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Santamaría Amorocho, Valentina

University of British Columbia

In the context of infection, a challenge for pathogenic bacteria is to maintain iron homeostasis with varying iron availability due to changes in pH, oxygen concentration, and host nutritional immunity. Organisms such as Campylobacter jejuni and uropathogenic Escherichia coli (UPEC) strains use the Fet system to acquire iron and overcome these changes. In C. jejuni, the Fet system is composed of eight genes (fetMP-fetABCDEH). In both species, FetM and FetP interact and are proposed to uptake ferric and ferrous iron. The role of fetABCDEH in iron uptake or as a mechanism to overcome stress has not been characterized in UPEC and is still not well understood. The iron/manganese-deficient strain ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH) was engineered to characterize the Fet system in a gain-of-function model. Using the chassis-independent recombinase-assisted genome engineering (CRAGE) system, fetMP, fetMP-fetABCDEH and fetAH were integrated into the genome of ECA458. The resulting strains (ECA-fetMP, ECA-fetMH, ECA-fetAH) were used for phenotypic characterization of the Fet system with E. coli K-12 W3110 (WT), ECA458 and ECA-LP (ECA458 with a landing pad in fliK) as controls. Growth on LB and iron-limited LB, response to oxidative and nitrosative stress, and tolerance to streptomycin were assessed for these strains. Strains ECA458 and ECA-LP had similar phenotypes such as limited growth, increased oxidative stress sensitivity and streptomycin tolerance compared to WT. The gain-of-function approach validated the role of FetMP in growth enhancement under iron restriction, increased sensitivity to oxidative stress and streptomycin compared to ECA458. The phenotypes for strains expected to express fetMP-fetABCDEH and fetABCDEH were similar to those seen in ECA458 and ECA-LP. In these strains, the expression of the fet genes in this model may be limited by unknown factors and further studies are required to define expression of fet genes by strains ECA-fetMH and ECA-fetAH. This work was a novel approach to assess the role of the Fet system in E. coli and support a role of FetMP in iron uptake and oxidative stress.

Text

2025-06-23

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/91409

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/