DELINEATING THE STRUCTURE-ACTIVITY RELATIONSHIP OF T7 BACTERIOPHAGE ENDOLYSIN

Abstract

Bacteria are single-celled prokaryotes capable of surviving in any habitat (air, water, and soil) and are the most common biological entities on the entire planet. The implication of bacteria in human health can be both beneficial and detrimental. For example, bacteria present in gut are advantageous to maintain human health. In contrast to this, pathogenic bacteria cause serious illnesses and life-threatening situations by secreting toxins (exotoxins or endotoxins) that can cause infection. The development of antibiotics and vaccines was a significant milestone in medicine. However, because of their immense metabolic capacity, bacteria have adapted to practically all ecological circumstances and become resistant. Therefore, one of the most serious microbiological issues of the twenty-first century is antibiotic resistance. This raises the questions regarding the usage of antibiotics in the future, as well as the possibility of other medicinal alternatives. Over the years, probiotics, plant extracts, essential oils, immunomodulators, and other options have been attempted as a replacement to widely used antibiotics. Unfortunately, no alternative treatment has able to match antibiotics' effectiveness. After years of research, vaccines and bacteriophages have been identified as the only viable choices, and owing to their host specific active bactericidal property and easy availability, phages are utilised in treatments to overcome antibiotic resistance developed against harmful bacteria such as Staphylococcus spp., Mycobacterium spp., and superbugs. phage mediated antibacterial treatment approach is convenient as they are less damaging to the natural micro flora of their particular host because of their specificity, show high efficacy and are cost effective as well. Bacteriophages (also known as phages) are diverse and prolific viruses that primarily infect bacteria (but they can also infect archaea and eubacteria). In addition to this, they also influence the genetic and ecological diversity of bacteria. Phages are parasites (lack self-replication machinery), that utilize the host's biosynthetic resources and system to replicate themselves and then trigger the host cell lysis to release their progeny virions at the end of the lytic cycle. At this opportunistic phase, phage encoded enzymes called endolysins start accumulating inside the host cell and access the bacterial cell membrane by means of holins (phage encoded membrane proteins that oligomerizes in the cytoplasmic membrane of the host) and swiftly breakdown the peptidoglycan layer of the host bacteria causing osmotic cell lysis and the escape the progeny viruses. The global proliferation of bacteria that are immune to existing antibacterial agents has spotlighted the endolysins as the most promising antibiotic alternatives due to their natural and powerful bactericidal activity that complements the conventional antibiotics. Despite its common lysis activity against peptidoglycan (PG) layer, endolysins possess biochemical and structural diversity. They are being used in various fields such as in human medicines, plant protection from pathogenic bacterial infection, food processing and preservation, biofilm eradication and biotechnologies are a few to mention. Considering the wide applications of endolysins, various protein engineering approaches including structure guided mutagenesis approach, rational designing, in silico computational modelling are being used to improve their stability, catalysis and specificity. Chimeolysins, artilysins, and gladskins are the flag bearers of successful engineering of efficient endolysins. Further phage-based has another arena in designing formulations and encapsulation of endolysins to for competent drug delivery methods. Endolysins can be globular or modular in structure depending on their origin. In most Gram-negative bacteria and a few Gram-positive bacteria, the peptide stems of peptidoglycan (PG) layers are joined by a direct interpeptide bond; however, in majority of the Gram-positive bacteria, peptide chain cross-linking occurs via an interpeptide bridge. The differences in PG types among bacteria are mostly due to differences in the peptide moiety, particularly in the amino acid content of the interpeptide bridge. Mycobacteriophages and Gram positive phages are modular in configuration, with two domains in most cases (enzymatic active domain and cell wall binding domain). Endolysins infecting Gram-negative hosts, on the other hand, have a globular structure with single domain (Enzymatic active domain). Endolysins belonging to the single domain family comprises T7L, T3, T4L, KP32, and K11. The murein cleavage specificity classifies endolysins into three groups: glycosidases, amidases, and endopeptidases. T7 bacteriophage endolysin (T7L), T4 bacteriophage lysozyme (T4L) are two well-studied endolysins that belong to amidases and glycosidases groups. T4L is a 164 amino acid (18.7 kDa) protein hydrolase which attacks the peptidoglycan in the cell wall of bacteria and hydrolyzes β1-4 linkage between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residue. T4 endolysin substrate binding mechanisms and mode of action is very similar to that of Hen egg white lysozyme (HEL, 129 aa, 14.4 kDa). Whereas, T7L is a 151 residue long (17 kDa) bifunctional amidase protein with a pI of 8.8. It has three α-helices (α1, α2, α3) and five β-sheets, out of these four are parallel (β1, β2, β3, β4, β5) and one is antiparallel (β3). Crystal structure of T7L also evidences that residues Lys128, Tyr46, and His17 play a critical role in catalysis. Keeping all these above-mentioned scenario and facts in mind, the research-work in the current thesis is designed to unravel the structure-stability-activity paradigm of T7 endolysin variants. The specific details of the thesis chapter (1-4) are as follows: Chapter 1 provides an overview of the bacterial diseases, antibiotic treatment, their mode of action, acquisition of antibiotic resistance among pathogenic bacteria and brief information about various alternative strategies applicable as antibiotics. The prevailing literature on history of bacteriophage, their types, structures, life cycles, and phage therapy with special emphasis on endolysins have been described in detail. The chapter also covers the structural detail of endolysins; their classification based on mode of action, and their wide range of applications. In this chapter, various engineering approaches for endolysin, such as, rational design, formulations of endolysins, list of patents and their significance have been provided in detail. Chapter 2 of the thesis delineates the combination therapy of bacteriophage endolysins (T7L, and T4L) with antimicrobial peptides (Colistin, Polymyxin B (PMB), Nisin). Due to their remarkable antimicrobial and anti-biofilm properties, bacteriophage endolysins have emerged as viable alternative to traditional therapies. To ameliorate the therapeutic efficacy of endolysins, activity studies were carried out alone and in conjunction with antimicrobial peptides. To accomplish that, bacteriophage endolysins T7L and T4L were purified using a combination of chromatograhic techniques. The antimicrobial activity were evaluated both with and without the combination of three AMPs against bacterial strains including Gram positive as well as Gram negative strains. PMB and colistin efficiently inhibited the planktonic growth of P.aeruginosa in combination with T7, and exhibited synergistic action. In addition to this, the combination of T4L and nisin showed synertistic affect against S.aureus. The synergisctic action of T4L, and T7L with the FDA-approved conventional drugs/ food grade antimicrobial agents can also potentially resolve the emergence of new food-borne pathogens causing serious food-borne illness. As many essential oils, natural substances extracted from aromatic plant possess antioxidant, antimicrobial activity henceforth, the combination of endolysins with all such antimicrobial agents can generate a novel basket of alternate therapeutic formulations. Chapter 3 unravels the role of coupled His network in structure-stability-activity paradigm of T7 endolysin. Protein folding is a dynamic process that involves diverse interactions among its constituent amino acids and depends on varied external factors such as pH, temperature, chemical environment and so forth. Biophysical, and NMR based studies have shown that T7L exhibits pH dependent conformational change from native to partially folded state, due to the protonation of the network of evolutionary conserved coupled His network (H18, H48, H69, and H123) present in the hydrophobic core of T7L protein. This chapter dissects the role of His residue(s) that trigger T7L protein’s partially folded state. Four variants (H18K, H48K, H69K, and H123K) were designed in which His was replaced with Lys exclusively, based on the assumption that Lys remains fully protonated at physiological pH (~7.0), and henceforth, its effect can be visualized on T7L structure. Various biophysical techniques such as size exclusion chromatography, fluorescence, CD and NMR were applied to characterize the T7L Lys variants. Fluorescence studies suggested the exposure of hydrophobic residues in variants, thus disrupting the tertiary structure. The studies established that only the mutant H123K showed similar activity and pH dependent reversibility to that of T7L-WT protein. Indeed, NMR based studies suggested that three variants H18K, H48K and H69K are very fragile, and also forms irreversible partially folded conformations. Among the designed variants, H48K is virtually a dead variant strongly suggesting that the His at this position (48th) is primarily responsible for core structure stability and pH dependent conformational switching of T7L. Urea denaturation-based CD studies at different pH establish the variable stability of the four variants in their native and partially folded conformations. All the observations strongly indicate H48K to be virtually a dead variant, strongly suggesting that the His at this position (48th) is primarily responsible for core structure stability and pH dependent conformational switching of T7L. The in-depth protein conformational characterization studies carried out in this chapter elucidates the structural intricacies of T7L protein structure and opens the gateways towards improved drug designing strategies and engineering endolysin(s) with enhanced activity and/or stability. Chapter 4 is devoted to delineate the lysis potency of the engineered T7L variant. To accomplish that, a structure guided mutagenesis approach has been used to engineer a T7 bacteriophage endolysin (T7L) by replacement of a non-catalytic gating residue (His 37). Two H37 variants (H37A and H37K) have been designed and characterized comprehensively using integrated biophysical and biochemical techniques to provide mechanistic insights into its structure-stability-dynamics-activity paradigm. Among the studied proteins, cell lysis data suggested that the obtained H37A variant exhibits enhanced amidase activity (~35%) to that of wild-type T7L endolysin (T7L-WT). In contrast to this, the H37K variant is highly unstable, aggregation prone and is less active. Comparative structural and dynamic analysis of H37A variant to that of T7L-WT evidenced that the alteration at the site of H37 resulted in long range structural perturbations with attenuated conformational heterogeneity and quenched μs-ms time scale motions. Stability analysis confirmed the altered stability of H37A to that of its WT counterpart. All the obtained results established that the H37A variant enhances the lysis activity by regulating the stability-activity trade-off. Chapter 4 provided deeper atomic level insights on the structure-activity relationship of endolysin proteins, thus aiding the researchers in rational designing of engineered endolysins with enhanced therapeutic properties. In summary, the current thesis work elucidated the structure-function characteristics of T7 phage endolysin. The evaluation of antibacterial activity of T7L in conjunction with antimicrobial peptides/conventional drugs suggested their synergistic action. The in-depth molecular level analysis unravelled the role of conserved residue(s) in conformational switching and stability- activity relationship of T7L endolysin. Henceforth, these findings could potentially be applicable in engineering next generation phage endolysin based enzybiotics with hyperactivity and/or hyperstability.