Broadening the spectrum of β-lactam antibiotics through inhibition of signal peptidase type I

Abstract

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Fig 1

β-Lactam synergists. Structure of krisynomycin (A), actinocarbasin (B), and M131 (C), a synthetic derivative of actinocarbasin.

Fig 2

Two-dimensional cluster analysis of S. aureus fitness test profiles. Actinocarbasin, krisynomycin, and M131(R,S-lysine) were compared to three compounds of known mechanism of action, amoxicillin, the translation inhibitor GE2270, and roxithromycin. Antisense strains that are significantly growth inhibited by the compound treatment are shown in magenta, and strain resistances are shown in cyan. The thresholds for including strains in the cluster analysis are a >5-fold reduction in strain abundance and a P value of ≤0.01 in three or more experiments. The profiles of strains that are reduced by actinocarbasin, krisynomycin, or M131(R,S-lysine) treatment cluster tightly together and are characterized by significant depletions of the spsB antisense strain at all compound concentrations tested.

Fig 3

Combinations of imipenem with the SpsB inhibitor M131. Combinations were tested using the checkerboard technique against MRSA strain COL and MSSA strain ATCC 29213. Fractional inhibitory concentrations (FIC) were determined for each combination by dividing the MIC in the presence of the other compound by MIC in its absence. Synergy is achieved when the sum of the FIC values for each agent (FIC index or FICI) is ≤0.5. Data points below the dashed diagonal line have an FICI of ≤0.5, demonstrating synergy.

Fig 4

Combinations of M131 with antibiotics of different classes and mechanisms. Combinations with the following were tested using the checkerboard technique with MRSA strain COL: carbapenems (A), cephalosporins (B), penicillins (C), cell wall inhibitors that are not β-lactams (D), and antibiotics that act by other mechanisms (i.e., inhibition of DNA replication, protein synthesis, RNA synthesis, fatty acid synthesis, folate metabolism, and membrane function) (E).

Fig 5

Killing curve analysis of imipenem-M131 combinations. Cultures of MRSA COL were treated with M131 at 1 μg/ml (1× MIC) with or without imipenem (4 μg/ml; 0.25× MIC), and viable bacterial counts (CFU/ml) were monitored over 24 h. M131 was also tested alone at 8 μg/ml (8× MIC).

Fig 6

In vivo activity of M131 combinations with imipenem on MRSA infection. (A) MRSA COL deep-thigh infection model. (B) MRSA COL systemic infection model. Both assays are performed with neutropenic female BALB/c mice. A single intraperitoneal dose of test agents were applied 2 h postinfection for the thigh model and 30 min postinfection for the bacteremia model. Linezolid (Lin) was employed as a positive control and dosed orally (20 mg/kg) at 2 h and 8 h postinoculation. Results are plotted as means of bacterial counts in infected tissue (n = 5); the error bars represent standard deviations. The dashed lines indicate the bacterial counts recovered from vehicle-treated MRSA-infected mice. Statistical significance was determined by one-way ANOVA with Bonferroni posttest: ϕ, P values of <0.05 for comparisons with uninfected mice; *, P values of <0.05 for comparisons with vehicle-treated mice.