Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

Microbial Siderophore Enterobactin Promotes Mitochondrial Iron Uptake and Development of the Host via Interaction with ATP Synthase.

Elucidating the benefits of individual microbiota-derived molecules in host animals is important for understanding the symbiosis between humans and their microbiota. The bacteria-secreted enterobactin (Ent) is an iron scavenging siderophore with presumed negative effects on hosts. However, the high prevalence of Ent-producing commensal bacteria in the human gut raises the intriguing question regarding a potential host mechanism to beneficially use Ent. We discovered an unexpected and striking role of Ent in supporting growth and the labile iron pool in C. elegans. We show that Ent promotes mitochondrial iron uptake and does so, surprisingly, by binding to the ATP synthase α subunit, which acts inside of mitochondria and independently of ATP synthase. We also demonstrated the conservation of this mechanism in mammalian cells. This study reveals a distinct paradigm for the "iron tug of war" between commensal bacteria and their hosts and an important mechanism for mitochondrial iron uptake and homeostasis.

An antioxidant role for catecholate siderophores in Salmonella.

Iron acquisition is an important aspect of the host-pathogen interaction. In the case of Salmonella it is established that catecholate siderophores are important for full virulence. In view of their very high affinity for ferric iron, functional studies of siderophores have been almost exclusively focused on their role in acquisition of iron from the host. In the present study, we investigated whether the siderophores (enterobactin and salmochelin) produced by Salmonella enterica sv. Typhimurium could act as antioxidants and protect from the oxidative stress encountered after macrophage invasion. Our results show that the ability to produce siderophores enhanced the survival of Salmonella in the macrophage mainly at the early stages of infection, coincident with the oxidative burst. Using siderophore biosynthetic and siderophore receptor mutants we demonstrated that salmochelin and enterobactin protect S. Typhimurium against ROS (reactive oxygen species) in vitro and that siderophores must be intracellular to confer full protection. We also investigated whether other chemically distinct siderophores (yersiniabactin and aerobactin) or the monomeric catechol 2,3-dihydroxybenzoate could provide protection against oxidative stress and found that only catecholate siderophores have this property. Collectively, the results of the present study identify additional functions for siderophores during host-pathogen interactions.

Catecholate siderophores protect bacteria from pyochelin toxicity.

BACKGROUND: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. METHODS AND PRINCIPAL FINDINGS: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity. CONCLUSIONS: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Production of aromatic compounds in bacteria.

The aromatic class of chemicals includes a large number of industrially important products. In bacteria and plants, the shikimate pathway and related biosynthetic pathways are a source of aromatic compounds having commercial value. Bacterial strains for the production of aromatic compounds from simple carbon sources as raw material have been generated by applying metabolic engineering and random/combinatorial strategies that modify central metabolism, aromatic biosynthetic pathways, transport, and regulatory functions. These strategies are complemented with heterologous gene expression and protein engineering. Engineered Escherichia coli and Pseudomonas putida strains are enabling the development of sustainable processes for the manufacture of 2-phenylethanol, p-hydroxycinnamic acid, p-hydroxystyrene, p-hydroxybenzoate, anthranilate, and cyclohexadiene-transdiols, among other useful chemicals.

Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system.

A chromosomal gene cluster encoding vanchrobactin biosynthesis and transport genes was identified in the Vibrio anguillarum serotype O1 strain, 775(pJM1), harbouring the anguibactin biosynthetic genes in the pJM1 plasmid. In this strain only anguibactin is produced as the vanchrobactin chromosome cluster has a RS1 transposition insertion into vabF, one of the vanchrobactin biosynthesis genes. Removal of this RS1 generating 775(pJM1)Delta tnp, still resulted in the detection of only anguibactin in specific bioassays. Surprisingly, when the pJM1 plasmid was not present as in the plasmidless strain H775-3, removal of the RS1 resulted in the detection of only vanchrobactin. These results thus can be interpreted as if presence of the pJM1 plasmid or of anguibactin itself is associated with the lack of detection of the vanchrobactin siderophore in bioassays. As high-performance liquid chromatography (HPLC) and mass spectrometry analysis demonstrated that both vanchrobactin and anguibactin were indeed produced in 775(pJM1)Delta tnp, it is clear that the pJM1-encoded anguibactin siderophore has higher affinity for iron than the vanchrobactin system in strains in which both systems are expressed at the same time. Our results underscore the importance of the anguibactin system in the survival of V. anguillarum 775 under conditions of iron limitation.

Iron metabolism at the host pathogen interface: lipocalin 2 and the pathogen-associated iroA gene cluster.

The host innate immune defense protein lipocalin 2 binds bacterial enterobactin siderophores to limit bacterial iron acquisition. To counteract this host defense mechanism bacteria have acquired the iroA gene cluster, which encodes enzymatic machinery and transporters that revitalize enterobactin in the form of salmochelin. The iroB enzyme introduces glucosyl residues at the C5 site on 2,3-dihydroxybenzoylserine moieties of enterobactin and thereby prevents lipocalin 2 binding. Additional strategies to evade lipocalin 2 have evolved in other bacteria, such as Mycobacteria tuberculosis and Bacillus anthracis. Targeting these specialized bacterial evasion strategy may provide a mechanism to reinvigorate lipocalin 2 in defense against specific pathogens.

Differences in excretion and efficiency of the aerobactin and enterochelin siderophores in a bovine pathogenic strain of Escherichia coli.

Secretion of aerobactin is thought to play an important part in the virulence of invasive Escherichia coli also capable of synthesizing enterochelin. Why, despite its markedly lower affinity for iron than that of enterochelin, aerobactin proves to be the predominant active siderophore for bacterial growth in transferrin was investigated. We studied the action of two iron chelators, 2,2'-dipyridyl and transferrin, in expression of the aerobactin and enterochelin genes. Specifically, we describe the sequential localization of the two siderophores in the cell compartments during bacterial growth under different iron limitation conditions. Our results demonstrated that, whatever the exogenous iron-chelating agent used, aerobactin was rapidly excreted, whereas enterochelin accumulated early in periplasm before its very belated release into the external medium. This work also showed that the advantage of aerobactin over enterochelin in competition with transferrin was not due to (i) lack of enterochelin activity, (ii) a cell-bound aerobactin-dependent mechanism, (iii) antagonism between the two siderophores, and probably (iv) genetic preferential induction of aerobactin. We propose that the superiority of aerobactin in competing with transferrin for iron(III) was a consequence of its more rapid excretion with respect to enterochelin. In contrast to transferrin, 2,2'-dipyridyl induced a greater efficiency of enterochelin, possibly by a more permanent function as iron-binding compounds in the bacterial envelope. In summary, unlike aerobactin, enterochelin appears to be a weakly secreted high-affinity iron ligand.

[Iron chelation. Biological significance and medical application]. / Eisenchelierung. Biologische Bedeutung und medizinische Anwendungen.

Iron, an essential element for all aerobic organisms, exists in a very insoluble form under physiological conditions. Therefore, most microorganisms secrete iron chelating compounds called siderophores which are able to sequester ferric ions from the environment. A vast number of such compounds has been isolated from cultures of microorganisms and tested for enhancement of iron excretion in experimental animals. Only one compound, deferrioxamine B, has been shown to be clinically effective and well tolerated in humans suffering from chronic iron overload. However, this drug can only be administered successfully by injection or slow infusion. In spite of considerable research it has not been possible to overcome this drawback by developing suitable formulations or derivatives which are orally active. Deferri-ferrithiocin, a novel type of siderophore, has recently been isolated from a streptomyces culture. This substance is well absorbed orally and has been shown to enhance the excretion of ferric ion in iron loaded rats. Further investigations are now necessary to establish acute toxicity levels and longterm tolerability before efficacy tests in man can be planned. Other recent developments in the field of metal chelation include experimental studies using deferrioxamine for the treatment of conditions resulting from toxic levels of iron or aluminium in chronically dialyzed patients. In addition, attempts are being made to administer chelation therapy in the treatment of various infections and chronic inflammation, as well as other conditions linked with disorders of iron metabolism.