The Klebsiella pneumoniae ter Operon Enhances Stress Tolerance

Abstract

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term-care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K₂TeO₃) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to radical oxygen species-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify the genes that are required to resist K₂TeO₃-induced and polymyxin B-induced stress, which suggested that K₂TeO₃-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K₂TeO₃ disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as a stress tolerance factor, thereby explaining its role in enhancing fitness in the gut and bladder.

Keywords: Klebsiella; tolerance; transposons; urinary tract infection.

FIG 1

TerC is necessary for complete fitness in the bladder during a urinary tract infection. Mice were transurethrally inoculated with approximately 10⁸ CFU of a 1:1 mix of WT NTUH-K2044 and NTUH-K2044ΔterC. (A) The bacterial burden in the urine and bladder was measured after 48 h, and (B) the log₁₀ competitive index (CI) of the mutant strain, compared to the WT strain, was calculated for each organ (N = 17; mean displayed; ****, P < 0.00005; one-sample t test). From 20 inoculated mice, CFU were recovered from 17 mice. The numbers above each column in panel A indicate the number of mice (of 17) with detectable CFU. Blue circles had no recoverable NTUH-K2044ΔterC. The CI was calculated using the limit of detection CFU value when no CFU were recovered.

FIG 2

TerC is necessary for tolerance to several stresses. The (A) K₂TeO₃ MIC (i: n = 5 independent experiments; median displayed; **, P < 0.005; ratio paired t test) was calculated for the NTUH-K2044 and NTUH-K2044ΔterC strains using broth microdilution. Kill curves (ii: n = 7 independent experiments; mean displayed ± SEM) were generated for these strains via the addition of a standard K₂TeO₃ concentration of 1 mM to overnight cultures. The (iii) AUC and (iv) MDK were calculated from these kill curves (n = 7 per group; *, P < 0.05; **, P < 0.005; ratio paired t test; ∞ indicates an incalculable MDK). Finally, the NTUH-K2044 pVector, NTUH-K2044ΔterC pVector, and NTUH-K2044ΔterC pTerZ-F survival was assessed at 4 h (240 min) post 1 mM K₂TeO₃ exposure (v: n = 6 to 7 independent experiments; median displayed; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; one-way ANOVA followed by Tukey’s multiple-comparison test). These experiments were repeated for ofloxacin (B), polymyxin B (PmB) (C), and cetylpyridinium chloride (CPC) (D). The standard concentrations for the killing assays for ofloxacin, polymyxin B, and cetylpyridinium chloride were 250 μg/mL, 500 μg/mL, and 25 μM, respectively. For subpanels iii, iv, and v, the connecting lines indicate paired biological replicates.

FIG 3

Systematic screen of K₂TeO₃ resistance. The KPPR1 strain, which lacks the ter operon, as well as the the NTUH-K2044 and NTUH-K2044ΔterC strains were cultured in increasing concentrations of K₂TeO₃ (A). The area under the curve (AUC) was calculated from these dose-response curves (B) (mean displayed ± SEM; ****, P < 0.0005; one-way ANOVA followed by Tukey’s multiple-comparison test). (C) 3,733 individual Tn insertion mutants were cultured in the presence of 1 μM K₂TeO₃ and were measured at OD₆₀₀. The blue line is the mean OD₆₀₀, and the red lines are ± 2 SD from the mean. Each symbol is an individual mutant, ordered by its gene number (VK055_#). (D) Exact K₂TeO₃ IC₅₀ values of validated Tn insertion mutants (n = 3 to 5 independent experiments). (E) KEGG BRITE functional hierarchies were assigned for the validated Tn insertion mutants. The highest order hierarchies are shown in the horizontal stacked bar chart, and the second-highest order hierarchies are shown in the unstacked bar chart. (F) The validated Tn insertion mutants were cultured in the presence of 0.5 μg/mL PmB (n = 3 independent experiments).

FIG 4

EtBr accumulates in K₂TeO₃ and polymyxin B treated Kp. Stationary-phase NTUH-K2044 pVector, NTUH-K2044ΔterC pVector, and NTUH-K2044ΔterC pTerZ-F were exposed to 1 mM K₂TeO₃ or 500 μg/mL PmB. EtBr accumulation (fluorescence) was measured at the baseline and at 1 h postexposure (n = 6 independent experiments; mean displayed; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; one-way ANOVA followed by Tukey’s multiple-comparison test).