Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq

Zhu，Qing1; Lin，Qiang2; Jiang，Yushan3; Chen，Shuyan4; Tian，Junxuan1; Yang，Shijin1; Li，Yuanchun1; Li，Mengjun3; Wang，Yuelin3; Shen，Chenguang3; Meng，Songdong5,6; Yang，Liang4,7,8; Feng，Youjun1,9; Qu，Jiuxin1

2023-10-31

10.1128/aem.00956-23

Applied and environmental microbiology   影响因子和分区

1098-5336

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. 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 K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA 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, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, 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 Klebsiella pneumoniae to identify genes that play critical roles in the fitness of antimicrobials in vitro and host immunity in vivo. The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in K. pneumoniae, 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.

antimicrobial target conditionally essential gene Klebsiella pneumoniae Mobile-CRISPRi-seq virulence gene

EI ; SCI

英语

第一

20234615069665

Antibiotics ; Bacteria

Biological Materials and Tissue Engineering:461.2 ; Medicine and Pharmacology:461.6

BIOLOGY & BIOCHEMISTRY

2-s2.0-85175586530

Scopus

1.Department of Clinical Laboratory,Shenzhen Third People's Hospital,National Clinical Research Center for Infectious Diseases,Second Affiliated Hospital of Southern University of Science and Technology,Guangdong Province,Shenzhen,China
2.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,Guangdong Province,Shenzhen,China
3.BSL-3 Laboratory (Guangdong),Guangdong Provincial Key Laboratory of Tropical Disease Research,School of Public Health,Southern Medical University,Guangdong Province,Guangzhou,China
4.Shenzhen Third People's Hospital,National Clinical Research Center for Infectious Diseases,Second Affiliated Hospital of Southern University of Science and Technology,Guangdong Province,Shenzhen,China
5.CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology,Center for Biosafety Mega-Science,Chinese Academy of Sciences (CAS),China
6.University of Chinese Academy of Sciences,China
7.School of Medicine,Southern University of Science and Technology,Guangdong Province,Shenzhen,China
8.Key University Laboratory of Metabolism and Health of Guangdong,Southern University of Science and Technology,Guangdong Province,Shenzhen,China
9.Departments of Microbiology and General Intensive Care Unit of the Second Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou,Zhejiang Province,China

Zhu，Qing,Lin，Qiang,Jiang，Yushan,et al. Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq[J]. Applied and environmental microbiology,2023,89(10).

Zhu，Qing.,Lin，Qiang.,Jiang，Yushan.,Chen，Shuyan.,Tian，Junxuan.,...&Qu，Jiuxin.(2023).Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.Applied and environmental microbiology,89(10).

Zhu，Qing,et al."Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq".Applied and environmental microbiology 89.10(2023).