Department of Clinical Laboratory, Shenzhen Third People's Hospital, National Clinical Research Center for Infectious Diseases, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong Province, China.
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, Guangdong Province, China.
Appl Environ Microbiol. 2023 Oct 31;89(10):e0095623. doi: 10.1128/aem.00956-23. Epub 2023 Oct 10.
is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent . The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for to identify genes that play critical roles in antimicrobial fitness and host immunity . Targeting 870 predicted CE genes in , Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes and under trimethoprim pressure. Our screening also identified genes and related to polymyxin and β-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, , and were demonstrated to contribute to the pathogenesis of . This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in , and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in to identify genes that play critical roles in the fitness of antimicrobials and host immunity . The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in , and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.
是一种普遍存在的人类病原体，其临床治疗面临两大挑战：多药耐药性和高毒力性病原体的发病机制。由于新型抗生素的发展受到限制，寻找和研究可以作为潜在抗菌靶点的条件必需（CE）基因一直是研究关注的焦点。然而，缺乏必需功能基因组数据阻碍了与抗菌药物敏感性相关的必需基因机制的研究。在这项研究中，我们开发了一种用于的群体 CE 基因移动聚类规则间隔短回文重复（CRISPR）干扰筛选方法（Mobile-CRISPRi-seq），以鉴定在抗菌药物适应性和宿主免疫中起关键作用的基因。针对 870 个预测的 CE 基因，Mobile-CRISPRi-seq 在甲氧苄啶压力下发现四氢叶酸合成途径基因 和 的耗竭。我们的筛选还通过应用基于 Mobile-CRISPRi-seq 和比较基因组学的筛选策略，鉴定了与多粘菌素和β-内酰胺敏感性相关的基因 和 。此外，使用小鼠感染模型和 Mobile-CRISPRi-seq，鉴定了多个毒力基因，其中基因 、 和 被证明有助于 的发病机制。这项研究为筛选潜在的抗菌靶点和 在 中的毒力基因提供了一个简单、快速和有效的平台，这个广泛适用的系统可以扩展到多种致病菌，特别是革兰氏阴性菌的高通量功能基因研究。重要性发现和研究可以作为潜在抗菌靶点的条件必需（CE）基因一直是研究关注的焦点，因为新型抗生素的发展受到抗菌靶点的限制。在这项研究中，我们在 中开发了一种群体 CE 基因广泛的移动聚类规则间隔短回文重复（CRISPR）干扰测序（Mobile-CRISPRi-seq）策略，以鉴定在抗菌药物适应性和宿主免疫中起关键作用的基因。数据表明，这是一种在群体基因敲低文库中筛选失活表型的强大工具，Mobile-CRISPRi-seq 可能会扩展到多种细菌，用于筛选和鉴定在抗菌药物适应性和宿主适应性方面起关键作用的基因。
