MOLECULAR INVESTIGATIONS AND APPLICATIONS OF SELECTED GRAS MOLECULES FOR ANTIBACTERIAL AND ANTICANCER EFFECTS

Abstract

The evolution of antibiotic resistant pathogens and slow-paced discovery of antibiotics have led to the requirement of new antibacterial therapies. Indiscrete and disproportionate use of antibiotics in livestock and poultry is contributing immensely to the development of antibiotic resistance in bacterial species associated with food contamination. In the light of exponential increase in bacterial antibiotic resistance, use of natural products (generally recognised as safe) in combination therapy can be a potent alternative strategy to restrain foodborne illnesses and contaminations. Bacterial strains belonging to the genera such as Vibrio, Salmonella, Escherichia and Listeria are major pathogens implicated in foodborne and gastrointestinal diseases. These organisms are a constant threat to mankind, and the spread of antibiotic resistance has compounded the problems. To strategize a combination for antimicrobial activity, traditionally used antimicrobial compounds were targeted in this study. Vitamins, metals and essential oils are well known for their antimicrobial activity, and have been used as food additives for improvement of metabolism in humans. The pro-oxidant properties of essential oil linalool and vitamin C in combination with copper (LVC) were exploited for combating multiple antibiotic resistant Vibrio fluvialis and a common foodborne pathogen Salmonella typhi, using 3-D checkerboard microdilution assay. Antibacterial propensity in terms of minimum inhibitory concentration revealed that the triple combination exerted synergistic effect (Fractional Inhibitory Concentration Index-0.48) as compared to individual constituents. The bactericidal effect of the triple combination was confirmed by Live/dead staining assay. Reactive oxygen species (ROS) measurements together with TUNEL assay and scanning electron microscopy imaging strongly suggested that the increase in ROS production upon the treatment is the underlying mechanism of enhanced antibacterial potency of the LVC combination. In addition, hypersensitivity of oxidative stress regulator mutants (oxyR, katG, ahpC and sodA) towards LVC corroborated the involvement of ROS in cell death. Furthermore, the LVC combination did not display toxicity to human cell lines as confirmed by MTT assay and could effectively reduce pathogens in food beverages. Antimicrobial peptides (AMP) are commonly used GRAS status constituents, utilized in food preservation and antimicrobial therapies. Similar to the antibiotics, AMPs are challenged with target modified resistant organisms, which abates the efficacy of the peptides. To address ii the problem, a food grade antimicrobial peptide; pediocin was bioconjugated with silver nanoparticles (AgNPs), to enhance the activity and antimicrobial spectrum of pediocin. Pediocin-AgNPs (PdSNPs) showed enhanced antimicrobial activity against foodborne pathogens; Listeria monocytogenes (13 folds) and V. fluvialis (10 folds) in comparison to citrate capped AgNPs (CSNPs) and pediocin, separately. The bactericidal effect of PdSNPs was confirmed by kill-kinetics and Live/ dead staining. Upon treatment with PdSNPs, both the pathogens displayed depolarised and damaged membrane, increased ROS, lesions in DNA and rapid permeabilization through outer membrane. It is hypothesised that PdSNPs could kill bacteria by effectively attaching to the cells, penetrating the membrane, and causing oxidative damage and leakage of ions. It was further demonstrated that PdSNPs could effectively eradicate mature Listerial biofilm, thus can contribute immensely in food and food manufacturing units. In addition, PdSNPs did not display any toxicity to mammalian cells and RBCs, thus could be effectively used as food preservative and in antimicrobial therapy. Hence, the present study, opens a new approach for combinatorial antimicrobial therapy with food grade constituents which may be promoted as effective preservatives and alternatives for antibiotics. GRAS status organisms and their bioactive compounds were also examined for their anticancer properties. One of the most important biotherapeutic proteins employed in leukemia is asparaginase. Conventionally used asparaginases are derived from pathogenic organisms, hence, have various side effects and toxicity. Thus, in order to mitigate the problems with conventional asparaginases, there is a desperate requirement for safer enzymes from benign sources. In this study, asparaginase (AnsLb) from Lactobacillus brevis NKN55 (a probiotic origin bacterium) was screened, isolated, cloned and expressed in E. coli BL21-AI followed by affinity purification. The purified protein was found to be a homodimer of 70 kDa, with monomeric size of approximately 35 kDa. The enzyme showed optimum activity between pH 7.0–8.5 and temperature 35–45°C, which is suitable for human use. AnsLb exhibited no inhibition with monovalent and divalent metal ions, which depicts it to be a cofactor independent protein. The apparent Km, Vmax and Kcat values were 0.833 mM, 4.12 mM/min and 3.045×10-3 sec-1 respectively. Structural studies using CD spectroscopy confirmed change in helical structure of the protein upon substrate (L-asparagine) binding. As a part of its utilisation in humans, antileukemic and acrylamide mitigation studies were performed. Antileukemic potential of AnsLb was explored using murine myeloid leukemic cells, where the enzyme displayed effective inhibition of the cancer cells. The IC50 value of AnsLb was iii determined to be 11.6 IU, which is comparable with other isolated asparaginases. AnsLb also exhibited acrylamide lowering property in fried potatoes, where 15 IU/ml could reduce almost 80% of acrylamide in 5 g of fried potatoes. AnsLb did not show any haemolytic activity, thus has been proved to be safe for human use. In order to increase the utilization spectrum of the foodgrade asparaginase AnsLb, a better thermotolerant mutant was generated by site specific mutagenesis, which displayed an increase of 5°C in melting temperature as observed in CD spectroscopy. Till date very few reports are available on asparaginases from lactic acid bacteria, and thus this finding can be a substantial step towards discovering safe, foodgrade antileukemic bioactive molecules. Overall, the work concludes utilization of food based bioactive molecules for inhibition of pathogenic bacteria and development of two food grade antibacterial regimens and their mode of actions for bacterial inhibition. An anticancer molecule was also screened and it's applicability for commercial utilization was also explored.