Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference.
Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference.
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沙眼衣原体是一种发育调节的专性细胞内细菌，编码三种σ因子：σ66，σ54和σ28。σ66是在早期和中期周期发育期间控制大多数转录起始的主要σ因子，因为感染性基本体（EB）转变为在细胞内的包涵体内复制的非感染性网状体（RB）。到目前为止，σ54和σ28这两个次要σ因子的作用还没有得到很好的表征;然而，有数据表明，随着RB向EB的过渡，它们在后期发育和次生分化中发挥作用。由于次级分化的过程本身的特点很差，澄清这些替代sigma因子的功能，通过确定由它们调节的基因，将进一步我们的衣原体分化的理解。我们推测σ54和σ28在启动衣原体晚期基因转录从而介导次级分化中具有非冗余和必需的功能。在这里，我们展示了每个次要西格玛因素在成功完成开发周期中的必要性。我们已经实施并验证了多路复用的规则间隔短回文重复序列（CRISPR）干扰技术，这是衣原体领域的新技术，用于检查单独和同时敲除每个替代西格玛因子的效果。同时，我们也过表达了每个sigma因子。与对照相比，改变任一种或两种替代西格玛因子的转录水平导致EB产生的严重缺陷。此外，RNA测序鉴定了替代西格玛因子失调期间差异表达的基因，表明了每个基因的推定调节子。这些数据表明，必须仔细调节替代σ因子的水平，以促进衣原体的生长和分化。 沙眼衣原体是一种重要的人类病原体，在发达国家和发展中国家。由于生物体独特的发育周期和细胞内生态位，基础研究一直缓慢而艰巨。然而，最近在衣原体遗传学的进展，使该领域取得重大进展，在实验询问衣原体的基本生理。一般来说，衣原体发展的驱动因素知之甚少，特别是关于发展的后期阶段是如何调节的。在这里，我们采用了一种新的遗传工具，用于衣原体，同时调查的影响失调的两个替代西格玛因子在生物体中，帮助控制转录起始。我们为sigma因子在晚期发育中的重要作用及其潜在的调节子提供了进一步的证据，为更深入的实验奠定了基础，以揭示衣原体分化所涉及的分子途径。
Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia. Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia. Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.
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