PMID:1444296

Citation	Brochu, A , Brochu, N , Nicas, TI , Parr, TR Jr, Minnick, AA Jr, Dolence, EK , McKee, JA , Miller, MJ , Lavoie, MC and Malouin, F (1992) Modes of action and inhibitory activities of new siderophore-beta-lactam conjugates that use specific iron uptake pathways for entry into bacteria. Antimicrob. Agents Chemother. 36:2166-75
Abstract	We describe here the mechanism of inhibition of two new siderophore-beta-lactam conjugates against Escherichia coli X580. One conjugate is a spermidine-based catechol siderophore-carbacephalosporin (JAM-2-263), and the other is an N5-acetyl-N5-hydroxy-L-ornithine tripeptide hydroxamate siderophore-carbacephalosporin (EKD-3-88). In an agar diffusion test, both conjugates produced large inhibitory zones against strain X580. Resistant strains (i.e., JAMR and EKDR) could be isolated after exposure of X580 to the conjugates JAM-2-263 and EKD-3-88, respectively. No cross-resistance was observed in these individual isolates. JAMR and EKDR were studied further to elucidate the mechanism of inhibition of each conjugated drug. The affinities of JAM-2-263 and EKD-3-88 for penicillin-binding proteins (PBPs) of isolated inner membranes were determined by a competition assay with 125I-penicillin V. JAM-2-263 targeted primarily PBPs 1A/B and 5/6, while EKD-3-88 targeted PBPs 1A/B and 3. Strains X580, JAMR, and EKDR showed similar PBP affinities for the conjugates. However, marked changes were observed in the iron-regulated outer membrane proteins of resistant isolates grown on agar plates depleted of iron. EKDR lost the expression of FhuA (78 kDa) and its sensitivity to phages T1 and T5, whereas JAMR lost the expression of Cir (74 kDa) and its sensitivity to colicin Ia. These results revealed the requirement of FhuA and Cir for the inhibitory activities of EKD-3-88 and JAM-2-263, respectively. In an antibiotic diffusion assay, ferrichrome (1 microM) strongly antagonized the activities of both conjugates against X580 and JAMR, including the residual activity of JAM-2-263 against JAMR. However, the susceptibility of strain EKDR lacking the ferrichrome receptor (FhuA-) to the two conjugates remained the same in the presence of ferrichrome. The antagonistic effect of ferrichrome on the activity of JAM-2-263 may also indicate a role for FhuA in the activity of this beta-lactam conjugate. A FhuA- Cir- double mutant confirmed this hypothesis, since it showed a higher level of resistance to JAM-2-263. To reproduce iron-restricted in vivo growth conditions, we grew X580 and EKDR cells in diffusion chambers implanted in the peritoneal cavities of rats. Strain EKDR showed impaired growth in such a cultivation system. This is the first report of beta-lactam drug transport into E. coli cells that involves the FhuA outer membrane protein.
Links	PubMed PMC245471
Keywords	Animals; Anti-Bacterial Agents/metabolism; Anti-Bacterial Agents/pharmacology; Autoradiography; Bacterial Outer Membrane Proteins/drug effects; Bacterial Proteins; Carrier Proteins/metabolism; Catechols/metabolism; Catechols/pharmacology; Cephalosporins/metabolism; Cephalosporins/pharmacology; Diffusion Chambers, Culture; Escherichia coli; Hexosyltransferases; Iron/metabolism; Lactams; Microbial Sensitivity Tests; Muramoylpentapeptide Carboxypeptidase/metabolism; Penicillin-Binding Proteins; Peptidyl Transferases; Rats; Siderophores/metabolism; Siderophores/pharmacology; beta-Lactams
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Materials and Methods Used

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Phenotype Annotations

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Phenotype of	Taxon Information	Genotype Information (if known)	Condition Information	OMP ID	OMP Term Name	ECO ID	ECO Term Name	Notes	Status
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: X580 NCBI Taxon ID: 83333	Genotype of Reference Strain: fhuA Genotype of Experimental Strain : fhuA(del)	Reference Condition:					Resistant to phage T5. See fig 5 & 9
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: X580 NCBI Taxon ID: 83333	Genotype of Reference Strain: fhuA Genotype of Experimental Strain : fhuA(del)	Reference Condition:					Resistant to phage T1. See fig 5 & 9
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: JAMR NCBI Taxon ID: 83333	Genotype of Reference Strain: cirA Genotype of Experimental Strain : cirA(del)	Reference Condition:					Resistance to Colicin Ia. See Table 1, Fig. 5 & 9.
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Notes

Phages

OMP term:Developing Resistance to Phage. T5 and T1 will be identified by taxon ID.

ECOID: Phage was measured by observing bacterial lawn in a Luria agar plates.

Colicins

OMP: Developing resistance to colicins

ECOID: Via Deferred Antagonism Test. From the paragraph it seems that it has something to do with overlay augar.

References

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