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

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.