Department of Developmental and Cell Biology, University of California, Irvinegrid.266093.8, California, USA.
Department of Microbiology and Molecular Genetics, University of California, Irvinegrid.266093.8, California, USA.
mBio. 2022 Aug 30;13(4):e0107622. doi: 10.1128/mbio.01076-22. Epub 2022 Jun 15.
Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progeny). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.
衣原体是专性细胞内细菌，存在于一个称为衣原体包含体的膜结合隔室内，位于真核宿主细胞内。这些病原体具有复杂的两相发育周期，包括在复制但非感染性的网状体（RB）和感染性的原始体（EB）之间的转换。已经报道小分子抑制剂对细胞内衣原体感染具有有害影响，但这些研究在测定和分析时间点方面通常受到限制。我们比较了已发表和新的抑制剂，并表明它们可以差异地改变包含体大小、衣原体数量和感染性 EB 的产生，并且这些效应可以在细胞内感染过程中发生变化。我们的结果为使用多种测定方法进行分析提供了依据，这些方法可以在感染结束时或在时间过程中进行。我们还表明，这种方法有可能确定抑制剂影响发育周期的特定步骤。我们进一步提出，通过使用称为最大后代产生（Progeny）的新值来最好地量化和比较抑制剂诱导的后代缺陷的程度。作为对这种系统方法有效性的证明，我们将其应用于 Akt 和 AMPK 的抑制剂，这两种抑制剂是参与脂质合成和胆固醇转运途径的宿主激酶。两种抑制剂都减少了 EB 的产生，但 Akt 阻断主要降低了 RB 到 EB 的转化，而 AMPK 抑制则反常地增强了 RB 的复制。衣原体是美国报告的细菌性性传播感染的最常见原因。这种细菌感染人类细胞，并通过一种不寻常的发育周期在细胞质包含体内繁殖，该周期涉及两种特殊的衣原体形式。已经报道小分子化合物会对包含体以及衣原体复制和感染性后代产生产生负面影响，但我们表明这些效应可能不一致，并在 48 至 72 小时的细胞内感染过程中发生变化。我们提出了分析这些非均匀效应的方法，包括在细胞内感染结束时进行测量，以及在发育周期过程中进行多个测定的更详细分析。然后，我们应用这种方法来研究和比较两种改变宿主脂质合成和胆固醇转运的抗衣原体效应抑制剂。
