Development of Host's Dependence on Intracellular Symbionts

9727911建议研究的长期目标是阐明感染微生物进入宿主细胞并在宿主细胞内生存的机制，其中一些微生物成为细胞内的共生体。这种集成可能导致为宿主细胞获取新的细胞组件。目前项目的目标是确定感染X细菌的细菌如何进入阿米巴虫并在阿米巴虫内生存，最终成为细胞内的共生体。在阿米巴和X-细菌的共生中，宿主阿米巴依赖共生体生存，而新感染的阿米巴在18个月内(约200代细胞)依赖共生体生存。因此，对于阿米巴来说，整合的X细菌成为新的细胞成分。两个重要而悬而未决的问题是：1)感染细菌如何避免其宿主细胞的破坏；2)阿米巴为什么或如何依赖细菌共生体生存。虽然从以前的研究中获得了对第一个问题的一些见解，但直到最近才发现了第二个问题的可行线索：X细菌阻止阿米巴产生一种关键的酶，但然后通过另一种途径提供这种酶。在不含共生体的D阿米巴中，含有共生体的xD阿米巴不再产生起S-腺苷甲硫氨酸合成酶作用的酶。在xD阿米巴中没有SAMS的原因是xD阿米巴因含有细菌共生体而未能转录相应的基因。这些结果表明，共生X细菌可能会导致它们的宿主变得依赖它们。在这项拟议的研究中，将检验以下假设：1)X-细菌抑制阿米巴看家基因SAMS的表达，但X-细菌提供基因产物SAMS，因此阿米巴最终会依赖共生体。PI有两个抗阿米巴SAMS的单抗用于我们的研究。2)X-细菌产生的蛋白(S)或X-细菌内质粒编码的蛋白作为调节因子抑制sAMS基因在xD阿米巴中的表达。这些研究将涉及使用DNA克隆、核苷酸测序、定点突变和基因组足迹等技术。这些研究的预期结果将为一般逃避感染微生物对宿主的破坏及其随后的生存机制提供进一步的洞察。这些结果还将有助于阐明建立和维持稳定的内共生机制，从而在真核细胞中获得新的细胞成分。

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.