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

Abstract

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.

Blunted blades: new CRISPR-derived technologies to dissect microbial multi-drug resistance and biofilm formation

“…CRISPRa in C. albicans identified Als1 as crucial for pathogenesis in a murine CAUTI model ( 63 ). CRISPRi has also been used in murine pneumonia models, which identified essential genes to virulence: exsA in P. aeruginosa ; pal , yciS , and rib in K. pneumoniae ; and purA in Streptococcus pneumoniae ( 48 , 50 , 62 ). Tailoring promoter strength for constitutive knockdown offers an alternative to induced CRISPRi, alleviating concerns over tissue penetration by some inducing agents ( 48 ).…”

“…This system has also proven useful in a screening strategy similar to Tn-Seq, to assess which genes or pathways have vital roles in antimicrobial resistance. Mobile-CRISPRi-Seq was applied to 870 predicted essential genes under antibiotic pressures at 25% MIC, which identified folB and folP as trimethoprim resistance genes ( 50 ). Additional genes of interest include waaE , a component of LPS biosynthesis and polymyxin B resistance, and fldA , an electron transport carrier and contributor to beta-lactam resistance ( 50 ).…”