Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa.
Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa.
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DOI：
10.1016/j.mex.2022.101941
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发表时间：
2023
期刊：
影响因子：
1.9
通讯作者：
van Houte, Stineke
中科院分区：
文献类型：
--
作者：
Pons, Benoit J.;Westra, Edze R.;van Houte, Stineke
用携带acr基因的噬菌体感染铜绿假单胞菌菌株。用CRISPR-Cas系统靶向的质粒和非靶向的质粒转化细胞，以测量质粒的相对转化效率。细菌具有广泛的防御机制来对抗细菌特异性病毒（噬菌体）和其他入侵的移动的遗传因子。在这些机制中，“CRISPR-Cas”（CRISPR相关的规则间隔短回文重复序列）系统记录先前的感染以防止再次感染，从而提供获得性免疫。然而，细菌对基于CRISPR的细菌免疫并非毫无防御能力。事实上，它们可以通过编码一种或几种抗CRISPR（Acr）蛋白来逃避CRISPR系统。Acr蛋白是噬菌体感染后最早产生的蛋白质之一，因为它们需要在CRISPR-Cas系统破坏噬菌体遗传物质之前快速抑制CRISPR-Cas系统。因此，Acr不能完美地保护噬菌体免受CRISPR-Cas系统的影响，感染往往失败。然而，即使感染失败，Acr也可以诱导CRISPR-Cas系统的持久失活。本文提出的方法旨在通过以下方式评估Acr蛋白诱导的铜绿假单胞菌中的持久CRISPR-Cas抑制：该方法可适于测量在不同培养条件下影响Acr诱导的免疫抑制的参数。
Infecting the P. aeruginosa strain with a phage carrying an acr gene. Making the cell electrocompetent while eliminating the phage Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the ‘CRISPR-Cas’ (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa induced by Acr proteins by: This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions.
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