Doctoral Dissertations

Eui Yul Choi

Date of Award

5-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Zoology

Major Professor

Kwang W. Jeon

Committee Members

Mary Ann Handel, John W. Koontz, Evans Roth, Alex Shivers

Abstract

The lack of lysosomal fusion with symbiosomes in symbiont-bearing xD amoebae may be due either to the presence of a component in the symbiosome membrane or to the absence of a component needed in the fusion process. In order to identify such a component(s) that may allow X-bacteria to survive inside the host cytoplasm, monoclonal antibodies (mAbs) against symbiosome-membrane components were obtained and used as probes. A total of 34 mAbs recognizing different epitopes of a high-molecular-weight protein first found on the symbiosome membranes of the xD strain of Amoeba proteus were obtained, of which seven stained cell membranes by indirect immunofluorescence. The membrane protein had two subtypes of 225 and 220 kDa and several mAbs cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. The amino-acid compositions of 225- and 220-kDa polypeptides were close to each other and were also similar to those of human erythrocyte and pig-brain spectrins. On the basis of these results, it was concluded that the protein is spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes. To study the function of amoeba spectrin, the mAbs were purified and injected into amoeba cytoplasm. Antigen-antibody complexes formed, and the precipitation of the complexes caused drastic changes in cellular shape and movement: amoebae became round and showed no formation of pseudopodia. Furthermore, some of the injected amoebae cytolyzed and their phagocytic activity was significantly reduced. However, the F-actin distribution was not affected by the microinjection. A 96-kDa protein was found on symbiosome membranes. The protein was present in X-bacteria but was not found on any cell component of symbiont-free amoebae, indicating that the protein is produced by symbionts and not by the host. Interestingly, the antigen was also found on some membranous structures of unknown nature inside symbiosomes but not on the X-bacterial cell surface. Lipopolysaccharides (LPS) were identified as other symbiosome-membrane components contributed by symbionts. Several mAbs were generated against X-bacterial LPS, and some of them stained symbiosome membranes when injected into xD amoeba cytoplasm, indicating that LPS are exposed on the cytoplasmic side of the membrane and that they are good candidates as 'fusion-preventing' factors. In order to test this possibility, the anti-LPS mAbs were injected and processed for double immuno-staining in conjunction with anti-amoeba lysosome mAbs. The injection of the mAbs caused symbiosomes to fuse with lysosomes. These results suggested that X-bacterial LPS are 'fusion-preventing' factors. X-Bacteria and Bradyrhizobium japonicum bacteroids in soybean-root nodules contained large amounts of 67-kDa and 65-kDa proteins, respectively, that were antigenically related to the groEL protein of E. coli. The proteins cross-reacted with an anti-hsp58 antibody (Tetrahymena). and mAbs prepared against the X-bacterial 67-kDa protein recognized groEL analogs from several different organisms. In symbiotic B. japonicum, the amount of the stress protein was more than 7 times greater than that found in their free-living counterparts. High level of stress proteins present in endosymbionts indicated that intracellular symbiosis is a stress condition even when the symbiotic relationship is considered to be 'mutually beneficial'.

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