A role for divalent cations in the cytotoxicity of Pasteurella Haemolytica Leukotoxin

Author

Donald G. Gerbig

Date of Award

8-1987

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Robert N. Moore

Committee Members

David A. Bemis, Michael A. Breider

Abstract

Pasteurella haemolytica (Biotype A, serotype 1), the primary causative agent of bovine pneumonic pasteurellosis, produces a potent ruminant specific leukotoxin that is believed to be a primary virulence determinant. The nature and mode of action of this toxin is poorly understood. Properties of Pasteurella haemolytica leukotoxin have been further elucidated using leukotoxin culture filtrates generated in RPMI medium 1640 (rpm/1640) with 1% horse serum. The BL-3 cell line was used as the leukotoxin target in an assay for cytotoxicity. Exposure of the target cells to the leukotoxin for as little as 5 min was sufficient to elicit cytotoxicity as assessed by neutral red cytotoxicity assays and thymidine incorporation assays. The leukotoxin killed target cells at 4°C and at 37°C and was not stabilized or destabilized in the presence or absence of serum. Estimated molecular weight of the leukotoxin, as determined by gel filtration chromatography, was approximately 75,000 daltons (d); however, some activity was detected in fractions with estimated molecular weights of 46,000 and 16,000 d.

Leukotoxin-target cell interactions were also investigated. The leukotoxin appeared to have a specific requirement for divalent cations. Using one hr neutral red cytoxicity assays, leukotoxic activity in concentrated filtrates was reduced following dialysis against divalent-cation free Hanks balanced salt solution (HBSS). Cytotoxicity was recovered by the addition of physiological concentrations of Ca²⁺, Ba²⁺, Co²⁺, and Sr²⁺, but not by Mg²⁺ or Mn²⁺. Furthermore, two Ca²⁺ chelators. Ethylenediamine tetraacetic acid (EDTA) and Ethyleneglycol-bis-(B aminoethyl ether) N,N'-tetraacetic acid (EGTA), abrogated the activity of leukotoxic filtrates. In addition, Verapamil and the ionophore A23187, both known to influence Ca²⁺ channels, reduced cytotoxicity in one hr assays when used with leukotoxin diluted in HBSS containing Ca²⁺ and Mg²⁺. The protective effect of Verapamil was overcome by increasing the Ca²⁺ concentration.

Results from additional experiments, however, indicated that divalent cations were not absolutely essential for cytotoxicity. Cytotoxicity was determined over time in RPMI 1640 with and without EGTA using neutral red cytotoxicity, trypan blue exclusion and chromium⁵¹ release assays. Cell death occurred in two phases: a rapid death requiring divalent cations and a prolonged death that was independent of divalent cations (specifically Ca²⁺). The cytotoxic effect of P. haemolytica leukotoxin, therefore, was influenced by the presence of divalent cations (specifically Ca²⁺). The requirement for Ca²⁺, however, was not absolute.

Recommended Citation

Gerbig, Donald G., "A role for divalent cations in the cytotoxicity of Pasteurella Haemolytica Leukotoxin. " Master's Thesis, University of Tennessee, 1987.
https://trace.tennessee.edu/utk_gradthes/13475