Optimizing Purification and Inactivation of Recombinant Corynebacterium pseudotuberculosis Phospholipase D

Abstract

Corynebacterium pseudotuberculosis is a Gram-positive pathogenic bacterium with pleomorphic forms. Two forms of the bacteria exist, which target different species: the equi biovar and the ovis biovar. Our research focuses on the equi biovar, which affects horses and cattle. The major exotoxin secreted is phospholipase D (PLD), which is critical in the pathogenesis of the bacteria. Three forms of the disease exist in horses: external abscesses, internal infection, and ulcerative lymphangitis. The most common type of external abscess is characterized by a swelling of the chest, which ultimately gives the disease its name: "pigeon fever." Transmission of the disease occurs through wounds, minor cuts in the skin, mucous membranes, and insect bites. PLD is instrumental in many methods employed in our lab to study the equine immune response to C. pseudotuberculosis . Therefore, we need to purify and inactivate large amounts of PLD. The PLD purification protocol was modified throughout the summer to improve yield and purity. These modifications include using a lower concentration of imidazole in washes and a higher concentration imidazole elution to improve the PLD concentration. Compared to our first purification, the concentration of our final PLD increased ten-fold. PLD must be inactivated in order to prepare for use in mouse model experiments. The currently used method, formalin-inactivation, chemically inactivates the PLD exotoxin. However, we are looking into site-specific mutagenesis as a more effective method of inactivating PLD.