Proteins involved in amoeba-bacteria symbiosis as studied using monoclonal antibodies

Author

Hyong Bai Kim

Date of Award

3-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Zoology

Major Professor

Kwang W. Jeon

Committee Members

G. L. Whitson, M. L. Pan, J. A. MacCabe, J. W. Koontz

Abstract

The protein composition of symbiotic (xD strain) and non-symbiotic (D strain) of Amoeba proteus and symbiotic X-bacteria was compared by two-dimensional polyacrylamide-gel electrophoresis and two prominent polypeptides were found in the amoeba cytosol. One was the xD-strain-specific polypeptide found only in xD amoebae and X-bacteria. It was called xD protein and its molecular weight was 29,000. The other polypeptide was the most prominent and common poly peptide among xD amoebae, D amoebae and X-bacteria. Its molecular weight was about 43,000.

To elucidate the origin of the xD protein, xD amoebae 3 3 were treated with antibiotics, and then labeled with ³⁵S-methionine. Results of radioautographic studies showed that the synthesis of xD protein was inhibited after chloramphenicol treatment and it was concluded that xD protein was synthesized in X-bacteria and transferred to host amoebae.

For further characterization of the xD protein, a monoclonal antibody against the protein was prepared. The native xD protein was purified using an Affi-Gel 10 column and the molecular weight of the native xD protein was found to be 87,000. The location of xD protein was examined using an immunogold-labeling technique by electron microscopy and it was found that the xD protein was a soluble cytoplasmic protein, contained in the amoeba cytoplasm randomly.

An antibody against the 43-kD polypeptide was prepared and the characteristics, location, and origin of the 43-kD polypeptide were checked with immunoblotting, immunofIorescent, and immunogoId-labeling techniques. The results suggested that the 43-kD polypeptide was actin and concentrated in X-bacteria vesicles. Its possible role seemed to be the prevention of lysosomal fusion with symbiotic vacuoles,

The lethal factor of xD amoebae against D amoebae was investigated using gel-filtration and microinjection techniques. The lethal factor was a cytoplasmic protein and its molecular weight was more than 200,000.

The protective effect of X-bacteria against the lethal factor and the time of appearance of the lethal factor in newly infected D amoebae were examined. X-Bacteria had a protective effect against the xD lethal factor, whether their injection preceded or followed that of the lethal factor. Newly infected D amoebae started to synthesize the lethal factor as a result of harboring X-bacteria.

Recommended Citation

Kim, Hyong Bai, "Proteins involved in amoeba-bacteria symbiosis as studied using monoclonal antibodies. " PhD diss., University of Tennessee, 1987.
https://trace.tennessee.edu/utk_graddiss/12086