results show that Mub plays an important role in the host-microbial cross-talk and possesses the potential for pathogen exclusion to a greater extent than mediated by L. plantarum cells. The functional and technological characteristics of Mubs5s6 make it suitable for breaking the host-pathogen interaction.
Highlights
Mucus layer of mammalian gut protects against pathogens by shedding off the bound bacteria by peristalsis from the gut[1,2]
In this study we report the adhesion of Mubs5s6 protein with different substrata and analyzed the factors which might be involved in its adhesion
The clarified cell lysate supernatant containing the soluble 82 kDa maltose binding protein (MBP)-Mubs5s6 fusion protein when used as sample in anion exchange chromatography led to its partial (~50%) purification (Fig. 1A)
Summary
Mucus layer of mammalian gut protects against pathogens by shedding off the bound bacteria by peristalsis from the gut[1,2]. Antimicrobial resistance has prompted intense research to find non-antibiotic based strategies to counter the pathogens In this context, alternative treatment like microbial interference therapy (MIT) based on adhesion property of probiotics has shown good promise[9]. Mub proteins represented in diverse species[18] is a peculiar surface adhesion protein restricted to only gut inhabiting species. These proteins contain repetitive Mub domains which are presumed to bind the mucin proteins present in the host mucosa. We cloned, expressed and purified last two domains including spacers (referred to as Mubs5s6) with the maltose binding protein (MBP) tag of this 2200 amino acid residues long protein from an indigenous probiotic L. plantarum Lp919,20. To further explore the potential of recombinant Mubs5s6 protein for oral delivery as an antibacterial agent, its stability profile under simulated gastrointestinal conditions was studied and the protective encapsulation was successfully achieved
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