Dynamics of intracellular bacterial replication at the single cell level
Dynamics of intracellular bacterial replication at the single cell level
复制标题
DOI：
10.1073/pnas.1000041107
复制
发表时间：
2010-02-23
影响因子：
11.1
通讯作者：
Holden, David W.
中科院分区：
文献类型：
--
作者：
Helaine, Sophie;Thompson, Jessica A.;Holden, David W.
几种重要的病原体通过在宿主细胞内生存和复制而引起疾病。细菌增殖是群体复制和杀灭的产物。然而，这些过程很难区分，通常通过测定净细菌负荷来一起评估。此外，净负荷的测量并不能揭示病原体种群内的异质性。这对于持续感染尤其重要，因为缓慢或不生长的细菌被认为具有重大影响。在此，我们报告了基于荧光稀释的报告系统的开发，该系统能够在群体和单细胞水平上直接定量小鼠巨噬细胞中鼠伤寒沙门氏菌血清型（S.Typhimurium）的复制动态。我们使用这种技术来证明鼠伤寒沙门氏菌毒力的主要决定因素，即沙门氏菌致病性岛 2 III 型分泌系统，是细菌复制所必需的，但对杀灭抵抗力没有重大影响。此外，我们发现，进入巨噬细胞后，许多细菌不会复制，而是似乎进入休眠状态。这些可能是持久性细菌的重要储存库。该方法可以扩展到其他病原体，以研究毒力和宿主抗性因子对复制和杀死的贡献，并识别和表征与慢性或潜伏感染相关的非复制细菌。
Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize non-replicating bacteria associated with chronic or latent infections.