Degree Type

Dissertation

Date of Award

2003

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Ricardo Rosenbusch

Abstract

The effects of Mycoplasma bovis on bovine lympho-proliferation and viability were studied. Incubation of bovine peripheral blood mononuclear cells (PBMC) in vitro with M. bovis induced lymphocyte apoptosis as monitored by Annexin V binding, propidium iodide incorporation, and DNA fragmentation. The induction of lymphocyte death was abrogated by chloramphenicol, indicating that lymphocyte death was dependent upon prokaryotic protein production.;In attempts to better define the lymphotoxic factors associated with M. bovis, the task of isolating the M. bovis lymphocyte inhibitory or cytotoxic factor was undertaken. Using size exclusion chromatography a fraction able to suppress bovine lympho-proliferatioe responses to ConA was isolated. MALDI-TOF analysis of the suppressive fraction revealed one dominant peak (M.W. of 2,970 daltons) unique to the suppressive fraction. N-terminal sequencing of the suppressive fraction revealed a 19 amino acid sequence homologous to the C-terminus of the M. bovis theoretical gene encoding Vsp-L (variable surface protein-L) as determined by a BLAST search against the NCBI national protein database. The sequence data was used to construct primers for PCR amplification of the vspL gene domain corresponding to the last 26 amino acids of the VspL's protein C-terminus. This PCR product was cloned and inserted into an expression plasmid as a GST-fusion protein engineered with a thrombin recognition cleavage site between GST and the recombinant peptide (to facilitate purification of the peptide). Upon purification, the peptide Mb-LIP (M. bovis lymphocyte inhibitory peptide) was tested for lympho-proliferatioe inhibition. The recombinant peptide inhibited bovine lymphocyte proliferation responses to ConA and CD3 stimulation in vitro. Further study indicated that incubation of PBMCs with the recombinant peptide decreased the number of viable lymphocytes in culture. The recombinant peptide Mb-LIP is therefore considered a lymphotoxic peptide. This marks the first described characterization of a mycoplasmal product able to inhibit naive lymphocyte function.

DOI

https://doi.org/10.31274/rtd-180813-11859

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Anthony J. Vanden Bush

Language

en

Proquest ID

AAI3118262

File Format

application/pdf

File Size

138 pages

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