9727911 Jeon The long-term goals of the proposed study are to elucidate the mechanisms whereby infecting microbes enter host cells and survive inside the latter, some of them becoming integrated as intracellular symbionts. Such integration may result in the acquisition of new cell components for a host cell. The objectives of the current project to determine how infecting X-bacteria enter amoebae and survive inside the latter, eventually becoming integrated as intracellular symbionts. In symbiosis between amoebae and X-bacteria, host amoebae are dependent on symbionts for survival and newly infected amoebae become dependent on symbionts within 18 months (about 200 cell generations). Thus, for amoebae, the integrated X-bacteria become new cell components. Two important and outstanding questions are 1) how infecting bacteria avoid destruction by their host cells and 2) why or how amoebae become dependent on bacterial symbionts for survival. While some insights to the first question were gained from previous studies, it was only recent that a viable clue to the second question was found: X-bacteria prevent the production of an essential enzyme by amoebae, but then supply the enzyme through an alternate route. Symbiont-bearing xD amoebae no longer produce an enzyme that functions as S-adenosylmethionine synthetase (SAMS) in symbiont-free D amoebae. The absence of SAMS in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. These results showed how symbiotic X-bacteria might cause their hosts to become dependent on them. In the proposed study, the following hypotheses will be tested: 1) That X-bacteria suppress the expression of an amoeba's housekeeping gene, sams, but X-bacteria supply the gene product, SAMS, so that amoebae become dependent on symbionts in time. The PIs have two monoclonal antibodies against the SAMS of amoebae to use in our studies. 2) That X-bacteria-produced protein(s) or a protein enco ded by a plasmid inside X-bacteria acts as a regulatory factor to suppress the expression of the sams gene in xD amoebae. These studies will involve the use of techniques such as DNA cloning, nucleotide sequencing, site-directed mutagenesis and genomic footprinting. The expected results from these studies will provide further insight into mechanisms for the general evasion of host destruction by infective microbes and their subsequent survival. The results will also enhance the elucidation of mechanism for the establishment and maintenance of stable endosymbiosis leading to the acquisition of new cell components in eukaryotic cells.
