Significance Gram-negative pathogens represent a major public health issue. With increases in antibiotic resistance and the limited availability of preventive strategies, new ways are needed to target these bacteria. Here, we propose a narrow-spectrum immunization strategy to block the ability of a gastrointestinal pathogen to acquire the essential metal nutrient iron. This approach involves the chemical synthesis of bacterial iron chelators, known as siderophores, and their conjugation to immunogenic carrier proteins. Mice immunized with these compounds developed antibodies against siderophores in the intestine, which hindered proliferation of the gut pathogen Salmonella. Because similar but distinct molecules are secreted by many bacterial and fungal pathogens, this synthesis and immunization strategy could be applied to a broad range of infectious agents.

Abstract Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB–siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections.

Gram-negative pathogens cause a range of human diseases, including foodborne illness, urinary tract infections, and sepsis. Infections with these organisms pose a serious global public health threat that is exacerbated by increasing antibiotic resistance, the dearth of new antibiotics in the drug pipeline, and the limited availability of vaccines (1). To slow the emergence of antibiotic resistance and to reduce the incidence of secondary infections, narrow-spectrum therapeutic strategies are needed, specifically ones that limit disruption of the gut microbiota, which comprises beneficial microbes that provide colonization resistance to pathogens (2). Many studies have elucidated molecular mechanisms by which pathogens thrive in the host, thus indicating potential targets for the prevention and treatment of infection. In recent years, bacterial metabolism has been proposed to be a key factor in promoting pathogenicity (3).

The vast majority of bacterial pathogens have a metabolic requirement for iron. Because the vertebrate host tightly controls the concentration of free iron (e.g., ∼10−24 M in serum), many microbes biosynthesize and export secondary metabolites called siderophores to scavenge iron from the host (4). These small molecules chelate ferric iron (Fe3+) with high affinity [e.g., enterobactin (Ent)] (5⇓⇓–8) (K d ∼10−25 M at neutral pH; Fig. 1A). Once a siderophore coordinates iron in the extracellular space, the iron-bound siderophore is recognized and transported into a microbial cell by a dedicated membrane receptor. Following cellular uptake, the iron is released from the siderophore to support microbial metabolism and replication. Siderophores are regarded as major virulence factors during infection with bacterial and fungal pathogens, examples of which include Salmonella enterica, uropathogenic Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, and Aspergillus fumigatus (9).

Fig. 1. Intranasal immunization with CTB-Ent induces the development of anti-Ent and antisalmochelin IgA antibodies in the gut mucosa. (A) Chemical structures of Ent (1) and salmochelin S4 (GlcEnt; 2) and a cartoon depicting the CTB-Ent (3) conjugate. Ent and GlcEnt are cyclic trimers of N-2,3-dihydroxylbenzoyl-l-serine. (B) Description and time line of the immunization protocol. The timing of intranasal immunization (day 0 and day 14) and the dose of antigen (100 μg/mL) are indicated. Feces were collected weekly. An ELISPOT assay was carried out at day 21 postimmunization. Mice were infected with S. enterica serovar Typhimurium per os between day 35 and day 51 postimmunization. (C) Anti-Ent IgA antibodies were quantified by using an in-house ELISA in fecal samples from mice immunized with either CTB (n = 5) or CTB-Ent (n = 7) during the indicated time course. Day 0 represents antibodies from naive mice before immunization. A twofold dilution was used for detection of fecal anti-Ent IgA (D) or anti-GlcEnt IgA (F) by ELISA in mice immunized with either CTB (n = 15–20) or CTB-Ent (n = 15–20) at day 21 postimmunization. Representative ELISPOT images of supernatant from B cells producing anti-Ent IgA (E) or anti-GlcEnt IgA (G) isolated from Peyer’s patches of mice immunized with either CTB or CTB-Ent at day 21 postimmunization are shown. The average number of spots detected in CTB- or CTB-Ent–immunized mice per 1 million Peyer’s patches cells (n = 5–7) is shown. (H) Twofold dilution for detection of fecal anti-CTB IgA in CTB (n = 6) and CTB-Ent (n = 6) by in-house ELlSA. (I) Representative ELISPOT images of CTB IgA from Peyer’s patches of mice immunized with either CTB or CTB-Ent at day 21 postimmunization. The average number of spots detected in the CTB or CTB-Ent–immunized mice per 1 million Peyer’s patches cells (n = 3) is shown. In C–I, bars represent the mean ± SE. Immunization experiments were repeated at least three times with different batches of CTB/CTB-Ent. ****P < 0.0001; ***P < 0.001;**P < 0.01. n.s., not significant; post-imm, postimmunization.

Ent (Fig. 1A) is a siderophore biosynthesized and deployed by commensal and pathogenic Enterobacteriaceae (10). In a process known as “nutritional immunity,” the host responds to microbial infection and inflammation by limiting the availability of essential nutrient metals, including iron (11). To prevent Ent-mediated microbial iron acquisition, epithelial cells and neutrophils secrete the host-defense protein lipocalin-2 (LCN2). This protein inhibits siderophore-mediated iron uptake by capturing ferric Ent in the extracellular milieu (12). By inhibiting the growth of Enterobacteriaceae that rely on Ent-mediated iron acquisition, LCN2 plays an essential role in preventing lethal infection by these organisms (12, 13). Nevertheless, various Gram-negative pathogens evade LCN2 by producing and using “stealth siderophores” that cannot be captured by this host-defense protein. For example, Salmonella spp. and strains of pathogenic E. coli overcome LCN2 by biosynthesizing a family of C-glucosylated Ent (GlcEnt) derivatives named salmochelins (14, 15) (Fig. 1A). These siderophores allow the pathogen to thrive in the inflamed gut in the presence of LCN2 and outcompete the microbiota (14⇓–16). Indeed, Salmonella mutants that lack the GlcEnt receptor IroN are susceptible to the LCN2-mediated host response; these mutants exhibit reduced colonization in the inflamed intestine and cannot outcompete the microbiota (17, 18). In the absence of intestinal inflammation or LCN2 expression, iron is more readily available and mutants in siderophore receptors are not attenuated (19).

Inspired by seminal studies exemplifying the importance of siderophore-mediated iron acquisition during infection with Salmonella as well as other pathogens (18, 20), we hypothesized that boosting host nutritional immunity by blocking siderophore-based iron acquisition would reduce microbial burden and improve the outcome of infection. To address this hypothesis, we designed and synthesized conjugates of a native siderophore used by Salmonella and an immunogenic carrier protein, and we immunized mice with the compounds to induce an antibody response against siderophores. Herein, we show that immunization of mice with the siderophore-carrier protein conjugate elicited an antibody response to siderophores in the intestinal mucosa and significantly reduced Salmonella colonization of the inflamed gut.

Summary and Conclusions For many years, siderophores, as well as the biosynthetic and transport machineries for these virulence factors, have garnered significant interest as targets for new antibiotics. Reported efforts include the design and application of siderophore–antibiotic conjugates for targeted drug delivery, the identification of small-molecule inhibitors of siderophore biosynthesis, and the inhibition of siderophore uptake by immunization against siderophore receptors or siderophores (9, 41⇓–43). A prior study described the development of antibodies against vibriobactin; however, that study did not evaluate whether the vaccination provided protection to the host during infection with Vibrio cholerae (44). Our work establishes that immunization against bacterial siderophores results in the production of anti-siderophore antibodies and affords reduced intestinal colonization (up to 20,000-fold) during infection with the enteric pathogen Salmonella. These results are particularly significant because Salmonella is known to thrive in the inflamed gut and to outcompete the microbiota in this environment. Our immunization shifted the balance in favor of the microbiota, promoting the expansion of beneficial microbes (e.g., Lactobacillus) that thrive in a similar environment. Moreover, because only mice that shed the highest level of Salmonella (the so-called “supershedders”) are able to achieve successful transmission of the pathogen to naive hosts (45), we reason that our strategy, which reduces fecal shedding, could be used to hinder Salmonella transmission. Because Salmonella intestinal colonization was most highly reduced in mice that developed the highest antibody titers, future studies will address further optimization of the immunization strategy to increase antibody production. In contrast to the mouse model, nontyphoidal Salmonella remains localized to the gut in most patients. We predict that anti-siderophore antibodies, either generated in the gut in response to immunization or therapeutically administered to patients infected with Salmonella, could reduce fecal shedding of the pathogen, thereby limiting its spread and reducing recovery time. Moreover, future immunization studies aimed at generating a systemic antibody response to siderophores in mice will determine whether this strategy could lead to greater protection at systemic sites, which would be informative for developing new preventive or therapeutic strategies for systemic salmonellosis. This immunization strategy could be further developed as a therapy for infections caused by pathogens that use other siderophores to thrive in the mammalian host. We expect that development of resistance against antibody-based immunization would develop only if a new siderophore biosynthesis gene cluster is acquired by horizontal gene transfer, and future studies will address this notion. In principle, our strategy can be applied to every siderophore, which would be useful in the event that resistance develops, or to target pathogens that use more than one siderophore. Moreover, the development of anti-siderophore antibodies will likely provide narrow-spectrum antimicrobial activity by targeting only microbes that use that subset of siderophores while having a minimal impact on the microbiota (in contrast to broad- and even narrow-spectrum antibiotics). Broadly, our work indicates that new preventive and therapeutic strategies for microbial infections should target siderophore-mediated iron acquisition, a virulence trait shared by almost all bacterial and fungal pathogens (9). Moreover, because recent studies demonstrate additional, nonclassical roles for siderophores (46, 47), including protection of bacteria from oxidative stress (48) and modulation of host gene expression (49), it is possible that antibody-mediated sequestration of siderophores may inhibit bacterial growth by iron-independent mechanisms and may provide additional benefits to the host.

Methods The preparation and characterization of CTB-Ent, Ent-PEG 3 -biotin, GlcEnt-PEG 3 -biotin, and native siderophores are described in SI Appendix. Six- to eight-week-old C57BL/6 female mice were purchased from Taconic. When indicated, 6- to 8-wk-old male or female C57BL/6 or C57BL/6 NRAMP1+ mice bred in our vivarium were also used for the experiments; 8 to 10 mice per experimental group were used for each experiment. The Institutional Animal Care and Use Committee at the University of California, Irvine approved all of the mouse experiments. Mucosal immunization with CTB, CTB-Ent conjugates, analysis of antibody production by ELISA and ELISPOT, infection models, histopathology, and analysis of gene expression and of the microbiota are described in SI Appendix.

Acknowledgments We thank Matthew Rolston (University of California, Davis, Host-Microbe Systems Biology Core) for processing samples for Illumina MiSeq analysis. This work was supported by the Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Disease [supported by Public Health Service (PHS) Grant U54AI065359] and by PHS Grants AI101784 and AI114625 (to M.R. and E.M.N.). Work in the laboratory of M.R. is also supported by PHS Grants AI105374, AI126277, AI121928, and DK058057. Work in the laboratory of E.M.N. is also supported by the Kinship Foundation Searle Scholar Award (to E.M.N.) and the Massachusetts Institute of Technology (MIT) Department of Chemistry. M.R. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. A.P.-L. was funded by a University of California Institute for Mexico and the United States (MEXUS) and El Consejo Nacional de Ciencia y Tecnología (CONACYT) (MEXUS-CONACYT) award. P.C. is a recipient of a Royal Thai Government Fellowship. NMR instrumentation in the MIT Department of Chemistry Instrumentation Facility is supported by National Science Foundation Grants CHE-9808061 and DBI-9729592.