| dc.description.abstract | Bacteria adopt several defense strategies to enable their survival against the environmental threats they encounter from time to time. Toxin-antitoxin (TA) systems are being understood as a key bacterial defense mechanism against invading viruses, antibiotics, and other environmental stress. TA systems consist of a pair of genes, usually under a common promoter, that code for a toxin and its cognate antitoxin . The toxin is usually a protein, that arrests cellular growth during stress, whereas the antitoxin can be a protein or a non-coding RNA, that inhibits the toxin. The TA systems are classified into six different types based on the mechanism of inhibition of toxin by antitoxin. In type III TA systems, the toxin is an endoribonuclease (RNase) that cleaves cellular RNAs when free, whereas antitoxin is a non-coding RNA. The toxin also processes its own precursor antitoxin RNA into smaller repeats and subsequently assembles with them to form an inactive TA complex. During normal growth conditions, the antitoxin RNA inhibits the toxin protein by forming the RNA-protein TA complex. However, when the bacteria encounter stress such as phage infection, the active toxin gets released from the complex and prevents phage replication.
Type III TA systems have been identified in several bacteria and classified into three different families - toxIN, cptIN, and tenpIN. However, type III systems have not been identified and well characterized in Escherichia coli. The identification and characterization of these systems in E. coli, which is the most commonly studied model organism with robust genetic manipulation tools available, would help in understanding them in detail for their functions and mechanism of action. In this thesis, by using protein sequence-based homology searches, we report the identification of ToxIN type III TA systems from several strains in E. coli. Multiple sequence alignment of the toxin protein sequences revealed that these systems could be further grouped in five different clusters and there are several conserved residue positions that could be vital for the toxin structure and function. Secondary structure analysis of representative sequences of antitoxin RNA repeats from five different clusters suggested that these RNAs have the propensity to form pseudoknot structure. Toxin-antitoxin functional assays performed using one of the identified TA systems from E. coli (strain 680) showed that the identified system indeed functions as a type III TA system.
Though type III TA systems are known to be found in several organisms, very few of them have been characterized structurally and biophysically. This is mainly due to the challenges in cloning the toxin proteins in expression vectors and the lack of standard protocols to express and purify the type III TA components. Hence, we decided to establish protocols for cloning and purification of type III TA components. Here, we report the large-scale expression and purification of the toxin, antitoxin and complex components from four different type III TA systems (three from toxIN and one from tenpIN families) in E. coli. This strategy involves cloning the toxin and antitoxin coding DNA sequences in two different co-expression compatible, commercially available expression vectors. Co-transformation and co-expression of toxin and antitoxin genes in laboratory strains of E. coli led to the expression and purification of type III TA complex. Using anion exchange chromatography, we could obtain separate fractions of toxin protein, antitoxin RNA, and complex components in significant quantities suitable for biophysical experiments.
Further, we were able to crystallize the type III TA complex from E. coli (strain 680) and solve the X-ray crystal structure at a resolution of 2.097 Å. This is the first reported structure of a type III TA complex from E. coli. The E. coli type III toxin and antitoxin were arranged in a cyclic heterohexameric assembly in the complex structure. This assembly was also verified in solution using SEC-MALS analysis. The toxin protein, which is an endoribonuclease, adopts a β-sheet containing core structure surrounded by α-alpha helices. The antitoxin RNA forms a pseudoknot structure with two stems and two loops and the 5′ and 3′ single-stranded regions interact with the toxin protein. The structure also uncovered the presence of several key interactions between the toxin and antitoxin and provided molecular basis for the substrate sequence specificity of the toxin. Mapping the amino acid residues which were conserved in all five clusters of E. coli toxIN, onto the structure of the E. coli type III TA complex showed that most of these residues were crucial for toxin folding and endoribonuclease activity. The multiple sequence alignment of antitoxin RNA sequences revealed that the core pseudoknot region was conserved for both sequence and structure across the five different clusters and the 5′ and 3′ single-stranded overhangs were variable, that could lead to specificity of the antitoxins to their cognate toxins.
The assembly of the type III TA complex has not been studied so far in terms of toxin-antitoxin binding affinity and free energy change of the complex formation. Hence, we characterized the binding of toxin protein and antitoxin RNA using isothermal titration calorimetry (ITC) experiments. The ITC experiments reveled that the toxin and antitoxin interact with each other with a very high binding affinity in a two-step binding event. The structure of the complex showed that toxin and antitoxin possess two non-identical binding sites for each other which leads to a two-step binding process. Using truncated antitoxin RNA mutants, we could simplify the two-step binding into two one-step binding events and estimate the binding contribution from each individual site. Based on our ITC experiments on the full-length antitoxin repeat and the truncated repeats, we have proposed a model for the assembly of toxin and antitoxin into a cyclic heterohexameric complex.
Using nuclear magnetic resonance (NMR) spectroscopy, we characterized the structure of the free antitoxin RNA repeat for its foldedness. The 1D 1H and the 2D 1H-1H NOESY NMR spectra showed that the free antitoxin RNA adopts a folded structure in solution. This was further confirmed by recording a 2D 1H-15N HSQC spectrum of the free antitoxin. The NMR spectra of only the core pseudoknot forming region of the antitoxin suggested that the antitoxin RNA could fold into a pseudoknot structure even in the absence of toxin protein. Perturbation of a noncanonical U-U base pair, which is part of a U:U:G triplet, in the antitoxin RNA significantly altered its structure indicating that noncanonical and tertiary interactions are crucial for antitoxin folding.
The thesis has been organized as follows: Chapter-1 provides a brief review on toxin-antitoxin (TA) systems and their classification with a special focus on type III TA systems, that have been studied in this work. Chapter-2 describes the identification and functional characterization of type III TA systems in Escherichia coli. Chapter-3 details the expression and purification of toxin, antitoxin, and complex components from four different type III TA systems in E. coli. In this chapter, we also propose a standard protocol for cloning, expression, and purification of type III TA components for biophysical experiments. In Chapter-4, we report the structure of the first type III TA complex in E. coli. Our studies on toxin and antitoxin binding using isothermal titration calorimetry (ITC) are also described in this chapter. Chapter-5 details the characterization of free antitoxin RNA by solution NMR spectroscopy. | en_US |
