Class IIb Microcin MccM Interferes with Oxidative Phosphorylation in Escherichia coli.

Dysbiosis of the human gut microbiota is linked to numerous diseases. Understanding the molecular mechanisms by which microbes interact and compete with one another is required for developing successful strategies to modulate the microbiome. The natural product Microcin M (MccM) consists of a 77-residue bioactive peptide conjugated to a siderophore and is a class II microcin involved in microbial competition with an enigmatic mode-of-action. In this work, we investigated the basis for MccM activity and leveraged bioinformatics to expand the known chemical diversity of class II microcins. We applied automated fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry to acquire MccM and demonstrated that its activity was bacteriostatic. We then used our synthetic molecule to ascertain that catecholate siderophore transporters in Escherichia coli K-12 are necessary for MccM import. Once inside the cell, we found that MccM treatment decreased the levels of intracellular ATP and interfered with gene expression. These effects were ameliorated in genetic mutants lacking ATP synthase or in conditions that support substrate-level phosphorylation. Further, we showed that MccM elevated the levels of reactive oxygen species within the target cell. We propose that MccM effects its bacteriostatic activity by decreasing the total energy level of the cell through inhibition of oxidative phosphorylation. Lastly, using genome mining, we bioinformatically identified 171 novel putative class II microcins. Our investigation sheds light on the natural processes involved in microbial competition and provides inspiration, in the form of new molecules, for future therapeutic endeavors.

Experimental methods for evaluating siderophore-antibiotic conjugates.

Siderophore-antibiotic conjugates (SACs) are of past and current interest for delivering antibacterials into Gram-negative bacterial pathogens that express siderophore receptors. Studies of SACs are often multifaceted and involve chemical and biological approaches. Major goals are to evaluate the antimicrobial activity and uptake of novel SACs and use the resulting data to inform further mode-of-action studies and molecular design strategies. In this chapter, we describe four key methods that we apply when investigating the antimicrobial activity and uptake of novel SACs based on the siderophore enterobactin (Ent). These methods are based on approaches from the siderophore literature as well as established protocols for antimicrobial activity testing, and include assays for evaluating SAC antimicrobial activity, time-kill kinetics, siderophore competition, and bacterial cell uptake using 57Fe. These assays have served us well in characterizing our Ent-based conjugates and can be applied to study SACs that use other siderophores as targeting vectors.

"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

Investigation of Siderophore-Platinum(IV) Conjugates Reveals Differing Antibacterial Activity and DNA Damage Depending on the Platinum Cargo.

The growing threat of bacterial infections coupled with the dwindling arsenal of effective antibiotics has heightened the urgency for innovative strategies to combat bacterial pathogens, particularly Gram-negative strains, which pose a significant challenge due to their outer membrane permeability barrier. In this study, we repurpose clinically approved anticancer agents as targeted antibacterials. We report two new siderophore-platinum(IV) conjugates, both of which consist of an oxaliplatin-based Pt(IV) prodrug (oxPt(IV)) conjugated to enterobactin (Ent), a triscatecholate siderophore employed by Enterobacteriaceae for iron acquisition. We demonstrate that l/d-Ent-oxPt(IV) (l/d-EOP) are selectively delivered into the Escherichia coli cytoplasm, achieving targeted antibacterial activity, causing filamentous morphology, and leading to enhanced Pt uptake by bacterial cells but reduced Pt uptake by human cells. d-EOP exhibits enhanced potency compared to oxaliplatin and l-EOP, primarily attributed to the intrinsic antibacterial activity of its non-native siderophore moiety. To further elucidate the antibacterial activity of Ent-Pt(IV) conjugates, we probed DNA damage caused by l/d-EOP and the previously reported cisplatin-based conjugates l/d-Ent-Pt(IV) (l/d-EP). A comparative analysis of these four conjugates reveals a correlation between antibacterial activity and the ability to induce DNA damage. This work expands the scope of Pt cargos targeted to the cytoplasm of Gram-negative bacteria via Ent conjugation, provides insight into the cellular consequences of Ent-Pt(IV) conjugates in E. coli, and furthers our understanding of the potential of Pt-based therapeutics for antibacterial applications.

Exploring the Antibacterial Activity and Cellular Fates of Enterobactin-Drug Conjugates That Target Gram-Negative Bacterial Pathogens.

Siderophores are secondary metabolites utilized by bacteria to acquire iron (Fe), an essential transition metal nutrient. Fe levels in the host environment are tightly regulated and can be further restricted to starve invading bacterial pathogens in a host-defense process known as nutritional immunity. To survive and colonize the Fe-limited host environment, bacteria produce siderophores and express cognate siderophore transport machinery. These active transport pathways present an opportunity for selective and efficient drug delivery into bacterial cells, motivating decades of research on synthetic siderophore-antibiotic conjugates (SACs) as a Trojan-horse strategy for the development of targeted antibiotics.Enterobactin (Ent) is a triscatecholate siderophore produced and utilized by many Gram-negative bacteria, including all Escherichia coli and Salmonella species. Within these species, pathogenic strains cause a variety of human diseases including urinary tract infections, gastroenteritis, and sepsis. Infections caused by these Gram-negative pathogens can be difficult to treat because of the impermeability of the outer membrane (OM). This impermeability can be overcome by utilizing siderophores as drug delivery vectors for targeting Gram-negative pathogens. Ent is a promising delivery vector because it undergoes active transport across the OM mediated by the Ent uptake machinery after scavenging Fe(III) from the extracellular environment. Despite the well-elucidated chemistry and biology of Ent, its use for SAC development was hampered by the lack of an appropriate functional group for cargo attachment. Our laboratory addressed this need by designing and synthesizing monofunctionalized Ent scaffolds. Over the past decade, we have used these scaffolds to explore Ent-based SACs with a variety of drug warheads, including ß-lactam and fluoroquinolone antibiotics, and Pt(IV) prodrugs. Investigations of the antibacterial activities of these conjugates and their cellular fates have informed our design principles and revealed approaches to achieving enhanced antibacterial potency and pathogen-targeted activity. Collectively, our studies of Ent-drug conjugates have provided discoveries, understanding, and invaluable insights for future design and evaluation of SACs.In this Account, we present the story of our work on Ent-drug conjugates that began about ten years ago with the development of monofunctionalized Ent scaffolds and the design and synthesis of various conjugates based on these scaffolds. We describe the antibacterial activity profiles and uptake pathways of Ent-drug conjugates harboring traditional antibiotics and repurposed platinum anticancer agents as well as studies that address cellular targets and fates. Finally, we discuss other applications of monofunctionalized Ent scaffolds, including a siderophore-based immunization strategy. We intend for this Account to inspire further investigations into the fundamental understanding and translational applications of siderophores and siderophore-drug conjugates.

Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a ß-Lactam to Gram-Negative Bacterial Pathogens.

Developing new antibiotics and delivery strategies is of critical importance for treating infections caused by Gram-negative bacterial pathogens. Hijacking bacterial iron uptake machinery, such as that of the siderophore enterobactin (Ent), represents one promising approach toward these goals. Here, we report a novel Ent-inspired siderophore-antibiotic conjugate (SAC) employing an alternative siderophore moiety as the delivery vector and demonstrate the potency of our SACs harboring the ß-lactam antibiotic ampicillin (Amp) against multiple pathogenic Gram-negative bacterial strains. We establish the ability of N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(2,3-dihydroxybenzamide) (TRENCAM, hereafter TC), a synthetic mimic of Ent, to facilitate drug delivery across the outer membrane (OM) of Gram-negative pathogens. Conjugation of Amp to a new monofunctionalized TC scaffold affords TC-Amp, which displays markedly enhanced antibacterial activity against the gastrointestinal pathogen Salmonella enterica serovar Typhimurium (STm) compared with unmodified Amp. Bacterial uptake, antibiotic susceptibility, and microscopy studies with STm show that the TC moiety facilitates TC-Amp uptake by the OM receptors FepA and IroN and that the Amp warhead inhibits penicillin-binding proteins. Moreover, TC-Amp achieves targeted activity, selectively killing STm in the presence of a commensal lactobacillus. Remarkably, we uncover that TC-Amp and its Ent-based predecessor Ent-Amp achieve enhanced antibacterial activity against diverse Gram-negative ESKAPE pathogens that express Ent uptake machinery, including strains that possess intrinsic ß-lactam resistance. TC-Amp and Ent-Amp exhibit potency comparable to that of the FDA-approved SAC cefiderocol against Gram-negative pathogens. These results demonstrate the effective application of native and appropriately designed nonnative siderophores as vectors for drug delivery across the OM of multiple Gram-negative bacterial pathogens.

Iron Sequestration by Murine Calprotectin Induces Starvation Responses in Pseudomonas aeruginosa.

Pathogen sensing by the mammalian host induces a pro-inflammatory response that involves release of the antimicrobial metal-sequestering protein calprotectin (CP, S100A8/S100A9 heterooligomer, MRP8/MRP14 heterooligomer) from neutrophils. Biochemical investigations on human CP (hCP) have informed the molecular basis of how this protein sequesters metal ions. Murine models of infection have provided invaluable insights into the ability of murine CP (mCP) to compete with bacterial pathogens for essential metal nutrients. Despite this extensive work, our knowledge of how mCP sequesters metals from bacterial pathogens and its impacts on bacterial physiology is limited. Moreover, whether mCP sequesters iron and induces iron-starvation responses in bacterial pathogens has not been evaluated. Here, we examine the ability of mCP to withhold iron from Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen that causes severe infections in immunocompromised individuals and cystic fibrosis patients. We demonstrate that mCP prevents iron uptake and induces iron-starvation responses in P. aeruginosa laboratory strains PA14 and PAO1 and the JSRI-1 clinical isolate from a cystic fibrosis patient. We also show that mCP prevents iron uptake and induces an iron-starvation response in the Gram-positive bacterial pathogen Staphylococcus aureus. The His6 site of mCP is the iron-sequestering site; it exhibits Ca(II)-dependent Fe(II) affinity and binds Fe(II) with subpicomolar affinity in the presence of excess Ca(II) ions. This work is important for understanding the structure, function, and physiological consequences of mCP and how the mammalian host and bacterial pathogens compete for essential metal nutrients.

Spectroscopic and computational investigations of Cobalt(II) binding to the innate immune protein human calprotectin.

Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8 kDa) and S100A9 (ß subunit, 13.2 kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His3Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His3Asp and His6 sites of CP were further characterized by utilizing Co2+ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co2+-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His3Asp and His6 sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.

Post-translational modifications on the metal-sequestering protein calprotectin.

Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant neutrophil protein that contributes to innate immunity by sequestering nutrient metal ions in the extracellular space. This process starves invading microbial pathogens of essential metal nutrients, which can inhibit growth and colonization. Over the past decade, fundamental and clinical studies have revealed that the S100A8 and S100A9 subunits of CP exhibit a variety of post-translational modifications (PTMs). This review summarizes PTMs on the CP subunits that have been detected and highlights two recent studies that evaluated the structural and functional consequences of methionine and cysteine oxidation on CP. Collectively, these investigations indicate that the molecular speciation of extracellular CP is complex and composed of multiple proteoforms. Moreover, PTMs may impact biological function and the lifetime of the protein. It is therefore important that post-translationally modified CP species receive consideration and integration into the current working model for how CP functions in nutritional immunity.

Siderophore Immunization Restricted Colonization of Adherent-Invasive Escherichia coli and Ameliorated Experimental Colitis.

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract and profound alterations to the gut microbiome. Adherent-invasive Escherichia coli (AIEC) is a mucosa-associated pathobiont that colonizes the gut of patients with Crohn's disease, a form of IBD. Because AIEC exacerbates gut inflammation, strategies to reduce the AIEC bloom during colitis are highly desirable. To thrive in the inflamed gut, Enterobacteriaceae acquire the essential metal nutrient iron by producing and releasing siderophores. Here, we implemented an immunization-based strategy to target the siderophores enterobactin and its glucosylated derivative salmochelin to reduce the AIEC bloom in the inflamed gut. Using chemical (dextran sulfate sodium) and genetic (Il10-/- mice) IBD mouse models, we showed that immunization with enterobactin conjugated to the mucosal adjuvant cholera toxin subunit B potently elicited mucosal and serum antibodies against these siderophores. Siderophore-immunized mice exhibited lower AIEC gut colonization, diminished AIEC association with the gut mucosa, and reduced colitis severity. Moreover, Peyer's patches and the colonic lamina propria harbored enterobactin-specific B cells that could be identified by flow cytometry. The beneficial effect of siderophore immunization was primarily B cell-dependent because immunized muMT-/- mice, which lack mature B lymphocytes, were not protected during AIEC infection. Collectively, our study identified siderophores as a potential therapeutic target to reduce AIEC colonization and its association with the gut mucosa, which ultimately may reduce colitis exacerbation. Moreover, this work provides the foundation for developing monoclonal antibodies against siderophores, which could provide a narrow-spectrum strategy to target the AIEC bloom in Crohn's disease patients. IMPORTANCE Adherent-invasive Escherichia coli (AIEC) is abnormally prevalent in patients with ileal Crohn's disease and exacerbates intestinal inflammation, but treatment strategies that selectively target AIEC are unavailable. Iron is an essential micronutrient for most living organisms, and bacterial pathogens have evolved sophisticated strategies to capture iron from the host environment. AIEC produces siderophores, small, secreted molecules with a high affinity for iron. Here, we showed that immunization to elicit antibodies against siderophores promoted a reduction of the AIEC bloom, interfered with AIEC association with the mucosa, and mitigated colitis in experimental mouse models. We also established a flow cytometry-based approach to visualize and isolate siderophore-specific B cells, a prerequisite for engineering monoclonal antibodies against these molecules. Together, this work could lead to a more selective and antibiotic-sparing strategy to target AIEC in Crohn's disease patients.

Heavy-Metal Trojan Horse: Enterobactin-Directed Delivery of Platinum(IV) Prodrugs to Escherichia coli.

The global crisis of untreatable microbial infections necessitates the design of new antibiotics. Drug repurposing is a promising strategy for expanding the antibiotic repertoire. In this study, we repurpose the clinically approved anticancer agent cisplatin into a targeted antibiotic by conjugating its Pt(IV) prodrug to enterobactin (Ent), a triscatecholate siderophore employed by Enterobacteriaceae for iron (Fe) acquisition. The l-Ent-Pt(IV) conjugate (l-EP) exhibits antibacterial activity against Escherichia coli K12 and the uropathogenic isolate E. coli CFT073. Similar to cisplatin, l-EP causes a filamentous morphology in E. coli and initiates lysis in lysogenic bacteria. Studies with E. coli mutants defective in Ent transport proteins show that Ent mediates the delivery of l-EP into the E. coli cytoplasm, where reduction of the Pt(IV) prodrug releases the cisplatin warhead, causing growth inhibition and filamentation of E. coli. Substitution of Ent with its enantiomer affords the d-Ent-Pt(IV) conjugate (d-EP), which displays enhanced antibacterial activity, presumably because d-Ent cannot be hydrolyzed by Ent esterases and thus Fe cannot be released from this conjugate. E. coli treated with l/d-EP accumulate ≥10-fold more Pt as compared to cisplatin treatment. By contrast, human embryonic kidney cells (HEK293T) accumulate cisplatin but show negligible Pt uptake after treatment with either conjugate. Overall, this work demonstrates that the attachment of a siderophore repurposes a Pt anticancer agent into a targeted antibiotic that is recognized and transported by siderophore uptake machinery, providing a design strategy for drug repurposing by siderophore modification and heavy-metal "trojan-horse" antibiotics.

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration.

Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His3Asp sites with high affinity. A similar His3Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.

Zinc sequestration by human calprotectin facilitates manganese binding to the bacterial solute-binding proteins PsaA and MntC.

Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. Streptococcus pneumoniae has a relatively high manganese requirement, and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, which contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.

Metal sequestration by S100 proteins in chemically diverse environments.

During infection, the mammalian host initiates a metal-withholding response against invading microbial pathogens to inhibit their growth and survival, a process often termed 'nutritional immunity'. The host-defense S100 proteins calprotectin (CP) (S100A8/S100A9 oligomer), S100A12, and S100A7 play key roles in the innate immune response by sequestrating essential transition metal nutrients from microbes in the extracellular space. Accumulating evidence suggests that the antimicrobial activity of these proteins varies between infection sites and may be affected by the local chemical environment. Herein, we discuss the interplay between host metal-withholding proteins and microbial pathogens in the context of the chemical complexity of infection sites and highlight recent advances in our understanding of how chemically diverse conditions affect the properties and functions of S100 proteins.

Bacterial Responses to Iron Withholding by Calprotectin.

Iron (Fe) plays important roles in both essential cellular processes and virulence pathways for many bacteria. Consequently, Fe withholding by the human innate immune system is an effective form of defense against bacterial infection. In this Perspective, we review recent studies that have established a foundation for our understanding of the impact of the metal-sequestering host defense protein calprotectin (CP) on bacterial Fe homeostasis. We also discuss two recently uncovered strategies for bacterial adaptation to Fe withholding by CP. Together, these studies provide insight into how Fe sequestration by CP affects bacterial pathogens that include Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. Overall, recent studies suggest that Fe withholding by CP may have implications for bacterial survival and virulence in the host, and further explorations that directly address this possibility present an important area for discovery.

Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αß heterodimeric apo protein into a Ca(II)-bound (αß)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αß heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αß heterodimer. These studies provide a structural model in which the apo αß heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αß heterodimer causes the active state to predominate, resulting in self-association and formation of the (αß)2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His3Asp and His6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His3Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix αIV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.

The Human Innate Immune Protein Calprotectin Elicits a Multimetal Starvation Response in Pseudomonas aeruginosa.

To combat infections, the mammalian host limits availability of essential transition metals such as iron (Fe), zinc (Zn), and manganese (Mn) in a strategy termed "nutritional immunity." The innate immune protein calprotectin (CP) contributes to nutritional immunity by sequestering these metals to exert antimicrobial activity against a broad range of microbial pathogens. One such pathogen is Pseudomonas aeruginosa, which causes opportunistic infections in vulnerable populations, including individuals with cystic fibrosis. CP was previously shown to withhold Fe(II) and Zn(II) from P. aeruginosa and induce Fe and Zn starvation responses in this pathogen. In this work, we performed quantitative, label-free proteomics to further elucidate how CP impacts metal homeostasis pathways in P. aeruginosa. We report that CP induces an incomplete Fe starvation response, as many Fe-containing proteins that are repressed by Fe limitation are not affected by CP treatment. The Zn starvation response elicited by CP seems to be more complete than the Fe starvation response and includes increases in Zn transporters and Zn-independent proteins. CP also induces the expression of membrane-modifying proteins, and metal depletion studies indicate this response results from the sequestration of multiple metals. Moreover, the increased expression of membrane-modifying enzymes upon CP treatment correlates with increased tolerance to polymyxin B. Thus, the response of P. aeruginosa to CP treatment includes both single- and multimetal starvation responses and includes many factors related to virulence potential, broadening our understanding of this pathogen's interaction with the host. IMPORTANCE Transition metal nutrients are critical for growth and infection by all pathogens, and the innate immune system withholds these metals from pathogens to limit their growth in a strategy termed "nutritional immunity." While multimetal depletion by the host is appreciated, the majority of studies have focused on individual metals. Here, we use the innate immune protein calprotectin (CP), which complexes with several metals, including iron (Fe), zinc (Zn), and manganese (Mn), and the opportunistic pathogen Pseudomonas aeruginosa to investigate multimetal starvation. Using an unbiased label-free proteomics approach, we demonstrate that multimetal withholding by CP induces a regulatory response that is not merely additive of individual metal starvation responses, including the induction of lipid A modification proteins.

Enterobactin- and salmochelin-ß-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073.

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-ß-lactam conjugates where the broad-spectrum ß-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG3) linker. These siderophore-ß-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-ß-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-ß-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine ß-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-ß-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-ß-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity.

Conjugation to Enterobactin and Salmochelin S4 Enhances the Antimicrobial Activity and Selectivity of ß-Lactam Antibiotics against Nontyphoidal Salmonella.

The pathogen Salmonella enterica is a leading cause of infection worldwide. Nontyphoidal Salmonella (NTS) serovars typically cause inflammatory diarrhea in healthy individuals, and can cause bacteremia in immunocompromised patients, children, and the elderly. Management of NTS infection poses a challenge because antibiotic treatment prolongs fecal shedding of the pathogen and is thus not recommended for most patients. In recent years, the emergence of antibiotic resistance in NTS has also become a major issue. Thus, new therapeutic strategies to target NTS are needed. Here, we evaluated whether six siderophore-ß-lactam conjugates based on enterobactin (Ent) and salmochelin S4 (digulcosylated Ent, DGE) provide antimicrobial activity against the two highly prevalent NTS serovars Typhimurium and Enteritidis by targeting the siderophore receptors FepA and/or IroN. The conjugates showed 10- to 1000-fold lower minimum inhibitory concentrations against both serovars Typhimurium and Enteritidis compared to the parent antibiotics under iron limitation and were recognized and transported by FepA and/or IroN. NTS treated with the Ent/DGE-ß-lactam conjugates exhibited aberrant cellular morphologies suggesting inhibition of penicillin-binding proteins, and the conjugates selectively killed NTS in coculture with Staphylococcus aureus. Lastly, the DGE-based conjugates proved to be effective at inhibiting growth of NTS in the presence of the Ent-sequestering protein lipocalin-2. This work describes the successful use of siderophore-antibiotic conjugates against NTS and highlights the opportunity for narrowing the activity spectrum of antibiotics by using Ent and DGE to target enteric bacterial pathogens.

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and patients with cystic fibrosis. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore-mediated ferric iron uptake, ferrous iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host-defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and iron-starvation responses. We extend our study to a second bacterial pathogen, S. aureus, and demonstrate that CP also inhibits iron uptake and induces iron-starvation responses by this pathogen. Similarly to P. aeruginosa, we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron-starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron-withholding strategies.