Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes.
Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes.
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DOI：
10.1128/mbio.00129-15
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发表时间：
2015-05-12
期刊：
影响因子：
6.4
通讯作者：
O'Toole GA
中科院分区：
文献类型：
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作者：
Heussler GE;Cady KC;Koeppen K;Bhuju S;Stanton BA;O'Toole GA
成簇规则间隔短回文重复 (CRISPR)/CRISPR 相关 (CRISPR/Cas) 系统是许多古细菌和细菌中存在的适应性免疫系统。 CRISPR/Cas 系统极其多样化，并且越来越多的证据表明 CRISPR/Cas 系统在不同于噬菌体免疫的细胞功能中发挥着作用。此前，我们的实验室报道了一种这样的替代功能，其中机会性病原体铜绿假单胞菌菌株 UCBPP-PA14（缩写为铜绿假单胞菌 PA14）的 1-F CRISPR/Cas 系统在细菌被噬菌体 DMS3 溶原时抑制生物膜形成和集群运动。在这项研究中，我们证明，铜绿假单胞菌基因组上仅存在 DMS3 原型间隔子和原型间隔子相邻基序 (PAM) 对于这种 CRISPR 依赖性的群体行为丧失是必要且充分的，而不需要额外的 DMS3 序列。我们还证明，CRISPR 系统与 DMS3 原型间隔子的相互作用通过核酸酶 Cas3 的活性和随后的 RecA 激活，诱导 SOS 调节的噬菌体相关基因的表达，包括充分表征的化脓菌素操纵子。此外，我们的数据表明，由于 CRISPR 接合菌株无法下调噬菌体相关基因的表达，噬菌体相关基因的表达导致表面上的细菌细胞死亡，而这些噬菌体相关基因对浮游条件下的生长和活力影响最小。噬菌体相关基因的删除可以恢复生物膜的形成和集群运动，同时仍然保持功能性的 CRISPR/Cas 系统，这表明这些群体行为的丧失是 CRISPR 自靶向的间接影响。在古细菌和细菌中发现的各种 CRISPR/Cas 系统极其多样化，对这些不同系统的复杂机制的了解的进展不仅增加了我们对宿主与病毒相互作用的了解，而且还导致了基因工程的重大进步。最近，越来越多的证据表明，除了对噬菌体感染的适应性免疫之外，细菌还可以利用 CRISPR 系统来发挥其他功能。这项研究检验了一种这样的替代功能，该报告描述了医学相关机会性病原体铜绿假单胞菌中 1-F 型 CRISPR 依赖性生物膜丢失和集群的机制。由于生物膜形成和集群运动对于铜绿假单胞菌的毒力都很重要，因此充分了解 CRISPR 系统如何调节此类群体行为对于开发新疗法至关重要。
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host-virus interplay but has also led to a major advancement in genetic engineering. Recently, increasing evidence suggested that bacteria can co-opt the CRISPR system for functions besides adaptive immunity to phage infection. This study examined one such alternative function, and this report describes the mechanism of type 1-F CRISPR-dependent loss of the biofilm and swarming in the medically relevant opportunistic pathogen Pseudomonas aeruginosa. Since both biofilm formation and swarming motility are important in the virulence of P. aeruginosa, a full understanding of how the CRISPR system can regulate such group behaviors is fundamental to developing new therapeutics.