Unraveling the mechanisms behind the enhanced efficacy of β-lactam-based sideromycins

Abstract

Previous studies have explored combining β-lactams with siderophores to create Trojan horse molecules that can penetrate the outer membrane of Gram-negative bacteria via TonB-dependent transporter (TBDT). While the main advantage explaining their enhanced antibiotic activity is believed to be improved membrane permeability, other factors remain underexplored. This study evaluates three siderophore-β-lactam compounds: a bis-catechol siderophore linked to ampicillin or loracarbef, and a mixed bis-catechol-mono-hydroxamate siderophore linked to cefaclor. Minimal inhibitory concentrations showed that siderophore conjugation could enhance β-lactam efficacy by over 8000-fold. Comparison with unconjugated β-lactams revealed a complex interplay between β-lactamase susceptibility, competition with endogenous siderophore, membrane uptake, and binding to penicillin-binding proteins (PBPs). Enhanced PBP binding, particularly in Escherichia coli, emerged as a key factor contributing to improved bacterial inhibition by siderophore-β-lactam conjugates. Overall, the study provides insights into how siderophore conjugation enhances β-lactam activity and the therapeutic potential of the conjugates as narrow or broad-spectrum antibiotics.

Fig. 1. Structure of conjugated β-lactams and molecules cited in this study.

Represented in orange are the siderophores and in purple the antibiotic components; a the natural antibiotic albomycin δ1 conjugates a trihydroxamate (ferrichrome-like) to a seryl tRNA inhibitor, b the synthetic antibiotic cefiderocol combines a mono-chloro-catechol to a ceftazidime/cefepime β-lactam moiety, c the siderophore fimsbactin A is an example of natural bis-catechol, d the bis-catechol (azotochelin)-ampicillin (BAMP), e the bis-catechol (azotochelin)-loracarbef (BLOR), and f the mixed ligand bis-catechol-mono-hydroxamate-cefaclor (MCEF), are also shown.

Fig. 2. Kinetic parameters of purified β-lactamases.

a Schematic representation of the purification steps for each β-lactamase. SHV-1 was purified by cloning the respective gene of K. pneumoniae ATCC 13883 into an expression vector and were purified using a Ni-NTA column. AmpC (PDC-5) from P. aeruginosa ATCC 27853 was partly purified from an extract in its native form using an affinity column containing aminophenylboronic acid. b Individual data points of the Michaelis–Menten curves for SHV-1 (n = 3) and AmpC (n = 2) against BLOR and LOR. Each curve were done with at least seven points. c Mean and standard deviation of the kinetic parameters of SHV-1 and AmpC. The deviation for AmpC represents the gap between the two kinetic parameters measured. The asterisk, * indicates that the reported parameters were measured indirectly as a Ki against nitrocefin (NCF); ND not determined.

Fig. 3. Antibiotic uptake assay with E. coli MC4100.

a, b Curves represent the net concentration in culture supernatants of a loracarbef (LOR) () or bis-catechol-loracarbef (BLOR) () and of b cefaclor (CEF) () or mixed bis-catechol-mono-hydroxamate-cefaclor (MCEF) (), in MHBCA (Fe+) and iron-deprived (ID)-MHBCA (Fe-), over a 24-h period. Net concentrations represent the levels of each antibiotic measured in the presence of E. coli, corrected by subtracting the corresponding concentrations measured in the absence of bacteria at each time point. Normality was verified with the Shapiro–Wilk’s test and the statistical analysis is a comparison of repeated measures ANOVA between each time point relatively to the 0-h time point (n = 3). BLOR showed a statistically significant uptake at 24 h (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001), but MCEF uptake was not significant (ns). The Δlog10 CFU/mL (bars with individual data points) was calculated by subtracting the bacterial count with drugs to the bacterial count without drugs (normal growth) at each time point. Panel c is the Δlog10 CFU/mL of LOR (blue) and BLOR (orange), and d is the Δlog10 CFU/mL of CEF (blue) and MCEF (orange).

Fig. 4. PBP binding of conjugated (SID-βL) compared to unconjugated (βL).

a Schematic protocol for measurement of apparent IC₅₀ values using bacterial membranes as the source of PBPs. b Apparent IC₅₀ value ratios (calculated from IC₅₀ values provided in Table S8) for PBPs 1 to 4 of the different bacterial species tested. The ratios (βL/SID-βL) are represented in a heat map with the intensity ranging from red (SID-βL loses affinity compared to βL) to green (SID-βL gained affinity compared to βL). NA not applicable, ND not determined. Gel images c, d show the fluorescent PBP profiles on SDS-PAGE obtained from E. coli MC4100 and P. aeruginosa ATCC 27853 membrane preparations, respectively. The arrow points to the apparent PBP3 IC₅₀ value measured with the Quantity One software (4.6.6). When two PBPs overlapped on gels, the IC₅₀ were measured together as one entity (e.g., E. coli or K. pneumoniae PBPs 1a and 1b). The concentrations of test drugs in each lane are given in μM and the CTL lane represents a membrane aliquot without test drug or Bocillin FL to highlight fluorescent proteins that are not PBPs (ns).

Fig. 5. β-lactams and conjugated β-lactams IC₅₀ and ratios values for purified P. aeruginosa PBP3 (PaPPB3).

a Schematic representation of the cloning and expression of PaPBP3 gene in E. coli. Purification was done using a Ni-NTA column. The purified PBP3 from Pseudomonas aeruginosa ATCC 27853 (PaPBP3) was used in the PBP assay with the test antibiotics and Bocillin FL. The IC₅₀ is the concentration of the test antibiotic needed to prevent the binding of 50% of Bocillin FL to PaPBP3. b Means and standard deviation of IC₅₀ values (uM) (n = 3) and IC₅₀ ratios for βL/SID-βL for P. aeruginosa PBP3 (either purified or in the membrane preparation). The ratios (βL/SID-βL) are represented in a heat map with the intensity ranging from red (SID-βL loses affinity compared to βL) to green (SID-βL gained affinity compared to βL). c Examples of assays with PaPBP3 on SDS-PAGE comparing a set of AMP concentrations to BAMP and of LOR concentrations to BLOR.