Doctoral Dissertations

Kee Jun Kim

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Zoology

Major Professor

Kwang W. Jeon

Committee Members

Mary Ann Handel, L. Evans Roth, John W. Koontz

Abstract

In the amoeba-bacteria symbiosis, unidentified Gram-negative bacterial endosymbionts, X-bacteria, reside within symbiosomes in the host cytoplasm and are known to avoid lysosome-symbiosome fusion for survival. However, the mechanism for the inhibition of lysosomal fusion is unknown, but several macromolecules were thought to be involved in the prevention of lysosomal fusion.

Among them, lipopolysaccharides (LPS) were characterized and their role was determined in this study. In order to characterize LPS of X-bacteria, the chemical composition was determined by using analytical methods. Eleven different kinds of sugars were identified in the polysaccharide part, including a rare sugar yersiniose A, which is found only in Yersinia sp. and Legionella sp. X-Bacterial LPS have a remarkably complex and distinct pattern of hydroxylated fatty acids, which is less common in that of Gram-negative bacteria. Glucosamine seems to be a sugar backbone connecting lipid A and polysaccharide parts.

For studying the role of LPS on symbiosome membranes, two kinds of monoclonal antibodies against X-bacterial LPS and lysosomal membrane proteins (LMP) were generated. Double immunostaining showed that some symbiosomes in xD amoebae microinjected with an anti-LPS antibody became stained with an anti-LMP antibody, implying that symbiosomes fused with lysosomes. Therefore LPS play a role in lysosome-symbiosome fusion prevention. LPS appeared on symbiosome membranes in newly infected D amoebae between 3 and 7 days after infection and reached a maximum level 21 days after infection. During a high-temperature treatment, the outer membranes containing LPS were released from X-bacteria 3 days after treatment, and LPS were dispersed in symbiosomes by the 5th day. At the same time active vesicular fusions were observed around symbiosome membranes and X-bacteria were digested. LPS disappeared from symbiosomes 14 days after treatment. How LPS actively block lysosomal fusion and how LPS might be incorporated into symbiosome membranes are discussed.

Since the identity and origin of X-bacteria are unknown, phylogenetic studies were performed. The X-bacterial 16S ribosomal DNA was amplified and sequenced by a polymerase-chained reaction method. When compared with that of other bacteria, X-bacteria appeared to belong to the gamma subclass of proteobacteria and have the highest nucleotide-sequence identity (97.1%) with Flavobacterium lutescens, which is a member of Pseudomonas superfamily.

A novel 85-kDa protein was found in inclusions of symbiosomes. This protein was synthesized in X-bacteria and was present in inclusions of symbiosomes. 85-kDa protein began to appear in symbiosomes 3 days after infection and the amount of the protein was indistinguishable with that of established xD amoebae 21 days after infection. Interestingly, the appearance time of 85-kDa protein was the same as that of LPS. Therefore 85-kDa protein is thought to be involved in the establishment of symbiosis.

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