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.

No abstract

No abstract

The emergence of multidrug-resistant Klebsiella pneumoniae has rendered a large array of infections difficult to treat. In a high-throughput genetic screen of factors required for K. pneumoniae survival in the lung, amino acid biosynthesis genes were critical for infection in both immunosuppressed and wild-type (WT) mice. The limited pool of amino acids in the lung did not change during infection and was insufficient for K. pneumoniae to overcome attenuating mutations in aroA, hisA, leuA, leuB, serA, serB, trpE, and tyrA in WT and immunosuppressed mice. Deletion of aroA, which encodes 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase class I, resulted in the most severe attenuation. Treatment with the EPSP synthase-specific competitive inhibitor glyphosate decreased K. pneumoniae growth in the lungs. K. pneumoniae expressing two previously identified glyphosate-resistant mutations in EPSP synthase had significant colonization defects in lung infection. Selection and characterization of six spontaneously glyphosate-resistant mutants in K. pneumoniae yielded no mutations in aroA. Strikingly, glyphosate treatment of mice lowered the bacterial burden of two of three spontaneous glyphosate-resistant mutants and further lowered the burden of the less-attenuated EPSP synthase catalytic mutant. Of 39 clinical isolate strains, 9 were resistant to glyphosate at levels comparable to those of selected resistant strains, and none appeared to be more highly resistant. These findings demonstrate amino acid biosynthetic pathways essential for K. pneumoniae infection are promising novel therapeutic targets.