Masters Theses

Date of Award

12-2000

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Robert N. Moore

Committee Members

David A. Bemis, Barry D. Bruce

Abstract

Bovine pneumonic pasteurellosis is the resultant acute respiratory disorder that arises from a variety of primary physical and infectious stressors with subsequent infection of the lower respiratory tract by Pasteurella species, the most common isolate being Pasteurella haemolytica serotype A1. Although this microbe possesses several virulence determinants, the primary virulence factor is a secreted, rummant-specific, pore forming cytotoxin called leukotoxin (LKT) As the name suggests, leukotoxin executes its effector function(s) on ruminant leukocytes. By reducing the population of functional infiltrating phagocytes, leukotoxin-induced damage leads to promotion of bacterial proliferation in the lower respiratory tract. Furthermore, leukocyte destruction enhances tissue injury via overexposure to proinflammatory agents. Given the significance of this cytotoxin, several studies have been conducted to 1) characterize this molecule on a structure/function basis and 2) devise and implement preventative measures against it. This laboratory has developed a panel of hybridomas secreting LKT-neutrahzmg monoclonal antibodies which includes a potent neutralizer, ltx-2 Previous studies have reported that ltx-2 prevents target cell association of LKT, and that the ltx-2-specific epitope(s) map(s) to the carboxy one-third of the molecule, a region which has not been demonstrated to be directly responsible for cytolysis. The primary objectives of this study were. 1) develop and characterize a leukotoxin-specific recombinant antibody molecule derived from ltx-2 mRNA and consisting of only the vanable regions of the light and heavy chains covalently linked by a flexible polyglycine linker, a single-chain fragment variable (ScFv), 2) compare the leukotoxin recognition and neutralization tendencies of the ScFv and ltx-2 using in vitro analyses in an effort to further define the mode of action of ltx-2, and 3) test the ability of both the ScFv and ltx-2 to recognize a panel of glutathione-S-transferase (GST) fused N-terminally deleted leukotoxin constructs generated from the carboxy one-third of the LKT molecule in order implicate the importance of this region m target cell association. A leukotoxin-specific 29.5 kDa ScFv was successfully constructed, isolated, and sequenced. The recombinant molecule was stably expressed in an Escherichia call bacterial cell line, readily detectable in both the culture supernatant and periplasm, and purified from periplasmic concentrate. Furthermore, the ScFv molecule, although five times smaller in size than the native ltx-2 antibody and without the benefit of Fc stabilization, was shown to bind to leukotoxin and the GST-fused LKT deletional constructs in-a fashion comparable to ltx-2. This study also demonstrated that the ScFv has the capacity to neutralize LKT. However, unlike the LKT recognition assays, the ScFv was shown to be approximately five times less efficient at LKT neutralization than its native counterpart. Finally, this set of experiments also revealed that 1) the region represented by ammo acids 766-939 contains all of the structural elements necessary for ltx-2/ScFv recognition and 2) in vitro, Itx-2/ScFv recognition of this carboxyl region closely resembles that observed for full length, native leukotoxin.

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