CHARACTERIZATION AND A DETAILED MECHANISTIC STUDY OF TYPE 1 LIPID TRANSFER PROTEIN FROM CITRUS SINENSIS

Abstract

Small proteins and peptides are a unique class of biomolecules having diverse functional properties and biotechnological applicability. The high specificity, selectivity, diversity and low host toxicity present small proteins and peptides as a promising alternative to the existing chemical drug treatments. Therapeutic proteins have become a growing field of interest to the scientific world due to the advancements in protein biochemistry, drug discovery, and medicine. These bioactive molecules represent a wide range of properties like antimicrobial, anticancer, antihypertensive, antioxidant, translation inhibitory potential, enzyme inhibitors, and insecticidal properties, which can be utilized for the treatment of plant diseases as well as human health care. The small proteins and peptides are naturally synthesized constitutively in plants, insects, animals, aquatic flora, and fauna or as a response to biotic and abiotic stress. Plants are the major source of unexplored small bioactive proteins/peptides. Continual exposure to various pathogens, insect pests, and viruses has resulted in the co-evolution of land plants by developing immune strategies. The plant immune system is a vast and complex network interconnected by diverse pathways involving signal molecules, defense proteins, enzymes, and phytochemicals. Plant defense proteins are a crucial set of proteins that act directly as antimicrobial agents or indirectly as signal molecules for activating the defense pathways and enzyme regulation. Plant defense proteins encompass a wide variety of proteins like defensins, protease inhibitors, thoinins, chitinases, endonuclease inhibitors, Lipid Transfer Proteins, pore-forming peptides, antioxidant proteins, ribosome-inactivating proteins, etc. It is a pre-requisite to study these bioactive proteins and their underlying mechanisms in order to understand their potential applications in plant disease control and human health. Non-specific Lipid Transfer Proteins belong to the protease/α-amylase inhibitor superfamily and are also known as Pathogenesis Related Protein -14 (PR-14). These are the basic, cytosolic, secretory proteins present in plant leaves, anthers, stems, bark, and fruit seeds. The ubiquitously present protein in different plant parts and their differential expression patterns at developmental stages signify its important role in plant physiology and defense. Non-specific Lipid Transfer Proteins are classified into five major subtypes namely LTP1, LTP2, LTPc, LTPd, LTPg and minor subtypes LTPe, LTPf, LTPh, LTPj and LTPk. These are the cysteine-rich, small-sized proteins classified based on the position of conserved introns, the amino acid sequence identity, and the spacing between the Cys residues. The role of LTPs was primarily hypothesized to be the transfer of lipids in plants as suggested by its name, but the presence of signal peptide directed their role in other physiological processes also. Since the discovery of LTP1, it has been observed to play a quintessential role in the development of anther, leaf elongation, plant defense, somatic embryogenesis, signal transduction, β-oxidation, and cuticle synthesis. The detailed understanding of their role in plant physiological processes and defense is yet to be understood which may give a novel perspective for the advancements in disease control. The present work includes the recombinant expression, characterization and functional studies of Type1 non-specific Lipid Transfer Protein from Citrus sinensis (CsLTP1). The physicochemical characterization identified the higher thermal and pH stability of the CsLTP1 along with the lipid binding phenomenon, indicating a higher affinity towards unsaturated lipids. The functional characterization revealed antimicrobial potential against bacterial and fungal pathogens by analyzing the 50% inhibitory concentrations, growth curve inhibition, and scanning electron microscopic observations to understand the effect of CsLTP1 on the physiology of pathogenic cells. Further, the in-planta antimicrobial potential of CsLTP1 by exogenous plant treatment against Xanthomonas oryzae affects rice crops, causing bacterial blight disease. The study identified the reprogramming of crucial metabolic pathways in rice crops as an effect of exogenous CsLTP1 treatment controlling the bacterial blight disease employing the Gass Chromatography-Mass spectrometry (GC-MS) technique. The insecticidal property of CsLTP1 has been evaluated against the polyphagous lepidopteran pest, Helicoverpa armigera, affecting cotton crop production. The significant inhibition of larval mid-gut α-amylase by CsLTP1 has been observed by using the DNSA method. The adverse effect on the growth and development of Helicoverpa larva and its potent insecticidal activity was identified by the in-vivo diet incorporation method. The study gave valuable insights into the potential application of CsLTP1 as an antimicrobial and insecticidal protein. Considering the diverse functional properties of CsLTP1, it also possesses a potential role in developing effective drug delivery systems for the treatment of human disease. The screening and evaluation of the drug delivery potential of CsLTP1 identified strong drug-binding properties of CsLTP1 and its transport into the cancer cells. It activated the intracellular oxidative stress and apoptosis of cancer cells. This work gives a detailed understanding and characterization of CsLTP1 protein, emphasizing its potential therapeutic applications in crop protection as well as drug delivery applications. Chapter 1 encompasses the journey and advancements towards the development of small proteins and peptides as therapeutics. It also includes the role of bioactive defense proteins/peptides from plants and their potential applications. Further, a detailed literature review of nsLTP1 proteins, classification, their role in plant physiology and defense, proposed mechanisms, and potential biotechnological applications have been performed. Chapter 2 describes the differential gene expression study of CsLTP1 in healthy and Candidatus Liberibater asiaticus (Clas) infected citrus plants. Thereafter, the chapter includes recombinant expression CsLTP1 in the prokaryotic system, purification, characterization and lipid binding studies of CsLTP1 using in-silico and in-vitro approaches. The gene expression study revealed the enhanced expression of CsLTP1 in the leaves of Clas-infected plants compared to the healthy control. The results highlighted the probable role of CsLTP1 in activating plant defense as a response to biotic stress. The CsLTP1 gene isolated from Clas-infected Citrus sinensis plants was successfully cloned into the pET32a vector, and recombinant expression was performed in Rosetta gami-2. The CsLTP1 with His-Thioredoxin tag was purified using Ni-NTA chromatography. The His-Thioredoxin tag was cleaved by TEV protease-mediated digestion in a dialysis system. The CsLTP1 was further purified using anion and cation exchange and size exclusion chromatography. The ~9.1 kDa CsLTP1 indicated higher thermal stability employing Differential Scanning Calorimetry. The Far-UV Circular Dichroism study identified the changes induced in the secondary structure of the protein at temperature ranges from 20 to 90 ℃ and pH from 3 to 11. The lipid binding study using in-silico and in-vitro investigations revealed a higher binding affinity of unsaturated lipids towards CsLTP1. Chapter 3 describes the functional characterization of CsLTP1. The work incorporates the evaluation of antimicrobial activity of CsLTP1 by in-vitro and in-planta experiments. The evaluation of larval mid-gut α-amylase inhibitory activity has also been studied, followed by the insect bioassay to assess the growth inhibitory potential against Helicoverpa armigera which severely affects the cotton crops. The study identified the potent antimicrobial activity of CsLTP1 against bacterial and fungal pathogens by disrupting the cell membrane integrity by employing in-vitro growth inhibitory experiments and FE-SEM. Further, the in-vivo antimicrobial efficacy of CsLTP1 by its exogenous application against Xanthomonas oryzae infection in rice crops has been performed. GC-MS analysis of rice plant metabolites identified the reprogramming of crucial metabolic pathways like amino acid metabolism, butanoate metabolism, inositol phosphate and t-RNA biosynthesis pathways attributed to CsLTP1 treatment for the management of bacterial blight disease. CsLTP1 showed potent insecticidal activity against Helicoverpa armigera by inhibiting its digestive enzyme α-amylase and larval developmental stages. The significant weight loss, rupture of larva, darkening, and disintegration-prone larval body drastically reduced the number of surviving larvae by 80% after 400 ppm CsLTP1 treatment. The results suggest that CsLTP1 possesses excellent antimicrobial and insecticidal potential in vivo, and it alters the plant metabolism to activate the defense response. Chapter 4 includes the study of the drug delivery potential of CsLTP1 using in-silico and in-vitro approaches. CsLTP1 possesses a long tunnel-like hydrophobic cavity that has the capability to accommodate hydrophobic compounds, and it is known for its function of membrane trafficking of lipids in plants. Hence, CsLTP1 was explored to assess the drug delivery in human breast cancer cell lines. Advanced biophysical techniques like SPR, Fluorescent spectroscopy, and CD have been employed to study the binding affinity and kinetic constants of the drug molecules. Subsequently, the ability of CsLTP1 to enhance the therapeutic efficacy of anticancer drugs has been evaluated using cell-based bioassays. The study demonstrated that CsLTP1 stably binds to the anticancer drug with high binding affinities to facilitate their transport. The cell-based bioassays indicated significantly enhanced apoptotic cells, intracellular oxidative stress, and loss of mitochondrial membrane potential in MCF-7 cells. CsLTP1-associated drug remarkably enhanced the expression of intrinsic apoptosis pathway markers, evidenced by qRT-PCR and western blot analysis. This study lays a foundation for the establishment of a potent drug delivery system using CsLTP1. Conclusively, this study characterized CsLTP1 with higher thermal and pH stability. It identified CsLTP1 as a potent in-vivo antimicrobial and insecticidal agent. CsLTP1 induced metabolic reprogramming in rice plants conferring resistance against attacking phytopathogen. CsLTP1 with α-amylase inhibitory potential hindered the most devastating larval stage of the Helicoverpa armigera life cycle. The drug delivery study proposed CsLTP1 as a promising candidate for the development of an efficient drug delivery system and related therapeutic interventions.