Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2

Abstract

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Fig. 1.

Glycosylated enterobactin but not yersiniabactin promotes growth in human serum. Overnight growth in 10% heat-inactivated human serum with or without 1.6 μM recombinant human Lcn2 was determined for the K. pneumoniae strains indicated (KP #). The means ± standard errors of the means for at least three independent experiments are shown as log₁₀ CFU/ml. The siderophore genotype of each strain is indicated by plus signs (+). For clinical isolates, the site of infection is noted as R (respiratory), B (blood), U (urine), or S (stool). *, growth is inhibited by rLcn2 as measured by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). &, growth is inhibited in serum alone compared to growth of KP7 by one-way analysis of variance (ANOVA) (&, P < 0.05; &&, P < 0.01; &&&, P < 0.001).

Fig. 2.

Siderophores are not required for growth in human urine. Overnight growth in pooled human urine with or without 1.6 μM recombinant human Lcn2 was determined for the K. pneumoniae strains indicated (KP #). The means ± standard errors of the means for at least three independent experiments are shown as log₁₀ CFU/ml. The siderophore genotype of each strain is indicated by plus signs (+). For clinical isolates, the site of infection is noted as R (respiratory), B (blood), U (urine), or S (stool). *, growth is inhibited by Lcn2 as measured by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). &&&, growth is inhibited in urine compared to growth of KP4 by one-way ANOVA (P < 0.001). ##, growth is stimulated by Lcn2 as measured by unpaired t test (P < 0.01).

Fig. 3.

Yersiniabactin is sufficient to evade Lcn2 and cause pneumonia but not sepsis. (A) Lung bacterial burden (log₁₀ CFU) at day 3 after retropharyngeal inoculation of 1 × 10⁴ CFU of the K. pneumoniae mutants indicated (KP #) was determined in C57BL/6 mice (n ≥ 10 mice per group). Box-and-whisker graph shows the median and interquartile ranges. *, P < 0.05 as determined by one-way ANOVA with Tukey's post test. ns, not significant. (B, C) Lung (B) and spleen (C) bacterial burdens at day 3 after retropharyngeal inoculation of 1 × 10⁴ CFU of the K. pneumoniae mutants indicated were compared between C57BL/6 (Lcn2^+/+) and isogenic Lcn2^−/− mice (≥5 mice per group). *, P < 0.05, and ***, P < 0.001, as determined by the Mann-Whitney test. Box-and-whisker graphs show the median and interquartile ranges. LOD, limit of detection. (D) The percentages of dead or moribund mice were determined at day 3 after inoculation. *, P < 0.05, and ***, P < 0.001, as determined by the log rank test. The siderophore genotype of each strain is indicated by plus signs (+). ns, not significant.