Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois, USA.
Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois, USA
Infect Immun. 2019 Oct 18;87(11). doi: 10.1128/IAI.00374-19. Print 2019 Nov.
It was previously determined that the type II secretion system (T2SS) promotes the ability of to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of intracellular infection.
先前已经确定 II 型分泌系统（T2SS）促进了在与变形虫共培养时生长的能力。在这里，我们通过比较野生型和 T2SS 突变体军团菌来辨别增强的细胞内感染阶段，以评估它们寄生的能力。尽管突变体在进入宿主细胞和随后逃避降解溶酶体方面表现正常，但它在复制能力方面存在缺陷，该缺陷在进入后约 9 小时首次显现。该复制缺陷最初通过三种方式进行了记录：通过确定从感染单层细胞的裂解物中回收的 CFU 数量，通过监测与表达绿色荧光蛋白（GFP）的细菌感染的变形虫单层相关的荧光水平，以及通过利用流式细胞术来定量个体变形虫中表达 GFP 的细菌数量。通过使用共聚焦显微镜和更新的成像技术，我们进一步确定了细菌空泡的体积和形状的进展，并发现 T2SS 突变体以较低的速度生长，并且不能达到最大的吞噬体大小。总体而言，与野生型菌株相比，T2SS 突变体的整个感染周期（即进入到退出）都明显较慢，并且该突变体的缺陷在多次感染循环中都得以维持。因此，T2SS 对于在变形虫内的空泡内生长到更多数量的是绝对必需的。将这些结果与我们最近对巨噬细胞感染的分析结果相结合，T2SS 显然是的细胞内感染的主要组成部分。
