Development of Host's Dependence on Intracellular Symbionts

9727911 Jeon拟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存活的机制，其中一些微生物成为细胞内共生体。 这种整合可以导致宿主细胞获得新的细胞组分。 当前项目的目标是确定感染X-细菌如何进入阿米巴原虫并在阿米巴原虫内存活，最终成为细胞内共生体。 在变形虫和X-细菌之间的共生中，宿主变形虫依赖于共生体生存，并且新感染的变形虫在18个月内（约200个细胞代）变得依赖于共生体。 因此，对于变形虫，整合的X-细菌成为新的细胞成分。 两个重要而突出的问题是：1）感染细菌如何避免被宿主细胞破坏; 2）阿米巴原虫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，但直到最近才发现第二个问题的可行线索：X细菌阻止阿米巴产生必需的酶，但随后通过替代途径提供酶。 携带共生体的xD阿米巴不再产生在无共生体的D阿米巴中起S-腺苷甲硫氨酸合成酶（SAMS）作用的酶。 在xD阿米巴中SAMS的缺乏可归因于xD阿米巴由于窝藏细菌共生体而不能转录相应的基因。 这些结果表明共生的X细菌可能会导致它们的宿主依赖它们。 在拟议的研究中，将测试以下假设：1）X-细菌抑制阿米巴管家基因SAMS的表达，但X-细菌提供基因产物SAMS，因此阿米巴原虫及时依赖于共生体。 PI有两种抗阿米巴SAMS的单克隆抗体用于我们的研究。 2)该X细菌产生的蛋白质或由X细菌内的质粒编码的蛋白质充当抑制xD阿米巴中sams基因表达的调节因子。 这些研究将涉及使用DNA克隆、核苷酸测序、定点诱变和基因组足迹等技术。 这些研究的预期结果将进一步深入了解感染性微生物对宿主破坏的一般逃避机制及其随后的生存。 这些结果也将有助于阐明真核细胞中建立和维持稳定的内共生关系，从而获得新的细胞组分的机制。

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.