Probiotic mediated NF-κB regulation for prospective management of type 2 diabetes.

Diabetes and other lifestyle disorders have been recognized as the leading cause of morbidity and mortality globally. Nuclear factor kappa B (NF-κB) is a major factor involved in the early pathobiology of diabetes and studies reveal that hyperglycemic conditions in body leads to NF-κB mediated activation of several cytokines, chemokines and inflammatory molecules. NF-κB family comprises of certain DNA-binding protein factors that elicit the transcription of pro-inflammatory molecules. Various studies have identified NF-κB as a promising target for diabetic management. Probiotics have been proposed as bio-therapeutic agents for treatment of inflammatory disorders and many other chronic clinical stages. The precise mechanisms by which probiotics acts is yet to be fully understood, however research findings have indicated their role in NF-κB modulation. The current review highlights NF-κB as a bio-therapeutic target for probable management of type 2 diabetes through probiotic intervention.

Expression of fibronectin-binding protein of L. acidophilus NCFM and in vitro refolding to adhesion capable native-like protein from inclusion bodies.

The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form. To study adhesion of its FBP (64 kDa), the fbp gene of L. acidophilus NCFM was cloned and expressed in E. coli. However, the fibronectin-binding protein expressed in soluble form could not be purified by Ni-NTA affinity chromatography possibly because of partially buried Histidine tag in the recombinant fusion protein. Therefore, the protein was expressed as inclusion bodies (IBs) at 37 °C and solubilized in urea followed by purification in denatured form by Ni-NTA affinity chromatography. The purified denatured protein was refolded in vitro to structurally stable and biologically active form. The conformational properties of the refolded protein were studied by circular dichroism, which showed prominence of α+ ß structural element. The refolded FBP also showed significant binding to human intestinal tissue sections. Our optimized refolding protocol from IBs of this recombinant probiotic FBP led into high amounts of biologically active protein. Our results help in increasing understanding of structure-function relation of surface adhesion proteins and host-microbial interactions.

Expression of recombinant truncated domains of mucus-binding (Mub) protein of Lactobacillus plantarum in soluble and biologically active form.

Mucins amount to 70% of total proteins present in mammalian mucus and serve as important substrata for bacterial adhesion. In probiotic bacteria such as Lactobacillus plantarum, surface adhesion proteins mediate its adhesion to mucus and adhesion is pivotal in bi-directional host-microbe interactions. Mucus binding (Mub) proteins are a group of bacterial surface adhesion proteins that bind to mucin proteins. The structural framework and functional role of these proteins needs immediate attention but is poorly understood because of their large size, low yield and lack of highly purified protein. The lp_1643 gene of L. plantarum encodes a large Mub protein of 240 kDa and has six mucus binding (Mub) domains in tandem. In this study, the fragment of lp_1643 containing the last two domains with their preceding spacers herein referred to as Mubs5s6 was cloned and expressed in E. coli for probing its functional role in the adhesion of L. plantarum. The protein was expressed with a solubility enhancing maltose binding protein (MBP) fusion tag, yet the MBP-Mubs5s6 protein expressed majorly (>90%) as biologically insoluble inclusion bodies. Thus, extensive optimization of culture conditions was carried out to achieve high level soluble expression (∼70%) of Mubs5s6 protein from its initial low level of solubility. The recombinant protein was purified up to homogeneity by affinity chromatography. Recombinant MBP-Mubs5s6 protein showed strong adhesion potential by binding with human intestinal tissue sections. Our results show a step-by-step hierarchical approach to improve the solubility of difficult-to-express extracellular surface proteins while retaining high functional viability.

Indian sewage microbiome has unique community characteristics and potential for population-level disease predictions.

Sewage wastewater pollutes water and poses a public health issue but it could also prove useful in certain research domains. Sewage is a complex niche relevant for research concerning 'one-health', human health, pollution and antibiotic resistance. Indian gut microbiome is also understudied due to sampling constraints and sewage could be used to explore it. Ostensibly, Indian sewage needs to be studied and here, we performed a cross-sectional pan-India sewage sampling to generate the first comprehensive Indian sewage microbiome. Indian sewage showed predominance of Burkholderiaceae, Rhodocyclaceae, Veillonellaceae, Prevotellaceae, etc. and has high representation of gut microbes. The identified gut microbes have overrepresentation of Veillonellaceae, Rikenellaceae, Streptococcaceae, and Bacillaceae. Imputed metagenomics of sewage microbiome indicated dominance of transport, motility, peptidases, amino acid metabolism, and antibiotic resistance genes. Microbiome-disease associations drawn using simple decision tree and random forest analysis identified specific microbes as potential predictors of diabetes and obesity in a city. Altogether, we generated the first Indian sewage microbiome and our non-invasive, high-throughput workflow could be emulated for future research, wastewater-based epidemiology and designing policies concerning public health.

Host-microbe interaction and pathogen exclusion mediated by an aggregation-prone surface layer protein of Lactobacillus helveticus.

Probiotic surface layer proteins (Slps) have multiple functions and bacterial adhesion to host cells is one of them. The precise role of Slps in cellular adhesion is not well understood due to its low native protein yield and self-aggregative nature. Here, we report the recombinant expression and purification of biologically active Slp of Lactobacillus helveticus NCDC 288 (SlpH) in high yield. SlpH is a highly basic protein (pI = 9.4), having a molecular weight of 45 kDa. Circular Dichroism showed a prevalence of beta-strands in SlpH structure and resistance to low pH. SlpH showed binding to human intestinal tissue, enteric Caco-2 cell line, and porcine gastric mucin, but not with fibronectin, collagen type IV and laminin. SlpH inhibited the binding of the enterotoxigenic E. coli by 70 % and 76 % and that of Salmonella Typhimurium SL1344 by 71 % and 75 % to enteric Caco-2 cell line in the exclusion and competition assays, respectively. The pathogen exclusion and competition activity and tolerance to harsh gastrointestinal conditions show the potential for developing SlpH as a prophylactic or therapeutic agent against enteric pathogens.

Milk-Derived Antimicrobial Peptides: Overview, Applications, and Future Perspectives.

The growing consumer awareness towards healthy and safe food has reformed food processing strategies. Nowadays, food processors are aiming at natural, effective, safe, and low-cost substitutes for enhancing the shelf life of food products. Milk, besides being a rich source of nutrition for infants and adults, serves as a readily available source of precious functional peptides. Due to the existence of high genetic variability in milk proteins, there is a great possibility to get bioactive peptides with varied properties. Among other bioactive agents, milk-originated antimicrobial peptides (AMPs) are gaining interest as attractive and safe additive conferring extended shelf life to minimally processed foods. These peptides display broad-spectrum antagonistic activity against bacteria, fungi, viruses, and protozoans. Microbial proteolytic activity, extracellular peptidases, food-grade enzymes, and recombinant DNA technology application are among few strategies to tailor specific peptides from milk and enhance their production. These bioprotective agents have a promising future in addressing the global concern of food safety along with the possibility to be incorporated into the food matrix without compromising overall consumer acceptance. Additionally, in conformity to the current consumer demands, these AMPs also possess functional properties needed for value addition. This review attempts to present the basic properties, synthesis approaches, action mechanism, current status, and prospects of antimicrobial peptide application in food, dairy, and pharma industry along with their role in ensuring the safety and health of consumers.

Allosteric regulation of glycogen breakdown by the second messenger cyclic di-GMP.

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria.

Mechanistic insights into the host-microbe interaction and pathogen exclusion mediated by the Mucus-binding protein of Lactobacillus plantarum.

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.