Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice

Abstract

Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae

“…KPPR1S produces the enterobacterial common antigen (ECA) and LPS. A recent report showed that mutants in LPS and ECA pathways had reduced HMV (determined by sedimentation) ( 34 , 37 ). To more clearly ascertain if either of these exopolysaccharides contributed to HMV, we tested a strain with a disruption in wecA , which encodes an undecaprenyl phosphotransferase; this mutant produces normal levels of UA but lacks both LPS and ECA and is attenuated in the pneumonia model ( 38 ).…”

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impactKlebsiella pneumoniaefitness

“…Even fuller datasets may be achieved by accessing the two other ordered K. pneumoniae transposon libraries, in addition to the one generated here. One such library contains 12,000 strains that correspond to 4,583 ORFs in strain KPN1H1 with the KPC-3 carbapenemase gene deleted and the other library has approximately 4,570 mapped transposon insertion site in ATCC 43816, although the number of unique ORFs disrupted is unclear [53, 54]. Altogether, these libraries represent invaluable genetic resources that will not only advance our understanding of K. pneumoniae pathogenesis and molecular biology within these specific strains, but can also be used as templates to generate insertional mutants in other strains or to study the contribution of individual domains to phenotypes of interest.…”

Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

“…Finally, there are some important common factors among the requirements for infection of G. mellonella , and for an hv Kp murine lung infection (Paczosa et al . 2020 ). Genes contributing to both infection types include some of those encoding ubiquitous virulence factors such as capsule or siderophore importers.…”