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Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.