Conditioning of uropathogenic Escherichia coli for enhanced colonization of host

Abstract

While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) encounters multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Here we show that UPEC, the primary cause of urinary tract infections, can be conditioned to grow at higher rates in the presence of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned UPEC bacteria are far more likely to establish an infection than nonconditioned bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several NsrR-regulated genes and other stress- and polyamine-responsive factors may be partially responsible for this effect. Compared to K-12 reference strains, most UPEC isolates have increased resistance to ASN, and this resistance can be substantially enhanced by addition of the polyamine cadaverine. Nitrosative stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also promote UPEC colonization of the bladder. These results suggest that UPEC interactions with polyamines or stresses such as reactive nitrogen intermediates can in effect reprogram the bacteria, enabling them to better colonize the host.

FIG. 1.

UTI89 adapts to growth in the presence of 3 mM ASN. (A) Diagram of the experimental protocol used. Colonies of UTI89 grown from a freezer stock were used to start overnight cultures in MES-LB broth without ASN. For each round, cultures were grown with shaking in tubes at 37°C and allowed to reach stationary phase before they were subcultured 1:100 into new media with or without ASN, as indicated. Culture growth was monitored by determining the OD₆₀₀. (B) Growth curves of cultures with ASN indicated in the diagram in panel A, demonstrating the difference in lag time between cultures conditioned with 3 mM ASN and cultures not conditioned with 3 mM ASN. The error bars indicate standard deviations from the means of five independent cultures.

FIG. 2.

Enhanced colonization of the bladder by ASN-conditioned UPEC. Adult female CBA/J mice were inoculated with 10⁷ CFU of UTI89 that had been grown to an OD₆₀₀ of 1.5 in MES-LB broth with or without 3 mM ASN. Bacterial titers in bladder homogenates were determined at 12 h or 5 days postinoculation. The horizontal bars indicate median values for the groups. The P values were determined using Fisher's exact test (n = 13 to 22 mice).

FIG. 3.

Functional classification of transcripts affected ≥5-fold by 3 mM ASN in the growth medium, as determined by microarray analysis. Some transcripts were identified by multiple probe sets listed in Table S1 in the supplemental material, but for clarity each transcript is shown only once.

FIG. 4.

Cadaverine stimulates UPEC growth in the presence of ASN and colonization of the bladder. (A) MG1655 (blue), 28 pyelonephritis isolates (orange), and 21 cystitis isolates (yellow) were diluted from overnight MES-LB broth cultures into fresh medium containing 3 mM ASN with or without 3 mM cadaverine. The top of each bar (− cad) indicates the time required for the individual isolate to reach an OD₆₀₀ of 0.5 in ASN-containing broth, while the bottom of each bar (+ cad) indicates the time required to reach the same OD₆₀₀ with both ASN and cadaverine present. (B) Data in panel A grouped to more clearly show the overall effect of cadaverine (cad) on growth of the UPEC isolates in the presence of 3 mM ASN. The P values were calculated using the Mann-Whitney U test. (C) Adult female CBA/J mice were inoculated with 10⁷ CFU of UTI89 that had been grown to an OD₆₀₀ of 1.5 in MES-LB broth with or without 3 mM cadaverine. Bacterial titers in bladder homogenates were determined at 12 h and 5 days postinoculation. The P values were determined using Fisher's exact test (n = 14 to 22 mice). The horizontal bars in panels B and C indicate median values for the groups.