Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy.

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Compared to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a host protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacteria's ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

"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.

ConspectusSiderophores 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.

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients, such as iron. Pathogens scavenge iron using siderophores, including enterobactin; however, this strategy is counteracted by host protein lipocalin-2, which sequesters iron-laden enterobactin. Although this iron competition occurs in the presence of gut bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron and sustains its resilience in the inflamed gut by utilizing siderophores produced by other bacteria, including Salmonella, via a secreted siderophore-binding lipoprotein XusB. Notably, XusB-bound enterobactin is less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. Because the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the host-pathogen interactions and nutritional immunity.

Adding siderophores: A new strategy to reduce greenhouse gas emissions in composting.

Microbial community is the primary driver causing the greenhouse gas emissions in composting. Thus, regulating the microbial communities is a strategy to reduce them. Here, two different siderophores (enterobactin and putrebactin) were added, which could bind and translocate iron by specific microbes, to regulate the composting communities. The results showed that adding enterobactin enriched Acinetobacter and Bacillus with specific receptors by 6.84-fold and 6.78-fold. It promoted carbohydrate degradation and amino acid metabolism. This resulted in a 1.28-fold increase in humic acid content, as well as a 14.02% and 18.27% decrease in CO2 and CH4 emissions, respectively. Meanwhile, adding putrebactin boosted the microbial diversity by 1.21-fold and enhanced potential microbial interactions by 1.76-fold. The attenuated denitrification process led to a 1.51-fold increase in the total nitrogen content and a 27.47% reduction in N2O emissions. Overall, adding siderophores is an efficient strategy to reduce greenhouse gas emissions and promote the compost quality.

Comparative Metabolomics Reveals a Bifunctional Antibacterial Conjugate from Combined-Culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596.

To investigate the potential for secondary metabolite biosynthesis by Streptomyces species, we employed a coculture method to discover natural bioactive products and identified specific antibacterial activity from a combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. Molecular networking using ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) data revealed a specific clade of metabolites in this combined-culture that were not detected in both monocultures. Using the chemical profiles, a previously unidentified conjugate between FabF inhibitor and catechol-type siderophore was successfully identified and named harundomycin A. Harundomycin A was a conjugate between the 2,4-dihydroxy-3-aminobenzoate moiety of platensimycin and N,N'-bis(2,3-dihydroxybenzoyl)-O-seryl-cysteine (bisDHBA-Ser-Cys) with a thioester linkage. Along with the production of harundomycin A, platensimycin, its thiocarboxylic acid form thioplatensimycin, enterobactin, and its degradation product N,N'-bis(2,3-dihydroxybenzoyl)-O-l-seryl-dehydroalanine (bisDHBA-Ser-Dha) were also induced in the combined-culture. Genomic data of S. hygroscopicus HOK021 and T. pulmonis TP-B0596 indicated that strain HOK021 possessed biosynthetic gene clusters for both platensimycin and enterobactin, and thereby revealed that T. pulmonis stimulates HOK021 and acts as an inducer of both of these metabolites. Although the harundomycin A was modified by bulky bisDHBA-Ser-Cys, responsible for the binding to the target molecule FabF, it showed a similar antibacterial spectrum to platensimycin, including against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, suggesting that the pharmacophore is platensimycin. Additionally, Chrome Azurol S assay showed that harundomycin A possesses ferric iron-chelating activity comparable to that of enterobactin. Our study demonstrated the transformation of existing natural products to bifunctional molecules driven by bacterial interaction.

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.

Hypervirulent Klebsiella pneumoniae infection presenting as endocarditis and liver abscess.

Hypervirulent Klebsiella pneumoniae infection, reported commonly from South-east Asia, is predominantly community-acquired and affects young healthy adults. Although abscesses of liver, brain and muscles, endophthalmitis or osteomyelitis have been reported, Infective endocarditis is a rare manifestation. This report illustrates a patient with uncontrolled diabetes mellitus who presented with clinical features of liver abscess with an incidental finding of infective endocarditis. Hypervirulent K. pneumoniae, which was isolated from blood culture of the patient carried the plasmid borne key virulence markers-rmpA and rmpA2 with enterobactin (entB), type 3 fimbriae (mrkD) and was of K1 type and ST3321, an uncommon clone of Hypervirulent K. pneumoniae. Transthoracic Echocardiography showed multiple mobile vegetations attached to mitral valve and posterior wall of left ventricle. With appropriate antibiotics blood cultures turned sterile, liver abscess and cardiac vegetations reduced in size. Mitral Valve replacement surgery was proposed. He declined treatment and succumbed to the infection subsequently.

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.

Outer Membrane Vesicles of Acinetobacter baumannii DS002 Are Selectively Enriched with TonB-Dependent Transporters and Play a Key Role in Iron Acquisition.

Outer membrane vesicles (OMVs) of Acinetobacter baumannii DS002 carry proteins which perform selective biological functions. The proteins involved in cell wall/membrane biogenesis and inorganic ion transport and metabolism occupied a significant portion of the 302 proteins associated with OMVs. Interestingly, the TonB-dependent transporters (TonRs), linked to the active transport of nutrients across the energy-deprived outer membrane, are predominant among proteins involved in inorganic ion transport and metabolism. The OMVs of DS002 contain TonRs capable of transporting iron complexed to catecholate, hydroximate, and mixed types of siderophores. Consistent with this observation, the OMVs were firmly bound to ferric-enterobactin (55Fe-Ent) and successfully transported iron into A. baumannii DS002 cells grown under iron-limiting conditions. In addition to the TonRs, OMVs also carry proteins known to promote pathogenesis, immune evasion, and biofilm formation. Our findings provide conclusive evidence for the role of OMVs in the transport of nutrients such as iron and show the presence of proteins with proven roles in pathogenicity and immune response. IMPORTANCE TonB-dependent transporters (TonRs) play a crucial role in transporting nutrients such as iron, nickel, copper, and complex carbohydrates across the energy-deprived outer membrane. Due to their unique structural features, TonRs capture nutrients in an energy-independent manner and transport them across the outer membrane by harvesting energy derived from the inner membrane-localized Ton-complex. In this study, we report the presence of TonRs capable of transporting various nutrients in OMVs and demonstrate their role in capturing and transporting ferric iron complexed with enterobactin into A. baumannii DS002 cells. The OMV-associated TonRs appear to play a critical role in the survival of A. baumannii, listed as a priority pathogen, under nutrient-deprived conditions.

A genome-wide screen reveals the involvement of enterobactin-mediated iron acquisition in Escherichia coli survival during copper stress.

Copper (Cu) is a key transition metal that is involved in many important biological processes in a cell. Cu is also utilized by the immune system to hamper pathogen growth during infection. However, genome-level knowledge on the mechanisms involved in adaptation to Cu stress is limited. Here, we report the results of a genome-wide reverse genetic screen for Cu-responsive phenotypes in Escherichia coli. Our screen has identified novel genes involved in adaptation to Cu stress in E. coli. We detected multiple genes involved in the biosynthesis and uptake of enterobactin, a siderophore utilized for high-affinity TonB-dependent acquisition of iron (Fe), as critical players in survival under Cu intoxication. We demonstrated the specificity of Cu-dependent killing by chelation of Cu and by genetic complementation of tonB. Notably, TonB is involved in protection from Cu in both laboratory and uropathogenic strains of E. coli. Cu stress leads to increased expression of the genes involved in Fe uptake, indicating that Fur regulon is derepressed during exposure to excess Cu. Trace element analyses revealed that Fe homeostasis is dysregulated during Cu stress. Taken together, our data supports a model in which lack of enterobactin-dependent Fe uptake leads to exacerbation of Cu toxicity, and elucidates the intricate connection between the homeostasis of Cu and Fe in a bacterial cell.

Enterobactin on a Bead: Parallel, Solid Phase Siderophore Synthesis Reveals Structure-Activity Relationships for Iron Uptake in Bacteria.

A solid-phase platform for the precise and sequential synthesis of enterobactin analogues is described. This chemistry unites the power of solid-phase peptide synthesis with the unique opportunities and applications offered by siderophore chemistry. Here, a series of hybrid enterobactin hydroxamate/catecholate (HEHC) analogues were synthesized using both catechols and amino acid derived hydroxmate chelators. The HEHC analogues were evaluated for their ability to bind free iron and to promote growth in siderophore-auxotrophic mutant bacteria. We find that, in contrast to S. aureus or E. coli, a number of HEHC analogues promote growth in P. aeruginosa and structure-activity relationships (SARs) exist for the growth promotion via HEHC analogues in this organism.

Enterobactin induces the chemokine, interleukin-8, from intestinal epithelia by chelating intracellular iron.

Iron is an indispensable nutrient for both mammals and microbes. Bacteria synthesize siderophores to sequester host iron, whereas lipocalin 2 (Lcn2) is the host defense protein that prevent this iron thievery. Enterobactin (Ent) is a catecholate-type siderophore that has one of the strongest known affinities for iron. Intestinal epithelial cells (IECs) are adjacent to large microbial population and are in contact with microbial products, including Ent. We undertook this study to investigate whether a single stimulus of Ent could affect IEC functions. Using three human IEC cell-lines with differential basal levels of Lcn2 (i.e. HT29 < DLD-1 < Caco-2/BBe), we demonstrated that iron-free Ent could induce a dose-dependent secretion of the pro-inflammatory chemokine, interleukin 8 (IL-8), in HT29 and DLD-1 IECs, but not in Caco-2/BBe. Ent-induced IL-8 secretion was dependent on chelation of the labile iron pool and on the levels of intracellular Lcn2. Accordingly, IL-8 secretion by Ent-treated HT29 cells could be substantially inhibited by either saturating Ent with iron or by adding exogenous Lcn2 to the cells. IL-8 production by Ent could be further potentiated when co-stimulated with other microbial products (i.e. flagellin, lipopolysaccharide). Water-soluble microbial siderophores did not induce IL-8 production, which signifies that IECs are specifically responding to the lipid-soluble Ent. Intriguingly, formyl peptide receptor (FPR) antagonists (i.e. Boc2, cyclosporine H) abrogated Ent-induced IL-8, implicating that such IEC response could be, in part, dependent on FPR. Taken together, these results demonstrate that IECs sense Ent as a danger signal, where its recognition results in IL-8 secretion.

Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB-dependent transport system.

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated Ton complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB-dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

The synergistic triad between microcin, colibactin, and salmochelin gene clusters in uropathogenic Escherichia coli.

A functional synergy was previously demonstrated between microcin, salmochelin and colibactin islands in Escherichia coli strains from B2 phylogroup. We aimed to determine this association prevalence in uropathogenic E. coli, and whether it was predictive of the infection severity in a collection of 225 E. coli strains from urinary samples. The high prevalence of this triad, even if it wasn't correlated with infection severity, suggested that it might not be a virulence factor per se within the urinary tract, but would promote its colonization. This triad would enable the strain to dominate the rectal reservoir with a minimal genetic cost.

EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058.

Extraintestinal pathogenic Escherichia coli (ExPEC) shows an enhanced ability to cause infection outside the intestinal tract. Avian pathogenic E. coli (APEC), one type of ExPEC, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide that is characterized by systemic infection. Some ExPEC strains as well as other pathogenic enterobacteria produce enterobactin, a catecholate siderophore used to sequester iron during infection. Here, we showed that disruption of enterobactin efflux via outer membrane protein TolC significantly decreased the pathogenicity of APEC strain E058. Furthermore, colonization and persistence assays performed using a chicken infection model showed that the ΔtolC mutant was obviously attenuated (p˂0.001). In contrast, disruption of enterobactin synthesis gene entE and/or the inner membrane transporter gene entS had little effect on pathogenicity. Analysis of growth kinetics revealed a significant reduction in the growth of triple mutant strain E058ΔentEΔentSΔtolC in iron-deficient medium compared with the wild-type strain (p˂0.001), while no growth impairment was noted for the E058ΔtolC mutant in either Luria-Bertani broth or iron-deficient medium. The E058ΔentEΔentSΔtolC mutant also showed significantly decreased virulence compared with single mutant strain E058ΔtolC. Low-copy complementation of strains E058ΔtolC and E058ΔentEΔentSΔtolC with plasmid-borne tolC restored virulence to wild-type levels in the chicken infection model. Macrophage infection assays showed that ingestion of E058ΔtolC by macrophage cell line HD11 cells was reduced compared with ingestion of the E058ΔentEΔentSΔtolC mutant. However, no significant differences were observed between the mutants and the wild-type in a chicken serum resistance assay. Together, these results suggest that EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058 in an iron-deficient environment.

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum ß-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.