Mapping Essential Genes in Klebsiella pneumoniae Using Transposon-Insertion Sequencing (Tn-seq)

Abstract

Klebsiella pneumoniae is a common cause of sepsis in adults and children. Given the emergence of multidrug-resistant isolates, there is an urgent need to develop novel therapeutic strategies. Essential bacterial genes and their products, which are critical to survival and replication, represent intrinsic vulnerabilities that can be leveraged as novel drug targets. A comprehensive, empirically derived essential genome required for growth under conditions relevant to human infection biology and conserved across a majority of clinically relevant strains of K. pneumoniae is currently lacking. To define the core essential genome of K. pneumoniae, libraries comprising hundreds of thousands of individual transposon-insertion mutants were generated across multiple strains using a transposon mutagenesis vector (pSC189). To determine the K. pneumoniae strain MGH_66 genes required for growth in laboratory media, approximately 150,000 unique mutants were grown on lysogeny broth agar (LBA), and their genomic DNA (gDNA) was extracted. The gDNA was fragmented, adapter-ligated, and PCR-amplified using custom primers targeting transposon–genome junctions. Libraries were sequenced on an Illumina MiSeq platform, and insertion sites were mapped to the K. pneumoniae MGH_66 genome using Bowtie2 software. Gene essentiality was analyzed using the statistical analysis software FiTnEss. The library of 150,000 K. pneumoniae MGH_66 transposon-insertion mutants saturated approximately 50% of all permissive transposon-insertion sites. Based on individual p values, 544 genes were predicted to be essential for K. pneumoniae MGH_66 growth on LBA, including gyrA, fusA, and argS; which encode subunit A of DNA gyrase, elongation factor G, and arginyl-tRNA synthetase, respectively. The present dataset was not powered to detect statistically significant essential genes after correction for multiple hypothesis testing. These data suggest 544 K. pneumoniae MGH_66 genes essential for growth on laboratory media. Future work will focus on increasing statistical power of the present dataset and using this workflow to identify genes essential for growth in host-specific environments (e.g., blood, urine) for MGH_66 and additional strains. The resulting core essential genome will comprise genes required for growth across all tested conditions and strains. These findings may inform the identification of potential therapeutic targets for multidrug-resistant K. pneumoniae.