Iron-regulated outer membrane proteins of Escherichia coli K-12 and mechanism of action of catechol-substituted cephalosporins

Abstract

Selected aminothiazolyl-oxime cephalosporin congeners substituted at C-3' with a catechol moiety were used to probe the basis of the enhanced antibacterial activity against Escherichia coli K-12 often associated with chemical modifications of this type. Evidence is presented for a tonB-dependent illicit transport of the compounds across the outer membrane of E. coli K-12, the process involving jointly and specifically the Fiu and Cir iron-regulated outer membrane proteins. Thus, both tonB and flu cir mutants showed a comparably reduced susceptibility to the probe compounds, whereas mutants singularly lacking any one of the six iron-regulated outer membrane proteins (Fiu, FepA, FecA, FhuA, FhuE, and Cir) or lacking any combination of any two of these proteins (except Fiu plus Cir) did not show this resistance. Mutants devoid of all six iron-regulated outer membrane proteins were no more resistant to the probe compounds than fiu cir or tonB strains. In addition to the latter genes, the products of the exbB and possibly the exbC loci were necessary for maximal antibacterial potency. A dependence of antibacterial activity on the level of expression of the uptake system components was noted. Comparison of penicillin-binding protein target affinity with antibacterial activity suggested a possible periplasmic accumulation of active compounds by E. coli K-12. Free vicinal hydroxyl groups of the catechol residue were a primary chemical requirement for recognition by the uptake pathway and thus for high antibacterial activity.Recent reports have described the enhanced antibacterial potency against members of the family Enterobacteriaceae and pseudomonads of ,B-lactam antibiotics variously substituted with catechol, derivatized catechol, and related moieties (2, 6, 29-31, 33, 34, 45). Using cephalosporin E-0702 (featuring a catechol derivative at C-7), Watanabe et al. (45) reported the isolation of spontaneous Escherichia coli K-12 mutants specifically resistant to this compound owing to a defect at the tonB locus. From the known functionality of the tonB gene product in the transport of iron-chelated siderophores by E. coli K-12, it was hypothesized that the potency of E-0702 resulted from the incorporation of the compound by a tonB-dependent siderophore transport route (45). Unpublished studies in this laboratory have shown that such (tonB) mutants of E. coli K-12 can be isolated irrespective of the chemical diversity of the catechol-substituted cephalosporin (catechol-cephalosporin) used. These mutants, like tonB mutants independently isolated as multiply colicintolerant variants of sensitive strains (3,24,25), are specifically cross resistant to catechol-cephalosporins as a chemical class (unpublished observations and this report).In view of the pivotal importance of the tonB gene product in determining the sensitivity of E. coli K-12 to these agents, we attempted to identify whether any of the known tonBdependent uptake processes of E. coli K-12 could be implicated in catechol-cephalosporin activity.The tonB gene pro...

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

“…Enterobactin is a tricatechol siderophore produced by several enterobacterial species. However, the FepA receptor does not seem to play a role in the transport of catechol-based siderophore-drug conjugates in E. coli (12,13), but it can transport beta-lactams linked to enterobactin (43,44).…”

“…This suggests that the two TBDRs are likely involved in the uptake of xenosiderophores, although the substrate(s) of PiuA has not been identified. The closest orthologue of PiuA in E. coli is Fiu (35% amino acid identity), a TBDR also involved in the uptake of cephalosporins conjugated to catechol siderophores (12,13).…”

“…The iron-binding moiety of beta-lactam siderophore conjugates is frequently a small catechol-type siderophore, such as dihydroxypyridone or a mixed catechol-hydroxamate (7). Such conjugates have been developed with aminopenicillins (8), cephems (KP-736 and S-649266) (9)(10)(11), and monocyclic beta-lactams (pirazmonam, BAL30072, and MC-1) (12)(13)(14)(15). In particular, the monobactam conjugates show potent activity against the Gram-negative nonfermenters P. aeruginosa and A. baumannii (15)(16)(17)(18).…”

The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN

“…The natural siderophore substrates of Fiu are catecholate siderophores (Hantke, 1990;Curtis et al, 1988). The receptor seems to accept very diverse substrates since catechol-cephalosporins are also accumulated by Fiu in a TonB-dependent manner (Curtis et al, 1988). However, fiu mutants were only partially resistant to colicins G and H. In E. coli, there is a second TonB-dependent receptor, Cir, which has nearly the same substrate specificity as Fiu (Hantke, 1990).…”

“…The Fiu protein belongs to the large family of TonB-dependent outer-membrane receptors that transport siderophores across the outer membrane of Gramnegative bacteria (Braun et al, 1998). The natural siderophore substrates of Fiu are catecholate siderophores (Hantke, 1990;Curtis et al, 1988). The receptor seems to accept very diverse substrates since catechol-cephalosporins are also accumulated by Fiu in a TonB-dependent manner (Curtis et al, 1988).…”