Siderophores from Fish Pathogenic Bacteria

Abstract

Aquaculture is an important food source worldwide but one of its main problems is the spread of infectious diseases caused by Gram-negative pathogenic bacteria that results in considerable economic losses. Since siderophores, low molecular weight organic compounds involved in the iron-uptake mechanisms of the bacteria, are critical for the growth and virulence of the producer pathogens, the bacterial iron acquisition systems are promising targets for the design of new antimicrobial strategies. In this chapter, the current chemical knowledge of the siderophores involved in the iron-uptake mechanisms of Gram-negative pathogenic bacteria Vibrio anguillarum, Photobacterium damselae subsp. piscicida, and Aeromonas salmonicida subsp. salmonicida, responsible for the main fish infectious diseases Vibriosis, photobacteriosis, and furunculosis, respectively, is summarized. The isolation, structural elucidation, and the chemical synthesis of the siderophores biosynthesized from those bacteria are displayed. Their involvement in the iron-uptake mechanisms, virulence importance, and the application in the development of new strategies to fight against the infectious diseases will be also discussed.

1 Introduction

The need for a safe, reliable, and economic supply of food to feed the high increase of human population is transforming aquaculture as an important food source worldwide. According to the Food and Agriculture Organization (FAO) of the United Nations, global fish consumption (from wild fishing and aquaculture) grew at an average of 3.1% yearly from 1961 to 2017, which is a rate almost twice that of the 1.6% annual world population increase. While wild fishing production has stabilized in the last 20 years, global aquaculture production increased more than five times in the last three decades (from 14.9 million tonnes in the period 1986/1995 to 82.1 million tonnes in 2018). Right now, more than 50% of fish for human consumption comes from aquaculture [1].

However, the production intensification increases the likelihood of pathogen outbreaks and the emergence of several infectious diseases which are causing severe economic losses in aquaculture (Fig. 1a) [2]. The extensive use of antibiotics has contributed to the spread of antimicrobial resistance (AMR), which has become a global health emergency. Moreover, fish are reservoirs of zoonotic pathogens such as Aeromonas hydrophila and Photobacterium damselae that can infect not only the animal host but also humans who are in contact with the aquaculture facility and via foodborne infections [3]. For all these reasons, the search for new strategies to prevent and control diseases in aquatic species is urgently needed. It is one of the main research objectives within the field of health in the next decade.

Fig. 1

(a) Turbot infected with photobacterioisis. (b) General iron uptake mechanisms in Gram-negative bacteria

For most bacteria, iron uptake ability during the naturally iron-limited conditions of an infection is a key virulence factor essential for multiplication within the host. Iron is a crucial nutrient in virtually all living organisms because it is needed in many metabolic processes, catalyzing a wide variety of indispensable enzymatic reactions. The wide range of properties of iron and its abundance, being the fourth most abundant element in the Earth’s crust, could explain why early microorganisms, from the beginning of evolution, used this element to develop their metabolic processes in an oxygen-poor atmosphere. However, when the atmosphere became rich in oxygen, ferrous iron was oxidized to ferric ion which precipitates to form ferric hydroxide which is highly insoluble at physiological pH. Since then, the bioavailability of free ion became very low and the iron acquisition uptake mechanisms to combat the extremely low iron availability became an indispensable process for most living organisms [4]. Now, a high competition for this scarce iron is taking place and so, iron sequestration systems have been identified as important virulence mechanisms in several pathogenic bacteria. In fact, potential hosts keep the level of free iron inside the body to a minimum as a defense mechanism.

One of the major strategies used by bacteria and fungi entailed the use of siderophores, a Greek term that means iron carriers. These are low molecular weight organic compounds that have extremely high affinity for ferric iron [5]. Thus, bacteria and fungi excrete “endogenous” siderophores into the culture medium where they are able to solubilize Fe (III) from its hydroxide or they can efficiently sequester the iron bound by transferrins and other iron-holding proteins within the host. Once siderophores are synthesized and secreted outside the bacterial cells, they bind Fe³⁺ ions to form a ferric-siderophore complex which is then internalized by the appropriate transport mechanism. In Gram-negative bacteria, ferric-siderophore complexes are recognized by specific outer membrane receptors (OMR). These proteins trigger the transport system across the outer and inner membranes, thus permitting the delivery of iron into the cells by an energy-dependent system. The components of such systems include a specific TonB-dependent ferric-siderophore transporter (TBDT) protein located in the outer membrane that is energized through the TonB system. The TBDT protein is coupled to an ABC transporter that catalyzes the final stages of ferric-siderophore transport through the cytoplasmic membrane from the periplasm to the cytosol [6]. Finally, the reduction of Fe³⁺ to Fe²⁺ by microbial-mediated processes is the most common mechanism for the release of the iron inside the cell (Fig. 1b) [7]. Additionally, some microorganisms are able to use the xenosiderophores, siderophores biosynthesized by other organisms, due to the presence of OMR that can recognize a wide spectrum of siderophores with the same type of chelating units.

On the basis of the bidentate groups involved in iron (III) binding, siderophores can be classified into four main classes: phenolates or catecholates, hydroxamates, α-hydroxycarboxylates, and mixtures with these functionalities (Fig. 2) [5].

Fig. 2

Examples of the main types of microbial siderophores

Iron restriction is an important host defense strategy; thus, successful pathogens must possess mechanisms to acquire iron from host sources in order to cause disease. During infection, a fierce battle of iron acquisition occurs between the host and bacterial pathogens. For that reason, siderophores are not only crucial in bacteria iron metabolism but also are key factors in the virulence to enhance the infection disease. In this way, the iron-uptake mechanism mediated by siderophores along with their structures provides an ideal target for therapeutic applications [4]. Indeed, the natural siderophore “desferal,” produced by Streptomyces pilosus, is used in clinics for the removal of excess iron in human blood, a disease known as thalassemia [8]. But one of the most promising applications of siderophores is for the development of new antibacterial treatments based on iron acquisition, such as novel antimicrobials and vaccines. Thus, iron-uptake mechanisms mediated by siderophores are providing the basis for the development of new antibacterial treatments: by the Trojan horse Strategy where the siderophore is coupled to a known antibiotic in order to overcome the permeability barrier of the Gram-negative outer membrane that uses a specific siderophore route of entry into the cell. The natural siderophore-antibiotic conjugates (sideromycins), such as albomycins and salmycins, inspired this methodology [9]. This approach seeks to avoid microbial resistance, thus enhancing sensitivity and specificity. In fact, the catechol based-siderophore cefiderocol (Fetcroja^®) is the first siderophore-drug conjugate approved by the U.S. Federal Drug Administration (FDA) [10] and the European Medicines Agency (EMA) [11]. Other applications imply the inhibition of biosynthesis of siderophore and the development of vaccines [4].

Vibriosis, photobacteriosis, and furunculosis, caused by Gram-negative marine bacteria, constitute the main infectious diseases in aquaculture [12]. In the present chapter, studies of the siderophores from the fish Gram-negative pathogenic bacteria involved in those infectious diseases and its application in fish farming will be presented. Special attention will be paid to the isolation methodology used, the synthesis of natural siderophores and their derivatives, the establishment of some Structure Activity Relationship (SAR), the biosynthetic proposals and the OMR involved.

2 Vibriosis by Vibrio anguillarum

Vibriosis is a group of diseases that cause a serious hemorrhagic septicemia associated with both wild and farmed marine fishes. This leads to severe economic losses in the aquaculture industry worldwide. Different Gram-negative bacterial species, mainly classified into the Vibrio genus, belonging to the family of Vibrionaceae are responsible for this infection [13]. The bacillus V. anguillarum is considered the etiological agent of classical vibriosis, being the most common and also the most extensively studied Vibrionaceae species. Its virulence in marine settings was reported as early as 1718 along the coastline of continental Europe and was referenced on record as the red disease in eel (Anguilla anguilla) in 1893, serving as the root of the name anguillarum. By 1970, it was known to have devastating effects on several marine organisms (cod, eel, finnock, oyster, salmon, trout, etc.) and now has a wide geographical distribution and host range. V. anguillarum is pathogenic to a variety of crustaceans, bivalves and mainly to warm- and cold-water fish of economic importance, including Pacific and Atlantic salmon, rainbow trout, turbot, sea brass, sea bream, among others, accounting for more than 90 susceptible aquatic species in at least 28 different countries [14]. A total of 23 different O serotypes of V. anguillarum isolates have been recognized. Among them, O1 and O2 are the main ones implicated in the infections, although serotype O3, and to a lesser extent O4 and O5, have increasing importance in vibriosis outbreaks in specific geographic areas [15]. The remaining serotypes are considered to be environmental strains and only on rare occasions are isolated as responsible for vibriosis in fish [16].

For a long time, just two different siderophore-mediated systems were described in V. anguillarum strains belonging to serotypes O1 and O2, the anguibactin (Ang, 1) and the vanchrobactin (Vcb, 2). More recently, a third one was reported, piscibactin (Pcb, 3), which turned out to be one of the most important virulence factors for this bacterium (Fig. 3). Additionally, V. anguillarum can also use xenosiderophores as iron sources, like enterobactin, ferrichrome or rhodotorulic acid.

Fig. 3

Structure of the siderophores biosynthetized by V. anguillarum

2.1 Anguibactin Iron-Uptake System

The first siderophore-mediated system described in this bacterium, called anguibactin system, was found in pathogenic strains of serotype O1, which bears the 65 kb pJM1 plasmid that harbors the genes for the biosynthetic machinery and utilization of the siderophore anguibactin (Ang, 1).

Ang (1) was purified from its supernatants of iron-deficient cultures by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column and reported in 1986 [17]. Three years later, its hybrid structure between a catechol and a hydroxamate type of siderophore was determined by ¹H- and ¹³C-NMR spectral analysis of its Ga(III)-complex, FAB-MS, chemical degradation, and single-crystal X-ray diffraction studies of its anhydro derivative, anhydroanguibactin (4) [18]. The crystal structure of the Ga(III)-complex of anguibactin was reported in 1998 [19]. However, the absolute configuration of the cysteine moiety in Ang (1) was not determined at that time.

The structure of Ang (1) is similar to that of preacinetobactin (PreAcb, 5), which contains instead an oxazoline ring. PreAcb (5) is the precursor of acinetobactin (Acb, 6), the siderophore produced by the Gram-negative pathogen Acinetobacter baumannii, which constitutes one of the major health concerns because it is responsible for a large number of hospital-acquired and nosocomial infections and by its increasing development of antimicrobial resistance (Fig. 4) [20].

Fig. 4

Similar structures to that of Ang (1)

Since the content of siderophores from marine bacteria is very low and they have potentially valuable applications, the development of chemical synthesis is a crucial step to further study their biological activities, mechanisms, absolute configurations, and/or structure-activity relationships. Furthermore, the synthesis of analogues gives us very important information, for instance, for the preparation of conjugates to be used as biosensors to study these mechanisms [21], for the development of novel therapies against iron-overload related disease [8], for the preparation of new antimicrobial agents that target bacterial pathways for the acquisition of iron [22], for a Trojan horse strategy to develop modified molecules with antimicrobial activity [9], or for the synthesis of bioconjugates of siderophores to be tested as antibacterial vaccines [23].

The total synthesis of Ang (1), published by H. J. Kim and co-workers in 2018, started with the preparation of N^α-benzyloxyhistamine (9) which was obtained from histamine dihydrochloride in three steps following the same procedure employed in the synthesis of Acb (6) [24]. The reaction of histamine dihydrochloride with NaNO₂ in acid media followed by treatment with SOCl₂ gave 7. Coupling of 7 with N-tert-butoxycarbonyl(Boc)-O-benzyloxyamine afforded imidazole 9 after deprotection with TFA. In parallel, the thiazoline fragment 12 was obtained by the coupling of aryl nitrile 10, prepared from o-vanillin, with L-cysteine.

Condensation of thiazoline acid 12 with N^α-benzyloxyhistamine (9) was one of the key steps of the synthesis in order to avoid the epimerization observed at position 8, because the coupling gives a near-racemic mixture. The lack of a substituent at position 9 in Ang (1), in relation to that in Pre-acb (5), seems to justify that racemization. The presence of an (R)-methyl group at C-9 in Pre-acb (5) could secure the sterically stable trans geometry with the (S)-configuration at C-8. The use of Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) in dichloromethane with a free amine form of 9 resolved this problem, giving the intermediate 14 with a stereoselective ratio of er = 98:2. The problematic removal of the two ether-type protecting groups present in 14 using boron trichloride (BCl₃) in the final step was solved by the addition of tetrabutylammonium iodide (TBAI), which allowed a clean removal of both benzyl and methyl groups. Using a similar procedure, the anguibactin analogue, 3-deoxyanguibactin (16), was also prepared (Scheme 1). The preparation of synthetic siderophore 1 made it possible to determine, by fluorescence titrations, a 2:1 binding stoichiometry between Ang (1) and Fe(III) in liquid phase. Furthermore, the iron delivery capability of Ang (1) and its thermal stability over PreAcb (5) led the authors to propose Ang (1) as a competent surrogate siderophore for Acinetobacter baumannii that can be useful for the future development of a siderophore-based antibiotic delivery system against that pathogen [25].

Scheme 1

Synthesis of Ang (1) and its analogue 3-deoxyanguibactin (15)

Evaluation of cytotoxic effects of Ang (1) on P388 murine leukemia cell lines displays an IC₅₀ value of <15 μM [26]. Moreover, Ang (1) has been patented for performing deferration therapy due to its properties for removing ferric iron from aqueous liquids. Among other properties, it was found that Ang (1) inhibits iron uptake by living cells, wrests iron from vertebrate tissues, removes iron from other siderophores and ferric hydroxide, and removes ferric iron from aqueous solutions, including cell-culture media [27].

Most biosynthetic genes of Ang (1) are located in the pJM1 plasmid and it is accomplished by a complex of non-ribosomal peptide synthetases (NRPS). However, its biosynthesis requires additional chromosomally encoded enzymes that are also involved in the production of Vcb (2) as the second siderophore described. The pJM1 plasmid series is present in most V. anguillarum O1 strains but is absent in serotype O2. The ferric iron-anguibactin complex is transported by the FatABCD proteins, all encoded by the pJM1 plasmid: the OM receptor FatA, the periplasmic binding protein FatB, and the inner membrane proteins FatC and FatD (ABC transporter) [28].

2.2 Vanchrobactin Iron-Uptake System

The second siderophore-mediated iron-uptake system reported in V. anguillarum was the vanchrobactin system. Vanchrobactin (Vcb, 2) is encoded by a gene cluster located in the chromosome that was first found in all serotype O2 strains and some plasmid-less serotype O1 strains. The terms of V. anguillarum (“van”), chromosome (“chro”) and the suffix “bactin” for chelating agent, were used to name this siderophore as vanchrobactin.

This compound was isolated for the first time from iron-deficient cultures of V. anguillarum serotype O2 strain RV22 by using a XAD-7 lipophilic resin, liquid–liquid fractionation, Sephadex LH-20, and RP-HPLC. Its planar structure was established by analysis of its 1D and 2D NMR, IR, UV, and mass spectral data as a dipeptide made up of the amino acids arginine and serine linked to a 2,3-dihydroxybenzoyl moiety [29]. A faster and a more convenient isolation procedure using Hydrophilic-Lipophilic Balance (HLB) resins followed by HPLC-HRMS analysis was developed and applied in the re-isolation of Vcb (2). This method, named as SPE-HLB/HPLC-HRMS, improved not only the efficiency of the isolation methodology but also the isolation time in relation to the existing methods [30].

The absolute configuration of Vcb (2) was established by synthesis of three out of four possible stereoisomers using 2,3-dihydroxybenzoic acid (DHBA), L/D-ornithine, and L/D-serine as starting materials and comparison to the natural product. Thus, 2,3-dibenzyloxybenzoic acid (17), prepared in three steps from DHBA, was coupled with N^δ-Cbz-D-ornithine-OMe (16a), obtained in four steps from D-ornithine, or with N^δ-Cbz-L-ornithine-OMe (16b), prepared from esterification of the commercial N^δ-Cbz- L-ornithine, to give acids 18a,b, respectively, after saponification. Then, tert-butyl esters of L/D-serine, synthetized from commercial amino acids, were coupled to acids 18a,b to afford 19a-c. Deprotection of the benzyl groups in 19a-c, followed by introduction of the guanidine functionality and deprotection of the tert-butyl group in acid media, yielded three out of the four possible stereoisomers of vanchrobactin. Comparison of the synthetic compounds with the natural sample by achiral HPLC, chiral capillary electrophoresis, along with NMR data, sign and value of the optical rotation allowed to establish the absolute structure of Vcb (2) as N-[N′-(2,3-dihydroxybenzoyl)-D-arginyl]-L-serine (Scheme 2) [31].

Scheme 2

Synthesis of Vcb (2) and its analogues DHBA-Arg/Orn 20–24

Two series of vanchrobactin analogues were synthetized using a similar strategy employed in the total synthesis of Vcb (2). The first one where the 2,3-dihydroxybenzoyl moiety is linked to Arg or Orn residues, compounds 20–24, (Scheme 2) and the second series where it was the serine residue which was linked directly to the aromatic moiety, compounds 27–29 (Scheme 3).

Scheme 3

Synthesis of Vcb analogues DHBA-Ser 27–29

The synthesized compounds were subjected to the chrome azurol-S (CAS) test and evaluated by growth promotion assay (siderophore activity test) with several bacteria that are well known for their ability to transport and use natural siderophores, specified in brackets: V. anguillarum RV22 (serotype O2) (Vcb (2) and Pcb (3)), V. anguillarum 775 (serotype O1) (Ang (1)), Salmonella enterica enb-1 (enterobactin), Erwinia chrysanthemi PPV20 (chrysobactin (34)), and V. alginolyticus TA15 (vibrioferrin). Most of analogues were positive in the CAS test and showed siderophore activity to both V. anguillarum serotypes tested. Although most of them displayed siderophore activity to S. enterica and E. chrysanthemi, none of them were able to restore the growth of V. alginolyticus. These results are in agreement with the catecholate structure of enterobactin and chrysobactin (34) and the non catecholate structure of vibrioferrin, an α-hydroxycarboxylate siderophore (Fig. 1). From the results, it was suggested that the aromatic ring in catecholate siderophores is crucial for the binding of the outer membrane receptors. Additionally, the results reveal the lack of stereoisomeric influence of the amino acid scaffold on the siderophore activity in both serotypes of V. anguillarum, although some differences can be observed in E. chrysanthemi PPV20 and S. enterica. This lack of specificity could be an evolutionary adaptation of bacterial pathogens to increase the number of possibilities to obtain iron from the environment. The low specificity of molecular recognition of the recipients of these strains could facilitate the use of those conjugates to be prepared. Analogues having ornithine (23–24) instead of arginine (20–22) residue maintain the siderophore activity to V. anguillarum, as the Vb analogue DHBA- D-Orn- L-Ser (24b), and they are of special interest since these molecules possess an appropriate functionality, an amino group, that can be used as an anchor group to attach it to other bioactive agents. For these reasons, these compounds were considered as potential antibiotic vectors for the delivery of known antibacterial agents via the bacterial iron-uptake systems [32].

Vcb (2) is structurally related to chrysobactin (30), isolated from the phytopathogenic bacterium E. chrysanthemi, which bears a lysine residue instead of arginine. This compound also has the L configuration for the serine residue, while the lysine residue has a D configuration as the arginine in Vcb (2) [33]. From an identified species of Vibrio, DS40M4 strain, a bis- and triscatechol amide analogues of Vcb (2), named divanchrobactin (31) and trivanchrobactin (32), were isolated. Vcb (2) and Ang (1) were also isolated from this bacterium and represented the first case that the two siderophores were isolated from the same strain. Their isolation was carried out using Amberlite XAD-2 resins and HPLC. The isolation of 31 and 32 raised the question whether linear trivanchrobactin is the real siderophore or comes from a hypothetical cyclic ester, a cyclic trivanchrobactin product, which was not detected or isolated due to the instability of the cyclic triserine ester or the reactivity of a possible esterase [34]. Curiously, cyclic trichrysobactin, linear trichrysobactin (33), and dichrysobactin (34) along with the monomeric siderophore unit, chrysobactin (30) were isolated from plant pathogen Dickeya chrysanthemi EC16 (formerly known as Pectobacterium chrysanthemi EC16 and E. chrysanthemi EC16) (Fig. 5) [26].

Fig. 5

Siderophores with similar structures to that of Vcb (2)

On the basis of the results of the siderophore activity evaluation of Vcb analogues, three conjugates 35–37 between the known antibiotic norfloxacin and Vcb analogues 23b and 24b were synthesized using acetate as a spacer arm. A four conjugate 38 was also prepared between norfloxacin and aminochelin (39), one of the siderophores produced by the Gram-negative free-living soil bacterium Azotobacter vinelandii [35]. Although aminochelin (39) displayed siderophore activity in V. anguillarum serotype O2, in contrast to the other Vcb two analogues, aminochelin does not use the OMR of Vcb (2) FvtA as the route of entry.

The coupling of 2,3-diisopropyloxybenzoic acid (40), obtained in a similar way as 9, with tert-butyl(4-aminobutyl)carbamate gave carbamate 41. The free amine obtained from the deprotection of the Boc group in 41 was coupled with chloroacetyl chloride and then with norfloxacin to give the aminochelin–norfloxacin conjugate 38 after the removal of isopropyl protecting groups with BCl₃. For the synthesis of DHBA-D-ornithine-norfloxacin conjugates 36 and 37, N^δ-Cbz-D-ornithine-O^tBu, prepared from the commercially available N^δ-Cbz-D-ornithine, was coupled with 2,3-diisopropyloxybenzoic acid (40) to give carbamate 42. Removal of the Cbz group in 42 by catalytic hydrogenation gave a free amine which was submitted to the same procedure as before to afford an intermediate that yielded conjugates 36 and 37, after removal of isopropyl protecting groups with BCl₃ in MeOH or H₂O, respectively. For the synthesis of DHBA-D-ornithine-norfloxacin conjugate 35, N^δ-Cbz-D-ornithine-OMe (16a, Scheme 2) was coupled first with 2,3-diisopropyloxybenzoic acid (40) and then, after saponification, with O-tert-butyl-L-serine tert-butyl ester to give dipeptide 43. Finally, the submission of the protected dipeptide 43 to the same sequence of reactions employed in the preparation of conjugate 38 from 41 gave conjugate 35 (Scheme 4). The four synthetic conjugates 35–38 gave a positive reaction in the CAS test indicating that the binding of the Vcb analogs to norfloxacin through the amino functionality did not affect the chelating ability of these compounds. However, the lower antibiotic activity displayed by the four conjugates 35–38 in relation to unconjugated norfloxacin indicates that they are not working as Trojan horse conjugates [36].

Scheme 4

Synthesis of the conjugates 35–38 between norfloxacin and Vcb analogues and aminochelin (39)

Vanchrobactin iron complexes studies using spectrophotometric and potentiometric methods were reported. Kinetic studies at different pHs showed that of free Vcb (2) is stable in the pH range from 4 to 10.4 while the presence of a single pK with a value of 6.79 was obtained by spectroscopic titration studies. The influence of pH on iron vanchrobactin complex formation, shown in its UV visible spectra, reflects the presence of three sequential complexation reactions, corresponding to the formation of the ferric mono-, bis-, and tris(Vcb) complexes. The color change from alkaline to acid media was a clear indication of a structural change under pH variation. The resulting potentiometric titration curve of ferric Vcb complexes indicates the stepwise formation of the ferric mono-, bis-, and tris(Vcb) complexes. A value of pFe(Vcb) of 20 (for enterobactin pFe for is 35.6) was obtained for Vcb (2) by modeling the Fe³⁺ uptake under physiological conditions [37].

The biosynthesis and transport of Vcb (2) in strain RV22 is encoded by the vab gene cluster, a c. 26 kb chromosomal region, containing 13 genes. The vabABCEF genes are the main biosynthetic components which encode the enzymes necessary for DHBA biosynthesis and activation, and vabF, a gene encoding an NRPS involved in the assembly of the siderophore components [38]. The biosynthetic pathway proposal was very helpful in elucidating its structure. Indeed, the first proposed structure, 2,3-dihydroxybenzoyl- serinyl -arginine, was discarded for the corrected one, [(2,3-dihydroxybenzoyl)arginyl]serine, where the position of serine and arginine was interchanged because the domain responsible for incorporating the arginine residue is located first [29].

Vanchrobactin transport to the cell is achieved by means of the TonB-dependent outer membrane receptor (OMR) FvtA, a 78 kDa protein, encoded by the fvtA gene linked to the biosynthetic genes. The synthesis of Vcb (2) along with their analogues described before and the preparation of a series of mutant strains were crucial in confirming the actual role of fvtA gene. The results indicated that FvtA seems to recognize primarily the catechol-iron center since it is not only the route of entry for Vcb (2) but also of the other Vb analogues [39].

2.3 Piscibactin Iron-Uptake System

When the complete genome sequence of the V. anguillarum RV22 strain was described [40], the in silico analysis of this genome showed that the second chromosome II contains an additional siderophore gene cluster that encodes Pcb (3), the siderophore responsible for the iron uptake of Photobacterium damselae subsp piscicida which will be discussed later in this chapter. The production of Pcb (3) in V. anguillarum RV22 wild type was demonstrated by chemical analysis using the SPE-HLB/HPLC-MS methodology of the cell-free supernatants of this bacterium, detecting its Ga (III) complex using the same methodology described in P. damselae subsp. piscicida. The analysis showed that both siderophores, Pcb (3) and Vcb (2), are produced simultaneously. In this way, it was demonstrated that Pcb (3) is actually the third siderophore produced by this Vibrio species [41].

2.4 Distribution of Each Siderophore Iron-Uptake System and Their Role in the Virulence

Although the ability of some pathogens to carry multiple iron acquisition systems seems redundant in laboratory culture conditions, the co-expression of siderophores with different chemical properties can play specialized roles at the host-pathogen interface. Indeed, the ability to produce more than one siderophore can enhance niche flexibility and pathogenesis of the bacteria.

The vanchrobactin biosynthesis and transport gene clusters (vabB and fvtA) are ubiquitous in both vanchrobactin- and anguibactin-producing V. anguillarum strains. However, anguibactin strains do not produce Vcb (2) because the vabF gene, which encodes the NRPS that assembles the siderophore molecule, is inactivated by the insertion of the transposable element encoded on the plasmid pJM1. On the other hand, piscibactin genes (irp-HPI) are not only widespread in V. anguillarum strains but also many other species of Vibrio [42]. Moreover, the piscibactin biosynthesis gene cluster is never present in anguibactin-producing strains. Most pathogenic V. anguillarum strains lacking the anguibactin system produce simultaneously both siderophores, Vcb (2) and Pcb (3).

It is assumed that the vanchrobactin mediated system is the ancestral iron-uptake mechanism mediated by siderophores of V. anguillarum. The anguibactin-mediated system could have been acquired later by V. anguillarum, likely by horizontal transfer, during the evolution of this species, leading to the inactivation of the vanchrobactin system, since anguibactin-mediated iron transport seems to be more efficient due to the Ang (1) stronger affinity for iron. The Highly Pathogenically Island encoding piscibactin mediated system (irp-HPI) could also have been acquired by horizontal transfer [16].

On the other hand, V. anguillarum is able to modulate the synthesis of Vcb (2) and Pcb (3) according to the surrounding temperature. Expression of piscibactin biosynthetic genes is three times higher at 18 °C than at 25 °C. Thus, Pcb (3) is preferentially produced at low temperatures, which are the temperature that V. anguillarum finds during infection of fish cultivated cold-water temperatures. In contrast, when V. anguillarum grows at warm-water temperatures (approx. 25 °C), piscibactin genes are down-regulated (fourfold lower) in relation to Vcb (2) [41].

It has been demonstrated that the ability to produce Ang (1) is a prerequisite for the virulence of V. anguillarum serotype O1 plasmid bearing strains and so, Ang (1) is a key virulence factor for those strains. In relation to the efficiency or virulence of Vcb (2) versus Ang (1), it was found that when an anguibactin-producing V. anguillarum strain co-infects a fish with a vanchrobactin producing strain, the former is the only one recovered from diseased fish in the first days of infection [16].

On the other hand, Pcb (3) contributes to a greater extent than Vcb (2) to virulence in fish and also, the ability to produce Pcb (3) seems to be sufficient to confer maximum virulence to V. anguillarum. In fact, piscibactin genes are associated with a highly virulent phenotype for several fish species. Pcb (3) plays a relevant role in the cell fitness and contributes to a greater extent than Vcb (2) to the virulence for fish [41]. Experimental infections in turbot showed that although both siderophores are simultaneously produced, Pcb (3) is a key virulence factor to infect fish, while Vcb (2) seems to have a secondary role in virulence. A recent analysis of 44 V. anguillarum genomes confirmed this hypothesis [15]. Thus, it is likely that Vcb (2) biosynthesis could be more related to persistence in a marine environment than to pathogenesis [43].

3 Photobacteriosis by Photobacterium damselae subsp. piscicida

Photobacteriosis, formerly known as fish pasteurellosis, is a septicemia caused by the Gram-negative pathogenic halophilic bacterium Photobacterium damselae subsp. piscicida (Vibrionaceae). The disease was originally named after the classification of this aforesaid agent as Pasteurella piscicida. It acquired its present name following the reassignment to the genus Photobacterium. The presence of white nodules in the internal viscera, particularly, spleen and kidney in the fish is the reason why it is also known as pseudotuberculosis. Since 1969, photobacteriosis has been one of the most important diseases in Japan, affecting mainly to yellowtail (Seriola quinqueradiata), and from 1990 became the major pathological problem in the culture of gilthead sea bream (Sparus aurata), seabass (Dicentrarchus labrax), and sole (Solea senegalensis and S. solea) in the European Mediterranean countries, including Spain. Additionally, this pathogen is also able to infect a wide variety of marine fish such as striped bass (Morone saxatilis), white perch (M. americana), and hybrid striped bass (a hybrid between the striped bass (M. saxatilis) and the white bass (M. chrysops)) in the USA, cobia (Rachycentron canadum) in Taiwan, and golden pompano (Trachinotus ovatus) in China. Now, photobacteriosis is now considered one of the most dangerous bacterial diseases in aquaculture worldwide due to its wide host range, high mortality rate, and ubiquitous distribution [44].

The structure of the siderophore responsible for the iron uptake in P. damselae subsp. piscicida, named as piscibactin (3), was predicted from the study of genes in the strain DI21 involved in its biosynthesis which are organized in a typical structure of a Pathogenicity Island (PAI) [45]. Database searches revealed that this PAI shows a high degree of homology to the High Pathogenicity Island (HPI) described in Yersinia spp., such as Yersinia pestis, which encodes the synthesis of the siderophore yersiniabactin (44) [46]. More specifically, the structural prediction was deduced from the differences between the domain composition of the multifunctional enzymes HMWP1 and HMWP2 of Y. enterocolitica and the corresponding Irp1 and Irp2 of strain DI21 of P. damselae subsp. piscicida. Thus, the presence of just one methyltransferase domain (MT) in Irp1 of P. damselae subsp. piscicida DI21 strain instead of the two MT in HMWP1 of Y. enterocolitica (Fig. 6) suggested the lack of some methyl groups in the structure of Pcb (3) in relation to that of yersiniabactin (44) (Fig. 7). Y. pestis is the causative agent of the bubonic plague or Black Death that killed one third of the European population in the Middle Age, while Y. enterocolitica causes severe enteric disorders in humans. Moreover, P. damselae subsp. piscicida can use yersiniabactin (44) as xenosiderophore [45].

Fig. 6

Comparison of the domain composition of the multifunctional enzymes HMWP1 and HMWP2 of Y. enterocolitica and the corresponding Irp1 and Irp2 of strain DI21 of P. damselae subsp. piscicida

Fig. 7

Structures of yersiniabactin (44) and prepiscibactin (PrePcb, 45)

Due to its high instability, Pcb (3) was isolated as its Ga³⁺ and Fe³⁺ complexes. Its Ga³⁺ complex made an NMR study possible, while a bioguided fractionation could be performed due to its Fe³⁺ complex. Thus, filtered supernatants of cell-free culture broth of P. damselae subsp. piscicida DI21 were incubated with the corresponding Ga³⁺ and Fe³⁺ salts and submitted to the SPE- HLB/HPLC-HRMS methodology where the use of acidic conditions being avoided at any time. Fractions containing the piscibactin Ga³⁺ complex were easily identified in the MS by the presence of the m/z cluster with the distinctive isotopic ratio of gallium (Mr = 69 and 71, ratio 3:2). The structure of Pcb (3) was established from the 1D/2D NMR and HR-ESIMS data analysis of its piscibactin-Ga(III) complex. Furthermore, a possible intermediate of its biosynthesis, called prepiscibactin (prePcb, 45), was also isolated in the process (Fig. 7) [47].

The stereoselective synthesis of prePcb (45), whose structure is characterized by the presence of four stereogenic centers and three heterocycle rings, was reported. The chiral sources for positions (9R) and (12R) were L- and D-cysteines while the secondary alcohol at position (13S) was generated by a stereoselective SmI₂-mediated Reformatsky reaction. The synthesis of prePcb (45) started with the conversion of L-cysteine to acid 46 which was converted to the thiazolidinic aldehyde 47 through the Weinreb amide and posterior reduction. The crucial SmI₂-induced Reformatsky reaction between the thiazolidinic aldehyde 47 and (R)-chloroacetyl-2-oxazolidinone 48 resulted in the formation of the desired secondary alcohol 49 as a single diastereoisomer with a very high yield. Removal of the chiral auxiliary, the acetonide and Boc protecting groups afforded the thiol amino alcohol ethyl ester 50. In parallel, condensation of D-cysteine and 2-hydroxy benzonitrile gave a thiazolinic carboxylic acid which was transformed to the thiazolidinic aldehyde 51 via the corresponding Weinreb amide followed by reduction. Finally, the coupling between the thiol amino 50 and the thiazolidinic aldehyde 51 in the presence of potassium acetate followed by refluxing in toluene gave prePcb (45) along with its epimer at C10 in a 1:1 ratio. The stereoselectivity of the required isomer prepiscibactin from 1:1 to 4:1 ratio was improved by addition of ZnCl₂ (Scheme 5) [48].

Scheme 5

Synthesis of PrePcb (45)

In the recently reported stereoselective convergent synthesis of Pcb (3), D-cysteine was used as a chiral source for the (9R) and (17S)-positions, L-cysteine was employed to generate the (12R)-position while Meldrum’s acid was utilized in the preparation of the secondary alcohol at the (13S)-position. One of the major challenges of this synthesis was the presence of a high sensitive β-hydroxy-2,4-disubstituted thiazoline moiety in Pcb (3) lacking the bulky gem-dimethyl groups at C-14 present in yersiniabactin (44) [49].

The synthesis started with the protection of the thiol and the primary amine of L-cys with Trt and Fmoc protecting groups, respectively, to give Fmoc-Cys(Trt)-OH (52) which was subsequently coupled with Meldrum’s acid to afford 53. Refluxing 53 in EtOAc followed by stereoselective reduction of the resulting tetramic acid with NaBH₄ gave lactam 54 where the stereogenic centers present in Pcb (3) at C-12 and C-13 positions were efficiently generated. Then, the selective basic cleavage of the lactam 54 in the presence of the Fmoc group to afford statine 55 was performed by using LiOH with added CaCl₂. Posterior protection of the hydroxyl group as triethylsilyl (TES) ether in 55 yielded the conveniently protected carboxylic acid 56. The next step implied the coupling of a freshly prepared solution of the methyl ester of (S)-α-azido-α-methylcysteine (57), obtained from D-cysteine following reported procedures, with statine 56, previously activated with EDC, to furnish thioester 58. The key step for the formation of the sensitive β-hydroxy-2,4-disubstituted thiazoline moiety implied the use of the mild and non-dehydrative conditions from the azide of thioester 58 via Staudinger reduction and the subsequent intramolecular aza-Wittig (S-AW process) with PPh₃ in 2,6-lutidine to give 59. A very mild method for removal of protecting groups in 59 was carried out by deprotection of the Trt group using I₂ in CH₂Cl₂, followed by treatment of the resulting disulfide 60 with NaN₃ in the presence of the scavenger octanethiol and ending with deprotection of the TES ether with TBAF to give the methylester thiazoline 62.

For the completion of the synthesis of 3, thiazolinic aldehyde 51, already described in the synthesis of prePcb (45), was coupled with methylester thiazoline 62, both freshly prepared, to afford an epimeric mixture of methyl esters 63a,b at C-9. Finally, basic hydrolysis of the ester mixture 63a,b with LiOH gave the very unstable acid 3 and its C-9 epimer which were subjected without purification to complexation with gallium Ga(acac)₃. The resulting product was submitted to chromatographic separation using RP-C18 cartridges followed by HPLC purification to deliver Pcb-Ga³⁺-complex 64 which resulted to be identical to the natural product. In this way, the absolute configuration of Pcb (3) could be determined as (9R,10R,12R,13S,17S)-3. Additionally, when the product obtained from the basic hydrolysis with LiOH of methyl esters 63a,b was subjected to complexation with gallium in CD₃OD furnished the deuterated Pcb-d₂ (65), where the two hydrogens at C-14 position were interchanged by two deuterium atoms. Pcb-d₂ (65) could be used as a new molecular tool to carry out further studies on the iron-uptake mechanisms.

Pcb (3) is a siderophore encoded by irp genes in a high-pathogenic island named irp-HPI [45] which is widespread among Vibrionaceae [42]. In P. damselae subsp. piscicida, the irp-HPI is part of a plasmid pPHDP70 [50]. It was suggested that this plasmid could be acquired by horizontal transfer, with an evolutionary origin similar to that of Yersinia HPI. The proposal of an assembly line for the biosynthesis of Pcb (3), showing the presence of just one methyltransferase (MT) domain in the multifunctional enzymes PKS/NRPS Irp1, agrees with the lack of the two methyl groups at C-14 in relation to that of yersiniabactin (44). Moreover, a cryptic metabolite was also predicted, although not detected yet, from the presence of additional module in Irp1 [47].

The plasmid pPHDP70 that encodes Pcb (3) biosynthesis could be transferred by conjugation from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus mutant strain, where it was abolished the production of its native siderophore, vibrioferrin [51]. Acquisition of plasmid pPHDP70 by conjugation restored the capacity of that mutant to grow under low-iron conditions since it could synthesize Pcb (3). Piscibactin production was demonstrated by chemical analysis of its culture supernatants using the same SPE-HLB/HPLC-MS methodology employed in the isolation of gallium (III) and iron (III) piscibactin complexes from P. damselae subsp. piscicida. These experiments confirmed not only that the plasmid pPHDP70 is responsible for the production of Pcb (3) but also showed that the horizontal transfer of that plasmid by conjugation can be very easy and common [50].

Piscibactin production was proven as one of the major virulence factors not only in V. anguillarum but also in Photobacterium damselae subsp. piscicida. The key role of Pcb (3) in the virulence of this bacterium was demonstrated by the fact that P. damselae subsp. piscicida strains cured of pPHDP70 not only were incapable of producing Pcb (3) and growing under iron-limited conditions but also exhibited markedly decreased virulence in fish [50].

The plasmid pPHDP70 encodes a hitherto uncharacterized TonB-dependent transporter named FrpA that would act as the Pcb (3) outer membrane receptor (OMR) [45]. In order to find a new vaccine formulation as alternative to the existence one based on the use of inactivated bacterial cells, FrpA was cloned, expressed in E. coli using an arabinose-inducible promoter, and the resulting rFrpA purified from its cultures. Evaluation of its effectiveness against photobacteriosis and immunogenicity as vaccine on sole (Solea senegalensis) showed that the rFrpA protein is immunogenic, displaying an immunity response that was equivalent to that observed in fish immunized with the corresponding bacterin, obtained from formalin-killed whole cells of P. damselae subsp. piscicida. Furthermore, experimental infection challenge trials showed that fish groups vaccinated with bacterin or rFrpA were protected against photobacteriosis with similar protection levels, reaching survival rates (RPS) of ca. 79% and 73%, respectively [52]. Indeed, rFrpA represents a promising antigen candidate for the development of novel more effective vaccines for the prevention of fish photobacteriosis. Although there are precedents for the use of OMR of siderophores from pathogenic bacteria as vaccines, this is one of the first applications against an infectious disease in fish [53].

4 Furunculosis by Aeromonas salmonicida

Furunculosis is a disease which causes economically devastating losses in cultivated salmonids in fresh and marine waters. It shows a widespread distribution. Although all salmonid species such as Atlantic salmon (Salmo salar), which is considered particularly susceptible, and rainbow trout (Oncorhynchus mykiss) can be affected, it also affects several non-salmonids species including turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus). The disease was first described by Lehmann and Neumann in 1896 and its name comes from the deep ulcerations that it causes on skin. Most Atlantic salmon infected by the disease presents a hemorrhagic septicemia with high mortality rates, in both juvenile and adult fish, within as little as 2 or 3 days [54].

The etiological agent of furunculosis is the non-motile rod Gram-negative γ-proteobacteria Aeromonas salmonicida subsp. salmonicida (A. salmonicida). This bacterium can be defined as biochemically, antigenically, and genetically homogeneous with no biotypes, serotypes, or genotypes. Three subspecies of A. salmonicida, masoucida, achromogenes, and smithia, named atypical strains, are also pathogenic [12].

The first studies of siderophore biosynthesis genes of A. salmonicida displayed the presence of a chromosomal gene cluster (asbGFDCBI) with high similarity to genes related to the synthesis of the siderophore Acb (6) from A. baumannii [55]. The completion of its genome sequence several years later revealed, by bioinformatic genomic analysis, the presence of a second gene cluster with high similarity to those of amonabactins (Fig. 8) from Aeromonas hydrophila. Finally, it was demonstrated that most A. salmonicida subsp. salmonicida strains produce two catechol siderophores simultaneously: Acb (6) and the four amonabactin variants that are denoted as P750, T789, P693, and T732 (66–69) according to the presence of either a Phe or Trp residue and their molecular weights (by the presence or absence of a Gly residue) [56].

Fig. 8

Structure of the amonabactins biosynthetized (66–69) by A. salmonicida subsp. salmonicida

The initial structure assigned for Acb as 5 when it was first described in 1994 from the human pathogen A. baumannii ATCC19606 [57] was corrected in 2009 as 6. It was suggested that 5, named as now preacinetobactin (preAcb, 5), is an unstable oxazoline which suffers an intramolecular nucleophilic substitution to give isoxazolidinone Acb (6) (see Scheme 7) [58]. A complete study of the Acb (6) isomerization at different pH was published in 2015 [59]. On the other hand, the four amonabactins (52–55) were previously isolated and characterized from the pathogenic bacterium of animals and humans A. hydrophila by Raymond and coworkers in 1994 [60].

Scheme 6

Synthesis of Pcb (3) and its Ga⁺³ complex 64

Acb (6) was isolated from the virulent strain A. salmonicida RSP74.1 which does not produce amonabactins due to a natural deletion in the amoG gene. The amonabactins were isolated and/or detected from the mutant strain A. salmonicida VT45.1ΔasbD. This mutant was obtained from another highly virulent strain, A. salmonicida VT45.1, which produces simultaneously both siderophores, where it was abolished the Acb biosynthesis (deletion of the asbD gene) to facilitate the isolation of the amonabactins. The methodology SPE-HLB/HPLC-HRMS [30] was successfully applied in both cases confirming its high efficiency to accelerate the isolation process of siderophores containing catecholate/salicylate moieties [56].

The structure of Acb as 6, including its absolute configuration, was confirmed by chemical synthesis developed by Takeuchi et al. [24]. This synthesis started from 2,3-dihydroxybenzaldehyde (70) which was transformed to the imidate 72 via the nitrile 71. Condensation of L-threonine benzyl ester (73) with imidate 72 gave an oxazoline intermediate which was submitted to hydrogenolysis to afford the oxazoline acid 74. Then, imidazole 9, whose preparation was described in the synthesis of Ang (1) displayed in Scheme 1, was coupled with acid 74 to yield the oxazoline protected 75. Finally, benzoyl deprotection by hydrogenolysis of 75 gave preAcb (5) which under reflux in MeOH afforded Acb (6) (Scheme 7).

Scheme 7

Synthesis of Acb (6)

Although the synthesis of the four natural amonabactins were first reported by Telford and Raymond in 1997 [61], an improved variation of that synthesis was published in 2019 [62]. The more recent synthesis of amonabactins started with the preparation of the activated building blocks 77 and 79. Thus, activation with N-hydroxysuccinimide (NHS) of the isopropyl-protected 2,3-dihydroxybenzoic acid 40 (see Scheme 4) affording 76 and posterior coupling with commercially available N^α-Boc-L-lysine gave, after activation of the carboxylic acid with NHS, the first building block 77. In parallel, activation of the carboxylic acid of commercially available N^α-Cbz-glycine with NHS and posterior coupling with N^α-Boc-L-lysine afforded, after hydrogenolysis of the Cbz group, the protected dipeptide 78. This dipeptide was coupled with the NHS activated isopropyl-protected 2,3-dihydroxybenzoic acid 76 followed by activation with NHS to give the second building block 79. Condensation of D-phenylalanine tert-butyl ester with the first building block 77 gave, after selective removal of the Boc protecting group, the protected dipeptide 80. Coupling of dipeptide 80 with building blocks 79 or 77 yielded amonabactins P750 (66) and P6903 (68) (bearing a Phe residue), respectively, after the simultaneous removal of the isopropyl and tert-butyl protecting groups with BCl₃. In a similar way, the commercially available D-tryptophan methyl ester was coupled with the first building block 77 to give, after Boc deprotection, the dipeptide 81. Dipeptide 81 was coupled with building blocks 79 or 77 to afford amonabactins T789 (67) and T732 (69) (bearing a Tyr residue), respectively, after basic hydrolysis of methyl ester with LiOH and removal of the isopropyl protecting groups with BCl₃ (Scheme 8).

Scheme 8

Synthesis of amonabactins (66–69)

The biosynthesis of both types of siderophores in A. salmonicida depends on entABCE genes, located within the amonabactin gene cluster, which encode the functions necessary for the synthesis of 2,3-dihydroxybenzoic acid (DHBA). Once DHBA is synthesized, asbBCD and amoFGH genes encode acinetobactin and amonabactins synthesis, respectively. Indeed, both siderophores share the first step of their biosynthesis, with the production of DHBA being the amonabactin gene cluster that supplies DHBA for both siderophores. Although both, acinetobactin and amonabactin, siderophore systems are present in all A. salmonicida strains, some isolates carry a mutation in the amoG gene that inactivates amonabactin synthesis. Interestingly, all A. salmonicida strains analyzed up to this point are able to use amonabactin as an iron source. The amonabactin biosynthesis genes are highly conserved in most Aeromonas species including the human pathogen A. hydrophila. On the contrary, Acb (6) is encoded by a gene cluster restricted to A. salmonicida. From the analysis of the genomic context of acinetobactin cluster, it seems clear that this gene cluster was likely acquired from other bacteria through horizontal gene transfer at some point during the speciation process of A. salmonicida while amonabactin must be the ancestral siderophore of the species [56].

Although FstB and FstC were previously identified as two outer membrane proteins specifically induced under iron deficiency and under in vivo conditions, their role in the internalization mechanisms of ferric-acinetobactin and ferric-amonabactin was recently demonstrated. The TonB-dependent outer membrane receptor (OMR) protein FstB was identified and characterized as the receptor protein of Acb (6) in A. salmonicida. Even though this protein was initially named FstA, FstA and FstB are probably two versions or alleles of the same protein [63]. On the other hand, FstC is the OMR of all four amonabactin forms. The FstC receptor is widespread in the genus Aeromonas, not only among A. salmonicida strains but also in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila [62].

Several analogues of Acb (6) and amonabactins (66–69) were prepared based on their simplicity in order to deduce some correlations between the structure and the siderophore activity to facilitate the preparation of conjugates for the development of novel applications of the bacterial iron-uptake mechanisms based on siderophores. In order to evaluate the influence of the stereogenic centers on the siderophore activity of Acb (6), its enantiomer, ent-Acb (82), was synthetized using a similar strategy employed in the synthesis of Acb (6) and Ang (1). Condensation of 2,3-diisopropyloxybenzoic acid (40) with L-threonine benzyl ester (73) gave amide 83 which was submitted to dehydrative-cyclization followed by debenzylation by hydrogenolysis to afford the oxazoline acid 84. An inversion of configuration of the oxymethine carbon at C-9 took place in 84 during the cyclization process. Coupling of N^α-benzyloxyhistamine (9) (Scheme 1) with oxazoline acid 84 using CDI in the absence of base gave the sterically stable trans-isomer 85 rather than the cis-isomer, probably due to epimerization of the activated acyl intermediate. Debenzylation of oxazoline 85 by hydrogenolysis followed by reflux in MeOH gave the expected rearrangement of oxazoline to isoxazolidinone which was treated with BCl₃ to remove the isopropyl groups to yield ent-Acb (82) (Scheme 9).

Scheme 9

Synthesis of ent-Acb (68)

With the aim of comparing the influence of the methyl group on the isoxazolidinone ring and the presence of similar heterocyclic rings, such as triazol instead of imidazole, on the siderophore activity of Acb analogues, four demethylacinetobactin derivatives (compounds 86–89) were prepared. They were synthetized from a common intermediate 90 which was prepared by coupling of 2,3-dibenzyloxybenzoic acid (17) (Scheme 2) and the commercially available D-cycloserine. N-alkylation of intermediate 90 with mesylate 91, prepared by mesylation of the commercial 3-benzyloxycarbonylaminopropanol, followed by subsequent removal of Cbz and Bn protecting groups by hydrogenolysis, gave the first demethylacinetobactin 86. The second demethylacinetobactin 87 was synthetized by N-alkylation of intermediate 90 with the substituted imidazole 7 (see Scheme 1) and posterior debenzylation by hydrogenolysis. Demethylacinetobactin derivatives 88 and 89 were synthetized by using the “click-chemistry” reaction involving the efficient Cu(I)-catalyzed azide-alkyne couplings. Thus, N-alkylation of 90 with the mesylate 92, obtained by mesylation of the commercial homopropargyl alcohol, gave the alkyne 93. Coupling of trimethysilyl azide or azide derivative 94, prepared by displacement of the mesylate group in 91 with sodium azide, with alkyne 93 afforded demethylacinetobactin analogues 88 and 89, respectively, after removal of the protecting groups by hydrogenolysis (Scheme 10).

Scheme 10

Synthesis of demethylacinetobactin derivatives 86–89

Several structure-activity relationships in relation to the OMR protein FstB implied in the internalization of ferric-Acb were deduced from the evaluation of the biological activity of synthetic ent-Acb (82) and four demethylacinetobactin analogues (86–89). All of them showed CAS values indistinguishable from those of Acb (6), indicating that they bind iron efficiently. The ent-Acb (82) displayed the same siderophore activity as the natural one, suggesting the lack of enantiomer preference on the molecular recognition by the protein receptor FstB. The recognition of Acb (6) by its cognate OMR protein FstB mostly resides in the presence of imidazole (87) or a similar heterocyclic ring (88) in its structure because the presence of a primary amino group (86) or a propylamino substituted triazole (89) was devoid of such activity. Moreover, removal of the methyl group at the isoxazolidinone, in combination with the presence of either an imidazole or a triazole ring (87 and 88), leads to higher levels of biological activity. Additionally, it was suggested an alternative route(s) of entry for analogues 87 and 88 since they displayed siderophore activity in the growth promotion assay of a mutant strain which is unable to produce or transport acinetobactin [63].

On the other hand, with the aim of evaluating the minimum structural requirements for amonabactin recognition by its OMR protein FstC, four simplified analogues of the amonabactins were synthetized, compounds 95–99 using a similar synthetic sequence as that described for the natural ones.

Coupling the activated NHS carboxylic acid intermediate 76 with the commercially available N-Boc cadaverine (99) gave amide 100 which was treated with BCl₃ for removal of isopropyl and Boc protecting groups to give monocatecholate amonabactin analogue 95. Removal of the Boc protecting group in 100 with TFA gave an intermediate which was coupled with activated NHS carboxylic acid intermediates 77 or 79 (Scheme 8) to give biscatecholate amonabactin analogues 97 and 98, respectively, after deprotection with BCl₃. Finally, activation of N^α-Cbz-glycine with NHS, posterior coupling with N-Boc cadaverine (99) followed by deprotection of the Cbz group by hydrogenolysis gave amide 101 which was coupled with activated NHS carboxylic acid 76 to yield monocatecholate amonabactin analogue 96 after treatment with BCl₃ (Scheme 11).

Scheme 11

Synthesis of amonabactin analogues 95–98

Important structure-activity correlations were deduced from the evaluation of the biological activity of each of the four natural amonabactins individually 66–69 and of the four synthetic amonabactin analogues 95–99. The lack of siderophore activity for monocatecholate amonabactin analogues 95 and 96 indicated that the presence of at least two catechol moieties is necessary to display biological activity. A quite different growth promotion activity was observed for each natural amonabactin form. The results suggested that natural amonabactins in which the length of the linker between the two iron-binding catecholamide units is 15 atoms, P750 (66) and T789 (67), instead of 12 atoms, P693 (68) and T732 (69), recognize more efficiently the OMR protein FstC. This was confirmed from the results obtained from the siderophore activity evaluation of the synthetic amonabactin analogues 97 and 98. Indeed, the higher biological activity displayed by the bis-catechol amonabactin analogue 98 with longer linker (15 atoms linker length) in relation to that of bis-catechol amonabactin analogue 97 with a shorter linker (12 atoms linker length) is in agreement with the hypothesis that the length of the linker backbone (presence or absence of glycine) between the two iron-binding catecholamide units is relevant to maximize ferric-siderophore acquisition. Even though the presence of Phe or Trp residues seems not be required for siderophore recognition, their presence could also participate in the recognition by the receptor since biscatecholate amonabactin analogues 97 and 98 show less biological activity than their natural counterparts.

In summary, three important conclusions were deduced from these studies. Firstly, the OMR protein FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. Second, the optimum linker length between the two catecholamide units for maximizing the siderophore activity in the amonabactin type-structures is 15 atoms, preferentially a Lys-Lys-Gly fragment which is present in the natural longer amonabactins 66 and 67 and analogue 98. Finally, the synthetic simplified analogues 97 and 98 could be used as vectors for the preparation of conjugates in the Trojan horse strategy to develop new effective antimicrobials against many different pathogens of the genus Aeromonas including the human pathogen A. hydrophila, and for the synthesis of fluorescence probes to study the iron-uptake mechanism in this bacterium [62].

5 Concluding Remarks

The iron-uptake pathway is a key process for microbial survival and virulence. The knowledge of the mechanisms of iron acquisition mediated by siderophores can be of great help for the design of new strategies against microbial infections.

Fish disease outbreaks, vibriosis, photobacteriosis, and furunculosis, are considered as the major threats alarming the aquaculture industry and food security. Chemical and biological studies of the Gram-negative bacteria responsible for these infectious diseases allowed not only the isolation, structural elucidation and synthesis of the siderophores involved in their iron-siderophore uptake mechanisms but also the identification of their corresponding OMR proteins and the gene cluster involved in their biosynthesis (Table 1). All this information is being used in the development of new antimicrobial and vaccines based on the iron-siderophore uptake mechanism of these bacteria.

Table 1 Siderophores from pathogenic bacteria involved in the main aquaculture infectious fish disease

It is worth mentioning the great structural similarity of the siderophores biosynthesized by both human and fish virulent pathogenic bacteria. The structure of yersiniabactin (44), the siderophore produced by Yersinia pestis, responsible for epidemics of high mortality throughout the history of mankind (the plague of Justinian, the Black Death and the third pandemic), is very similar to that of Pcb (3) whose production confers maximum virulence in both V. anguillarum and P. damselae subsp. piscicida. Moreover, pyochelin, one of the siderophores produced by the human multidrug resistant bacteria pathogen Pseudomonas aeruginosa, is also structurally related to Pcb (3). One of the siderophores produced by A. salmonicida, Acb (2), is also biosynthesized by the human pathogen A. baumannii. This bacterium has been identified as one of the group of pathogens with a high rate of resistance to antibiotics and responsible for the majority of nosocomial infections. Moreover, the structure of Acb (2) is closely related to that of Ang (1) produced by some virulent strains of V. anguillarum. The fact that the anguibactin and piscibactin iron-uptake systems in V. anguillarum and the acinetobactin iron-uptake system to A. salmonicida had been probably acquired by horizontal transfer from other pathogenic bacteria seems to confirm the high efficiency of these iron absorption pathways.