Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can be only activated when exposed to specific cell-surface proteases produced by a specific cell type. We ultimately want to target breast cancer cells that differentially secrete or express proteases such as matrix-metalloproteases (Stautz D et al., 2012; Zhang, M et al. 2013). To engineer this protein we took advantage of the N-terminus of ClyA. The N-terminus of ClyA, which is highly hydrophobic (Oscarsson J et al), undergoes a conformational change to insert into the target cell membrane (Oscarsson J et al). This conformational change allows ClyA to penetrate the target membrane to form a transmembrane domain of ClyA. The hydrophobic nature of lipid membranes makes it highly unfavorable for any charged residues to cross the membrane (Hunt J 1997). With this in mind, we hypothesize that negative charges inserted into the N-terminus of ClyA will inhibit it from inserting into the membrane. Thus, we mutated the N-terminus of the ClyA protein by inserting an inactivation site composed of negatively charged amino acids that we hypothesize would prevent insertion into the plasma membrane of the target cell. Once we confirmed that this construct was an inactive ClyA mutant, we inserted a thrombin cleavage site right after the inserted negative charges. This site should allow us to remove the negative charges once the protein is exposed to thrombin. Once the negative charges are removed, the protein should recover its activity. This approach will allow us to create a version of ClyA that is protease-switchable.

Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.