Opposing roles for iron transport systems in gallium tolerance in extraintestinal pathogenic <i>Escherichia coli</i>

Gallium is a promising antibacterial candidate because it displaces iron atoms inside bacterial cells but does not undergo redox cycling. It inhibits growth by disrupting essential iron-dependent processes. However, <i>Escherichia coli</i> are naturally less sensitive to gallium than many other bacteria, and the mechanisms that control gallium tolerance are not completely understood. We performed a genome-wide transposon sequencing (TnSeq) screen to identify genes important for the survival of an extraintestinal pathogenic <i>E. coli</i> isolate (M12) in gallium nitrate. The TnSeq results indicated that inactivation of enterobactin siderophore-related genes (<i>entS</i>, <i>fepD</i>, <i>fes</i>, and <i>fepB</i>) enhances bacterial survival in gallium, while disrupting the ferric dicitrate transport system increases susceptibility. We validated these findings through targeted gene knockouts and gallium sensitivity experiments. Our findings suggest that enterobactin can complex with gallium for cellular uptake, but that the ferric citrate receptor FecA can discriminate between gallium citrate and iron citrate. Expression of <i>fecA</i> increased with gallium exposure, showing that gallium induces FecA-mediated iron uptake. Gallium also increased intracellular levels of manganese in the Δ<i>fecA</i> strain. Supplementation with iron or manganese restored growth of M12 Δ<i>fecA</i> in gallium, suggesting that gallium sensitivity is linked to both iron starvation and oxidative stress. As the ferric dicitrate transport system is an important virulence factor in several extraintestinal infection sites, our results suggest that targeting FecA may increase <i>E. coli</i> susceptibility to gallium while also suppressing virulence.IMPORTANCE<i>Escherichia coli</i> extraintestinal infections that are resistant to traditional antibiotics are associated with more deaths than any other species. Gallium-based therapies may represent a non-antibiotic approach for treating extraintestinal pathogenic <i>E. coli</i> strains that affect both humans and animals. Our results are significant as they show that the enterobactin siderophore and the ferric dicitrate iron transport systems expressed by these bacteria have opposing roles in <i>E. coli</i> gallium sensitivity. These findings could be leveraged to enhance the efficacy of gallium therapeutics.