Sideromycins: tools and antibiotics

Abstract

Sideromycins are antibiotics covalently linked to siderophores. They are actively transported into gram-positive and gram-negative bacteria. Energy-coupled transport across the outer membrane and the cytoplasmic membrane strongly increases their antibiotic efficiency; their minimal inhibitory concentration is at least 100-fold lower than that of antibiotics that enter cells by diffusion. This is particularly relevant for gram-negative bacteria because the outer membrane, which usually forms a permeability barrier, in this case actively contributes to the uptake of sideromycins. Sideromycin-resistant mutants can be used to identify siderophore transport systems since the mutations are usually in transport genes. Two sideromycins, albomycin and salmycin, are discussed here. Albomycin, a derivative of ferrichrome with a bound thioribosyl-pyrimidine moiety, inhibts seryl-t-RNA synthetase. Salmycin, a ferrioxamine derivative with a bound aminodisaccharide, presumably inhibts protein synthesis. Crystal structures of albomycin bound to the outer membrane transporter FhuA and the periplasmic binding protein FhuD have been determined. Albomycin and salmycin have been used to characterize the transport systems of Escherichia coli and Streptococcus pneumoniae and of Staphylococcus aureus, respectively. The in vivo efficacy of albomycin and salmycin has been examined in a mouse model using Yersinia enterocolitica, S. pneumoniae, and S. aureus infections. Albomycin is effective in clearing infections, whereas salmycin is too unstable to lead to a large reduction in bacterial numbers. The recovery rate of albomycin-resistant mutants is lower than that of the wild-type, which suggests a reduced fitness of the mutants. Albomycin could be a useful antibiotic provided sufficient quantities can be isolated from streptomycetes or synthesized chemically.

Fig. 1

Albomycin and ferrichrome transport system of E. coli. Both compounds are transported by the same proteins: FhuA in the outer membrane, FhuD in the periplasm, and FhuB in the cytoplasmic membrane. FhuC is the ATPase that provides the energy for transport across the cytoplasmic membrane. The proteins TonB, ExbB, and ExbD form a complex that provides energy for transport across the outer membrane. The transmembrane topology of the cytoplasmic membrane proteins but not the stoichiometry of TonB, ExbB, and ExbD is shown. The complete crystal structures of FhuA (red) and FhuD (green) loaded with albomycin and the crystal structures of periplasmic portions of TonB and ExbD are shown. The 22 anti-parallel β-strands of FhuA form a β-barrel (blue-green) that is closed by a globular domain (yellow). The TonB box of FhuA forms with TonB a four-stranded β-sheet. The structure of the linkage between the membrane segment and the crystallized domain of TonB is unknown

Fig. 2

Chemical structures of ferrichrome, albomycin, ferrioxamine B, and salmycin. The co-crystals of ferrichrome and albomycin with FhuA reveal their structures in the protein-bound form (Fig. 1)

Fig. 3

Arrangement of the fhu genes on the genome of S. pneumoniae and S. aureus. The number of nucleotides (nt, nucleotides; mnt, million nucleotides) between the genes is indicated; a negativenumber indicates that the genes overlap

Fig. 4

Competitiveness of the albomycin-resistant S. pneumoniae mutant D39T1 with the wild-type parent strain D39 in mice. Mice (n = 12) were infected with a mixture of 1,000 CFU of strain D39 and 14 CFU of strain D39T1. After 12 h post-infection, six mice received placebo (PBS) and six mice received albomycin (5 mg/kg body weight, thrice a day). In 8 h intervals, blood samples were collected from the tail vein of each mouse and the bacterial cells were counted. All PBS-treated mice died within 26 h post-infection. Open squares, CI_mutant from PBS-treated mice; filled triangles, CI_mutant of albomycin-treated mice. CI_mutant = percentage of mutant CFU in the total CFU/percentage of mutant CFU in the inoculum CFU. A CI_mutant of 1 indicates that the mutant proliferates as well as the wild-type, and a CFU_mutant <1 indicates that the mutant multiplies less than the wild-type

Fig. 5

Salmycin treatment of infected mice. Mice were infected with S. aureus on day 0, and antibiotics were administered 6 and 24 h post-infection. The bacterial numbers (CFU) were determined in the kidney 72 h post-infection. For comparison, infected mice were treated with a mixture of rifamycin and vancomycin. n, number of mice. Dotted line indicates detection limit