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

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.

Keywords: Klebsiella pneumoniae; amino acid biosynthesis; aroA; glyphosate; multidrug resistant; neutropenic.

FIG 1

Neutrophil-depleted mice have a higher bacterial burden despite similar limited bioavailability of amino acids in the lungs. (A to D) Mock-depleted (circles) and αGr1 (squares) mice were infected with 1 × 10³ CFU of WT K. pneumoniae or PBS (baseline) and sacrificed at 40 hpi. (A and B) BAL fluid was analyzed by NMR, and the percentage of each metabolite normalized to the total measured metabolites in the lung is reported. “^Tyr” is conditionally essential in the absence of Phe in mammals. (C) CFU/g lung for each mouse. (D) PMN population (Ly6G⁺ CD11b⁺) assessed by flow cytometry. Each symbol represents an individual mouse, and the bar represents the geometric mean. Each condition was tested with two to four mice per cohort in at least two independent experiments. The data are combined from all experiments. Statistical significance was determined using two-way ANOVA on log-transformed values with Bonferroni’s posttest (A and B) and the Student t test on log-transformed (C) and (D) nontransformed values. **, P < 0.01; ***, P < 0.001.

FIG 2

K. pneumoniae genes encoding enzymes required for amino acid biosynthesis are attenuated in the lungs of WT and neutropenic mice. (A to D) Mock-depleted and αLy6G mice were infected with 2 × 10⁴ CFU of a minilibrary of K. pneumoniae Tn mutants and sacrificed at 33 hpi. (A) CFU/g lung for each mouse. (B) Percent PMN population (Ly6G⁺ CD11b⁺) per live cells. (C and D) Abundance of each Tn strain in the output versus the input pool in mock-depleted (C) and αLy6G (D) mice. (E) CFU in lungs of mice infected with 1 × 10³ CFU of the indicated strains and sacrificed at 45 hpi. (F) CFU in lungs of Mock-depleted (circles) and αGr1 (squares) mice infected with 2 × 10³ CFU of the indicated strains and sacrificed at 40 hpi. (G) CFU of mice infected with 2 × 10⁴ CFU of the indicated strains and sacrificed at 33 hpi. Each symbol represents a mouse; open symbols indicate no CFU were recovered and limit of detection, and dashes represent geometric mean (A, C to G) or mean (B). Each strain was tested in two to four mice per cohort in at least two independent experiments. The data are combined from all experiments. Statistical significance was determined by Student t test (A and B) on log transformed (A) and nontransformed (B) values or by one-way ANOVA with Dunnett’s posttest (C and D) on log-transformed values by comparing each mutant against the neutral mutants present at 5.8% in the input pool. To assess whether Tn mutants had statistically significant fitness differences in αLy6G mice compared to WT, the fitness of each mutant in WT mice was compared to the fitness of the same mutant in αLy6G mice using one-way ANOVA with Dunnett’s posttest. No mutants had a significantly higher value in αLy6G mice than WT. (E to G) One-way ANOVA on log-transformed values using Dunnett’s (E) or Tukey’s (F and G) posttests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

aroA inhibition attenuates K. pneumoniae growth in the lungs. (A) The indicated strains were grown in L with or without iron-chelator DIP (0.25 or 1 mM) for 16 h. (B) WT K. pneumoniae was grown in M9+glucose for 16 h in a dose curve of glyphosate. The OD₆₃₀ after 16 h of growth is shown. (C) Strains were grown in M9+glucose with 10 mM phenylalanine (F), tryptophan (W), or tyrosine (Y) and/or 250 μg/ml glyphosate (G) as indicated. (D and E) Swiss-Webster (D) or αLy6G (E) mice were infected with 2 × 10⁴ CFU of WT and mock treated with PBS (circles) or 0.5 mg of glyphosate (triangles) at 6 and 24 hpi and harvested at 30 hpi, and the CFU from the lungs were determined. Each symbol represents a mouse; the bar represents the geometric mean. Experiments were performed independently at least twice with three technical replicates (A to C) or at least three times (D and E) with two to four mice per cohort. The data are from representative experiments (A to C) or combined from all experiments (D and E). Statistical significance was determined by one-way ANOVA with Sidak’s posttest on EC₅₀ values (A), Student t test on log-transformed values (D), or Mann-Whitney test (E). *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 4

Glyphosate-resistant mutants remain susceptible to glyphosate in infection. (A) The indicated strains were grown in M9+glucose for 16 h. (B and C) The indicated strains were grown in M9+glucose with various glyphosate, and the OD₆₃₀ after 16 h (B) or 24 h (C) of growth is shown. (D and E) The aroA mRNA copy number of the indicated strains was quantified using qRT-PCR after growth to mid-log phase in M9+glucose. (F and G) CFU counts in lungs of mice infected with 2 × 10⁴ CFU of the indicated strains, mock treated with PBS (circles), or treated with 0.5 mg of glyphosate (triangles) at 6 and 24 hpi and harvested at 30 hpi. Each symbol represents a mouse; open symbols indicate that no CFU were detected at limit of detection; bars indicate the geometric means. Experiments were done independently at least twice with groups of two to three mice. The data are from a representative experiment of three performed in triplicate (A to C) or are combined from all experiments (D to G). Significance was determined using one-way ANOVA on log-transformed values with Dunnett’s (D and E) or Fisher LSD (F and G) posttest. NS (not significant), P > 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 5

Sensitivity of clinical blood and respiratory K. pneumoniae isolates, including ESBL- and KPC-producing strains, to glyphosate. (A and B) Clinical K. pneumoniae strains were grown in M9+glucose containing 250 μg/ml glyphosate (black, non-EBSL/KPC; red, ESBL; or blue, KPC) or M9+glucose alone (gray, K. pneumoniae 43816; see also Fig. S3C and D in the supplemental material) for 20 h. (C) Mutations in aroA from B3 and B19 were introduced into the K. pneumoniae 43816 ΔaroA strain and grown in M9+glucose with the indicated concentrations of glyphosate for 16 h. (D) The indicated strains were grown in M9+glucose with glyphosate; the OD₆₃₀ at 16 h is shown. Experiments were performed at least twice with three technical replicates. The data are combined from all experiments (A to C) or are from a representative experiment (D). Statistical significance was determined by using one-way ANOVA with the Fisher LSD test (A and B) or Sidak’s posttest (C). *, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.