Strain-specific proteins in symbiotic and non-symbiotic amoebae

The characteristics of X-bacteria as an infective agent, the establishment of symbiosis and its kinetics were investigated in D amoebae newly infected with X-bacteria by the use of poly-L-lysine as an inducer of phagocytosis. Symbiotic (xD strain) Amoeba proteus originated from non-symbiotic D strain as a result of spontaneous infection by a large number of X-bacteria in 1966. The xD amoebae have different cell characteristics from those of D amoebae. Amoebae phagocytosed over 30,000 X-bacteria by induced infection, but infected amoebae lost half of ingested X-bacteria within 12 hours. On day 4, amoebae hod only about 5% (average number 1600 per amoeba) of the initially ingested X-bacteria. After 4 days, the number of X-bacteria increased and their mean generation time was 2.52 days (k=0.1 19). The infected amoebae had a maximum number of X-bacteria (43,000 per amoeba) on day 16.

The protein composition of symbiotic xD amoebae and non-symbiotic D amoebae was compared by two-dimensional gel electrophoresis in order to characterize strain differences. When the proteins were stained with silver, more than 500 peptide spots were detected on a typical gel. There were minor differences in several peptide spots between the two amoeba strains, but one prominent polypeptide with an isoelectric point of 5.5 and a molecular weight of 29,000 was present only in xD amoebae. That spot, referred to as the xD protein, was also present in isolated X-bacteria, suggesting that the xD-specific protein may have been synthesized in X-bacteria and transferred to the host amoebae.

In order to determine the relationship between X-bacteria and the xD protein, proteins were extracted from xD and D amoebae cultured under different experimental conditions and compared by two-dimensional PAGE. First, the disappearance of the xD-protein spot from xD amoebae was examined after removing X-bacteria from xD amoebae cultured at a higher-temperature. In the higher-temperature culture, the D-amoeba-protein pattern did not change. but the size of xD-protein spot decreased gradually in xD amoebae with the decreasing number of X-bacteria.

Second, the appearance of xD-protein spot was investigated after induced infection. After infection, xD protein could be detected from day 6 when the average number of X-bacteria per amoeba was about 4600. The size of the xD-protein spot and density increased in newly infected D amoebae according to the increasing number of X-bacteria. These results suggested the xD protein was synthesized in X-bacteria and transferred to the host amoebae.