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

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.

Amino Acid Biosynthetic Pathways Are Required for Klebsiella pneumoniae Growth in Immunocompromised Lungs and Are Druggable Targets during Infection.

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.

Lactosylceramide-induced apoptosis in primary amnion cells and amnion-derived WISH cells.

Amnion apoptosis is part of a programmed process of fetal membrane remodeling leading to weakening and rupture. The apoptotic agent lactosylceramide is elevated in amniotic fluid of premature infants with rupture of membranes. We have shown that apoptosis in WISH cells, induced by staurosporine, cycloheximide, or actinomycin D, can be blocked by cyclooxygenase inhibitors, suggesting a relationship between prostaglandin production and apoptosis. Cyclic adenosine monophosphate (cAMP) is known to inhibit prostaglandin release in amnion and WISH cells. This study was undertaken to determine the apoptotic potential of lactosylceramide and the effect of cyclooxygenase inhibitors and cAMP activators on lactosylceramide-induced apoptosis in primary amnion and WISH cells. Primary amnion cells and WISH cells were incubated with lactosylceramide to determine apoptosis and prostaglandin E(2) (PGE(2)) release. Apoptosis was confirmed by agarose gel electrophoretic DNA fragmentation analysis, nuclear matrix protein (NMP), and nucleosome enzyme-linked immunosorbent assay. In some studies, cells were preincubated with cyclooxygenase inhibitors or cAMP activators. Lactosylceramide induced a 20-fold increase in NMP (measure of cell death) in both cell types. Apoptosis was confirmed by the studies listed in methods. Lactosylceramide increased PGE(2) release in parallel with apoptosis. Cyclooxygenase inhibitors as well as cAMP activators inhibited both PGE(2) release and apoptosis. Lactosylceramide-induced apoptosis in both amnion and WISH cells. Parallel PGE(2) release was demonstrated with apoptosis. Cyclooxygenase inhibitors and cAMP activators blocked both processes.