Less is more: Enterobactin concentration dependency in copper tolerance and toxicity.

The ability of siderophores to play roles beyond iron acquisition has been recently proven for many of them and evidence continues to grow. An earlier work showed that the siderophore enterobactin is able to increase copper toxicity by reducing Cu2+ to Cu+, a form of copper that is more toxic to cells. Copper toxicity is multifaceted. It involves the formation of reactive oxygen species (ROS), mismetallation of enzymes and possibly other mechanisms. Given that we previously reported on the capacity of enterobactin to alleviate oxidative stress caused by various stressors other than copper, we considered the possibility that the siderophore could play a dual role regarding copper toxicity. In this work, we show a bimodal effect of enterobactin on copper toxicity (protective and harmful) which depends on the siderophore concentration. We found that the absence of enterobactin rendered Escherichia coli cells more sensitive to copper, due to the reduced ability of those cells to cope with the metal-generated ROS. Consistently, addition of low concentrations of the siderophore had a protective effect by reducing ROS levels. We observed that in order to achieve this protection, enterobactin had to enter cells and be hydrolyzed in the cytoplasm. Further supporting the role of enterobactin in oxidative stress protection, we found that both oxygen and copper, induced the expression of the siderophore and also found that copper strongly counteracted the well-known downregulation effect of iron on enterobactin synthesis. Interestingly, when enterobactin was present in high concentrations, cells became particularly sensitive to copper most likely due to the Cu2+ to Cu+ reduction, which increased the metal toxicity leading to cell death.

Antimicrobial activity of MccJ25(G12Y) against gram-negative foodborne pathogens in vitro and in food models.

The use of bacteriocins is a promising alternative to improve food security through the biocontrol of food pathogens and spoilage microorganisms. Gram-negative produced microcin J25(G12Y), known as (MccJ25(G12Y)) is a variant of the well-studied and characterized antimicrobial peptide, microcin J25 (MccJ25). In the present work, we explored the activity of this microcin against Gram-negative bacteria linked to foodborne diseases. We evaluated the in vitro antimicrobial activity of MccJ25(G12Y) in solid medium against a collection of pathogenic and food-altering strains and studied its activity and stability in meat and dairy food systems. We show that MccJ25(G12Y) exhibited the same in vitro antimicrobial spectrum as its parental microcin (MccJ25) against different Gram-negative foodborne pathogens and spoilage strains. We highlight that low concentrations of MccJ25(G12Y) between 0.45 and 29.4 µM were able to inhibit a substantial number of pathogens, including Salmonella, Escherichia, Shigella and Enterobacter genus. We also demonstrate the antimicrobial effectiveness of the peptide against Escherichia coli O157:H7 NCTC 12900, Enterobacter cloacae CECT 194, and Salmonella enterica CECT 4396 in fish and beef burgers and yogurt. MccJ25(G12Y) was added or not to food matrices inoculated with the foodborne pathogens at 105 CFU/g or mL. Afterward, food products were stored at 4 °C and selective media for the specific enumeration were used to determine the antimicrobial susceptibility of each pathogen to MccJ25(G12Y). The viability of the three pathogens was significantly reduced in the different food biological environments. In yogurt, the peptide decreased E. coli numbers on day 5 by about 4 log 10 CFU/mL as compared to non-treated samples. For S. enterica and E. cloacae no viable cells were detected at the end of the treatment. Adding MccJ25(G12Y) to fish burgers decreased E. cloacae numbers during storage 2 log10 CFU/g on the first day, reaching a difference of about 5 log 10 CFU/g after 10 days compared to non-treated control. Finally, the peptide decreased E. coli O157:H7 numbers on the beef burgers samples during storage on day 10 by about 3 log 10 CFU/g as compared to non-treated samples. The stability analysis demonstrated that MccJ25(G12Y) is capable of remaining active in these food matrices for a considerable time during the storage at refrigeration temperatures. These results reinforce the studies on the potential applicability of this microcin as a biopreservative in the food industry.

Enterobactin as Part of the Oxidative Stress Response Repertoire.

Microorganisms produce siderophores to facilitate iron uptake and even though this trait has been extensively studied, there is growing evidence suggesting that siderophores may have other physiological roles aside from iron acquisition. In support of this notion, we previously linked the archetypal siderophore enterobactin with oxidative stress alleviation. To further characterize this association, we studied the sensitivity of Escherichia coli strains lacking different components of the enterobactin system to the classical oxidative stressors hydrogen peroxide and paraquat. We observed that strains impaired in enterobactin production, uptake and hydrolysis were more susceptible to the oxidative damage caused by both compounds than the wild-type strain. In addition, meanwhile iron supplementation had little impact on the sensitivity, the reducing agent ascorbic acid alleviated the oxidative stress and therefore significantly decreased the sensitivity to the stressors. This indicated that the enterobactin-mediated protection is independent of its ability to scavenge iron. Furthermore, enterobactin supplementation conferred resistance to the entE mutant but did not have any protective effect on the fepG and fes mutants. Thus, we inferred that only after enterobactin is hydrolysed by Fes in the cell cytoplasm and iron is released, the free hydroxyl groups are available for radical stabilization. This hypothesis was validated testing the ability of enterobactin to scavenge radicals in vitro. Given the strong connection between enterobactin and oxidative stress, we studied the transcription of the entE gene and the concomitant production of the siderophore in response to such kind of stress. Interestingly, we observed that meanwhile iron represses the expression and production of the siderophore, hydrogen peroxide and paraquat favour these events even if iron is present. Our results support the involvement of enterobactin as part of the oxidative stress response and highlight the existence of a novel regulation mechanism for enterobactin biosynthesis.

The alternative role of enterobactin as an oxidative stress protector allows Escherichia coli colony development.

Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress.