Characterization of Siderophore-Host Interactions Emma Worden-Sapper Thesis Defense Date April 8, 2019 Thesis Advisor: Min Han, MCDB Honors Council Representative: Christy Fillman, MCDB Other Committee Member: Stephanie Renfrow, EBIO Abstract The role of the gut microbiome in influencing human health has received increasing attention in the past decade, with many of the interactions between symbiont and host being elucidated. A recent study found that the siderophore enterobactin, secreted by E. coli to scavenge iron from its environment, is an essential metabolite for Caenorhabditis elegans (C. elegans) development. The benefit of enterobactin, which appears to be conserved in mammals, carries potential for treating iron deficiency disorder. This thesis focuses on characterizing the nature of the interaction of C. elegans with siderophores other than enterobactin, to assess the uniqueness of the benefit enterobactin conveys to the host. By performing a series of assays with C. elegans, I provide evidence that yersiniabactin, ornibactin, and bacillibactin do not promote C. elegans growth, implying that these siderophores do not convey a benefit similar to enterobactin. 1. Introduction The increasing attention towards the health impacts of human microbial residents (the microbiome) has produced many surprising contradictions to the previous notions of the nature of host-microbe interactions. The microbiome has been found to impact mental health (Dinan and Cryan 2017; Hsiao et al. 2013; Sharon et al. 2014), the development of the immune system (Brestoff and Artis 2013; Hooper, Littman, and Macpherson 2012; Ellermann and Arthur 2017), and metabolism (Sharon et al. 2014; Ellermann and Arthur 2017) among many other physiological functions. One of these previous notions, that bacterial residents of the gastrointestinal (GI) tract “steal” iron from their hosts (Ellermann and Arthur 2017), has recently been challenged (Qi and Han 2018), suggesting a new interaction between host and resident that may carry implications for anemia treatments (Eschner 2018). Iron, as a metabolite that is essential for many biological processes, generates a conflict between host and resident, with both essentially fighting for its uptake (Ellermann and Arthur 2017). To this end, most bacteria secrete siderophores, small molecules which bind ferric (III) iron. After their reuptake by the cell—or by a different cell that is able to pirate the siderophore—the ferric iron is released from the siderophore and reduced to soluble ferrous (II) iron (Miethke and Marahiel 2007; Ellermann and Arthur 2017; Andrews, Robinson, and Rodríguez-Quiñones 2003). Siderophores have been thought to be key measures of bacterial virulence (Miethke and Marahiel 2007; Visser et al. 2004; Cassat and Skaar 2013). This paradigm is supported by the response of the mammalian host, which is to produce and secrete a protein (lipocalin-2) that binds and sequesters the E. coli produced siderophore enterobactin (Ellermann and Arthur 2017). The model organism Caenorhabditis elegans (C. elegans), a roundworm with a well-documented microbiome (Berg et al. 2016; Zhang et al. 2017; Shapira 2017), has provided a useful platform for testing this interplay between host and microbial resident. (Qi and Han 2018) recently revealed an interaction between C. elegans and one of its GI tract residents, E. coli, that challenged this canonical idea of siderophores as agents of virulence. Qi previously found that heat killed E. coli does not provide C. elegans with key nutrients required for normal growth (Qi, Kniazeva, and Han 2017), but that growth was rescued when a small amount of live, wild type E. coli was provided to the worms as a supplement. Thus, the live bacteria provide the worms with essential metabolites. However, replacing the live wild type E. coli with a live mutant unable to synthesize enterobactin yielded severely attenuated worm growth, implying that one of the key nutrients E. coli provides to its C. elegans host is enterobactin. Both worm growth and iron levels were rescued by the addition of enterobactin to this assay. This result carries great significance for the treatment of iron deficiency if it is conserved in mammals. Anemia affects roughly a quarter of the world’s population, especially the elderly (Tettamanti et al. 2010), children, pregnant women, and the populations of poorer countries (de Benoist B et al., eds. 2008; Pena-Rosas, Rogers, and Stevens 2015). Enterobactin, if it has the same effect in humans as in worms, could be used as a more effective treatment for this condition. The current treatment, iron supplements, poses several health threats, such as damage to the liver, heart, and other tissues that respond poorly to oxidative stress, due to the high reactivity of iron (Fisher and Naughton 2004; Wessling-Resnick 2017). There is still much unknown about the interaction between host and microbial residents, however, which must be elucidated prior to the use of treatments such as enterobactin. One of these unknowns is if siderophores other than enterobactin have the same beneficial effect on the host. This result would shed light on the role enterobacteria play in the gut as symbionts, and would potentially offer further explanation of the mediation of iron homeostasis in the host, an important clarification to make before introducing enterobactin to the host as a treatment for iron deficiency. If there is an interplay between beneficial siderophores, the host, and the symbionts in the GI tract, it would not be ideal to potentially interrupt this homeostasis with an abundance of a particular siderophore. Ruling out other siderophores would also allow for increased focus on enterobactin as the principal drug target. In this work, I addressed the question: Are siderophores other than enterobactin necessary or beneficial for C. elegans growth and/or iron levels? I tested C. elegans due to its previous use in the work in which the beneficial effect of enterobactin was discovered (Qi and Han 2018), making it the most likely organism to use other siderophores. In addition, C. elegans has a well- documented gut microbiome (Berg et al. 2016; Zhang et al. 2017; Shapira 2017). Knowing the types of bacteria likely to be present in the guts of wild-type worms narrows down the list of potentially beneficent siderophores, since if the worm does not live with a certain bacteria as a canonical symbiont, it is not likely to have evolved a way to use its siderophore. With this model, I performed the same assay as described by Qi (Qi and Han 2018) to test four siderophores, yersiniabactin, ornibactin, salmochelin, and bacillibactin. Of these, salmochelin requires further investigation, and the rest did not provide a benefit to the host. 2. Methods and Materials 2.1 C. elegans maintenance, strains, and preparation for plating C. elegans were grown on full bacterial lawns of OP50 (wild-type E. coli), on NGM (nematode growth medium) plates (20 mL petri dishes containing 10 mL NGM) at 20o C. Two different strains of C. elegans were used in this work, N2 (wild type) and an XA 6901 pftn-2::gfp reporter strain. This strain fluoresces with high iron levels (Qi and Han 2018), so it was used in order to analyze iron in the worm. While the reporter strain does not reach the same volume as the N2 strain (Fig. S2), the relative volumes are conserved. The N2 worms were used for the initial volumetric assays (Fig. 1, 2), and the reporter strain was used for the assay involving ampicillin (Fig. 3) and the assay performed over three days with full lawns of B. subtilis and Ent A- (Fig. S4), due to availability. The worms were plated at the L1 (juvenile) stage, roughly 300 worms per plate, roughly 5 plates per group. The worms were synchronized to L1 via bleaching of well-fed gravid adults the day before plating. 2.2 Characterization and maintenance of bacteria Four types of E. coli were used in this work: OP50, K12, Ent A-, and Ent F-, the former two being wild-type, the latter two being members of the Keio collection (Baba et al. 2006), a library of single knockouts of K12 genes. Ent A- and Ent F- are unable to synthesize enterobactin (Qi and Han 2018). Five types of B. subtilis were used in this work: wild-type B. subtilis, BKK 31990 ΔdhbC, BKK 31980 ΔdhbE, BKK 31970 ΔdhbB, and BKK 31960 ΔdhbF. The mutants are all knockouts for genes that encode enzymes that catalyze reactions in the bacillibactin biosynthesis pathway, and are thus unable to synthesize bacillibactin (Pi and Helmann 2018; May, Wendrich, and Marahiel 2001; Caspi 2008). BKK 31990 ΔdhbC is a knockout of ΔdhbC, BKK 31980 ΔdhbE is a knockout of ΔdhbE, BKK 31970 ΔdhbB is a knockout of ΔdhbB, and BKK 31960 ΔdhbF is a knockout of ΔdhbF. The bacterial mutants were streaked onto LB (lumina broth) plates with kanamycin, and the wild-type strains were streaked onto regular LB plates. 2.3 Preparation of bacteria Full lawns of bacteria were spotted onto NGM plates from an overnight culture in LB at 37o C. Roughly 0.5 mL of culture was used per plate. Small food spots for the assays with heat killed bacteria were prepared similarly in LB. The mutants were incubated in LB treated with 0.05 mg/mL kanamycin, while the wild type strains were incubated in untreated LB. Optical density was measured with a wavelength of 600 nm. In the case of the assays testing bacillibactin and yersiniabactin (Fig. 1B, 1C), the ODs were not synchronized. In the case of the assay testing ornibactin and salmochelin (Fig. 1D), the ODs of these food spots were synchronized to roughly 1, while in the case of the assay with ampicillin and the B. subtilis mutants (Fig. 3) the ODs were synchronized to roughly 0.7. The bacteria was heat killed as previously described by Qi (Qi and Han 2018; Qi, Kniazeva, and Han 2017).