Salmonella Gene Expression in Complex Environments

描述（由申请人提供）：沙门氏菌每年导致数千万人感染，它已被用作生物恐怖武器。这种微生物必须与高度复杂的肠道微生物群相抗衡，才能感染宿主，而微生物群的特征已知会影响这种感染过程的成功。事实上，在正常肠道菌群存在的情况下，沙门氏菌的传染性降低了10万倍。病原体与共生或共生菌群相互作用的遗传过程是什么？如果没有报告方法，很难对这些环境进行调查。这两种方法可用于研究其他生物种群中的病原体，将以沙门氏菌为模型进行比较和对比。在每一种方法中，沙门氏菌基因组中的所有基因或启动子都将被标记为报告系统。每个细菌携带一个标签，然后将细菌混合物引入三组小鼠：非生物、非生物和正常。在这些环境中生长后，沙门氏菌基因或启动子中所有标签分布的变化将在我们建立的沙门氏菌特异性微阵列上进行监测。要测试的方法包括：(i)通过转座子标记基因组来选择环境中需要的基因，以及（ii）通过筛选启动子质粒库中无启动子荧光标记的表达来鉴定环境中诱导但不一定需要的基因。通过微阵列对基因进行标记和随后的分析，可以监测其他方法难以或不可能监测的复杂环境中的基因表达。这些方法可能会揭示新的途径，从而更好地理解与微生物群的相互作用，以及揭示潜在的新弱点，可以利用这些弱点来对抗这些病原体。作为一类方法，这些方法应该适用于研究任何细菌或真核微生物群落的基因表达。

DESCRIPTION (provided by applicant): Salmonella causes tens of millions of infections in humans each year and it has been used as a bioterror weapon. This organism must contend with a highly complex gut microbiota in order to infect the host and the characteristics of the microbiota are known to affect the success of this infection process. Indeed, infectivity by Salmonella is reduced by 100,000-fold in the presence of normal gut flora. What are the genetic processes that underlie the interaction of a pathogen with commensal or symbiotic microbiota? These are difficult environments to investigate without reporter methods. Two methods, which could be used for studying pathogens in a population of other organisms, will be compared and contrasted using Salmonella as a model. In each of these methods, all the genes or promoters in a Salmonella genome will be tagged, en mass, with reporter systems. The mixture of bacteria, each individual carrying a single tag, will then be introduced into three groups of mice: abiotic, gnotobiotic, and normal. After growth in these environments, changes in the distribution of all the tags in Salmonella genes or promoters will be monitored on a Salmonella-specific microarray that we have built. The methods to be tested involve (i), selection for genes that are required in an environment by transposon tagging of the genome, and (ii), identification of genes that are induced, but not necessarily required, in an environment by screening for expression of a promoterless fluorescent marker in a promoter plasmid library. Tagging of genes and subsequent analysis by microarrays should allow monitoring of gene expression in complex environments that would be difficult or impossible to monitor by other methods. The methods may reveal new pathways that will lead to a better understanding of interactions with the microbiota, as well as revealing potential new vulnerabilities that can be exploited to fight these pathogens. As a class, these methods should be applicable to studying gene expression in any bacterial or eukaryotic microbial community.