Abstract
Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.
Highlights
Urinary tract infections (UTIs) are one of the most common infections worldwide, affecting nearly half of all women at least once in their lifetime [1]
Because 500 μM CuSO4 did not affect the growth of either EC958 or E. coli K-12 strain MG1655 in Lysogeny broth (LB) (SI Appendix, Fig. S1B), we attribute this difference in copper sensitivity to the different media used
The zinc toxicity antibacterial pathway was shown to be operational in the soil amoeba Dictyostelium discoideum [40], indicating that this is an evolutionarily conserved innate immune response
Summary
Urinary tract infections (UTIs) are one of the most common infections worldwide, affecting nearly half of all women at least once in their lifetime [1]. To investigate mechanisms contributing to differential susceptibility of zinc-sensitive EC958 versus MG1655 mutants to killing by macrophages (Fig. 3), we generated a corresponding MG1655 zinc stress reporter strain (MG1655 pQF50-zntA-mCherry).
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