Development of Host's Dependence on Intracellular Symbionts

该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。这种整合可能导致为宿主细胞获得新的细胞成分。当前项目的目标是确定感染x细菌如何进入变形虫体内并在后者体内生存，最终成为细胞内的共生体。在变形虫与x细菌的共生中，宿主变形虫依赖共生体生存，新感染的变形虫在18个月内（约200个细胞代）对共生体产生依赖。因此，对于变形虫来说，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)变形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，但直到最近才发现了对第二个问题的可行线索：x细菌阻止变形虫产生一种必需的酶，但随后通过另一种途径提供这种酶。共生的xD变形虫在无共生的D变形虫体内不再产生s -腺苷甲硫氨酸合成酶（SAMS）。xD变形虫中SAMS的缺失可归因于xD变形虫由于携带细菌共生体而无法转录相应的基因。这些结果显示共生的x细菌可能会使它们的宿主变得依赖它们。在拟进行的研究中，将检验以下假设：1)x -细菌抑制变形虫的管家基因sams的表达，但x -细菌提供基因产物sams，从而使变形虫及时依赖共生体。在我们的研究中，PIs具有两种针对变形虫SAMS的单克隆抗体。2) x细菌产生的蛋白或由x细菌内的质粒编码的蛋白作为抑制xD变形虫中相同基因表达的调节因子。这些研究将涉及使用诸如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揭示感染微生物普遍逃避宿主破坏及其随后存活的机制。这一结果也将进一步阐明真核细胞内稳定共生关系的建立和维持，从而获得新的细胞成分的机制。

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.