In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1–A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.
The biochemical properties of CMY-32, a class C enzyme possessing a single amino acid substitution in the Ω loop (Gly214Glu) were compared to the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (kcat/Km) by 50-70% against “good” substrates (i.e., cephalothin) while increasing kcat/Km against “poor” substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C6 hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of -SO3-2. Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining increased cefotaxime hydrolysis. When docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight: i) the impact of single amino acid substitutions on protein evolution in clinically important AmpC enzymes; and ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 β-lactamases.
BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072.
Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.
New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC β-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need.
We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time–kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057).
In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2–8-fold. In time–kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2–8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo.
These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.
MICs; time–kill assays; drug susceptibility testing; multidrug resistant
BAL30376 is a triple combination comprising a siderophore monobactam, BAL19764; a novel bridged monobactam, BAL29880, which specifically inhibits class C β-lactamases; and clavulanic acid, which inhibits many class A and some class D β-lactamases. The MIC90 was ≤4 μg/ml (expressed as the concentration of BAL19764) for most species of the Enterobacteriaceae family, including strains that produced metallo-β-lactamases and were resistant to all of the other β-lactams tested. The MIC90 for Stenotrophomonas maltophilia was 2 μg/ml, for multidrug-resistant (MDR) Pseudomonas aeruginosa it was 8 μg/ml, and for MDR Acinetobacter and Burkholderia spp. it was 16 μg/ml. The presence of the class C β-lactamase inhibitor BAL29880 contributed significantly to the activity of BAL30376 against strains of Citrobacter freundii, Enterobacter species, Serratia marcescens, and P. aeruginosa. The presence of clavulanic acid contributed significantly to the activity against many strains of Escherichia coli and Klebsiella pneumoniae that produced class A extended-spectrum β-lactamases. The activity of BAL30376 against strains with metallo-β-lactamases was largely attributable to the intrinsic stability of the monobactam BAL19764 toward these enzymes. Considering its three components, BAL30376 was unexpectedly refractory toward the development of stable resistance.
Burkholderia pseudomallei is an intrinsically antibiotic-resistant Category B priority pathogen and the aetiological agent of melioidosis. Treatment of B. pseudomallei infection is biphasic and lengthy in order to combat the acute and chronic phases of the disease. Acute-phase treatment preferably involves an intravenous cephalosporin (ceftazidime) or a carbapenem (imipenem or meropenem). In this study, the anti-B. pseudomallei efficacy of a new monosulfactam, BAL30072, was tested against laboratory strains 1026b and 1710b and several isogenic mutant derivatives as well as a collection of clinical and environmental B. pseudomallei strains from Thailand. More than 93% of the isolates had minimal inhibitory concentrations (MICs) in the range 0.004–0.016 μg/mL. For the laboratory strain 1026b, the MIC of BAL30072 was 0.008 μg/mL, comparable with the MICs of 1.5 μg/mL for ceftazidime, 0.5 μg/mL for imipenem and 1 μg/mL for meropenem. Time–kill curves revealed that BAL30072 was rapidly bactericidal, killing >99% of bacteria in 2 h. BAL30072 activity was not significantly affected by efflux, it was only a marginal substrate of PenA β-lactamase, and activity was independent of malleobactin production and transport and the ability to transport pyochelin. In summary, BAL30072 has superior in vitro activity against B. pseudomallei compared with ceftazidime, meropenem or imipenem and it is rapidly bactericidal.
Burkholderia pseudomallei; Melioidosis; Therapy; Monosulfactam; Efflux; Siderophore
BAL30072 is a new monocyclic β-lactam antibiotic belonging to the sulfactams. Its spectrum of activity against significant Gram-negative pathogens with β-lactam-resistant phenotypes was evaluated and was compared with the activities of reference drugs, including aztreonam, ceftazidime, cefepime, meropenem, imipenem, and piperacillin-tazobactam. BAL30072 showed potent activity against multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter sp. isolates, including many carbapenem-resistant strains. The MIC90s were 4 μg/ml for MDR Acinetobacter spp. and 8 μg/ml for MDR P. aeruginosa, whereas the MIC90 of meropenem for the same sets of isolates was >32 μg/ml. BAL30072 was bactericidal against both Acinetobacter spp. and P. aeruginosa, even against strains that produced metallo-β-lactamases that conferred resistance to all other β-lactams tested, including aztreonam. It was also active against many species of MDR isolates of the Enterobacteriaceae family, including isolates that had a class A carbapenemase or a metallo-β-lactamase. Unlike other monocyclic β-lactams, BAL30072 was found to trigger the spheroplasting and lysis of Escherichia coli rather than the formation of extensive filaments. The basis for this unusual property is its inhibition of the bifunctional penicillin-binding proteins PBP 1a and PBP 1b, in addition to its high affinity for PBP 3, which is the target of monobactams, such as aztreonam.
BAL19403 is a macrolide antibiotic from a novel structural class with potent activity against propionibacteria in vitro. The antibacterial spectrum of BAL19403 covers clinical isolates with mutations in the 2057 to 2059 region of 23S rRNA that confer resistance to erythromycin and clindamycin. The basis of this improved activity was investigated by ribosome binding assays and by a coupled transcription and translation assay. The latter was specifically developed for the use of ribosomes from Propionibacterium acnes. BAL19403 inhibited protein expression by ribosomes from erythromycin-sensitive and erythromycin-resistant P. acnes with similar potencies if the resistance was due to G2057A or A2058G mutations. BAL19403 showed a >10-fold higher activity than erythromycin against ribosomes from a strain with the erm(X) gene. Erm(X) confers high levels of macrolide and lincosamide resistance by dimethylation of A2058. Assays with such ribosomes showed that BAL19403 was potent enough to inhibit half of the total activity with a 50% inhibitory concentration very close to the value measured with erythromycin-sensitive ribosomes. We concluded from our data that the P. acnes strain with the erm(X) gene had a mixed population of ribosomes, with macrolide-sensitive and macrolide-resistant species.
The interactions of ceftobiprole with purified β-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent β-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 β-lactamase, the class A TEM-1 β-lactamase, and the class C AmpC β-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum β-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than β-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the family Enterobacteriaceae.
Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.
The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains.
Streptococcus pneumoniae M22 is a multidrug-resistant mutant selected after exposure of capsulated wild-type S. pneumoniae NCTC 7465 (strain M4) to ciprofloxacin. DNA microarray analysis comparing the gene expression profiles of strain M22 with those of strain M4 showed that strain M22 constitutively expressed 22 genes at levels higher than those observed in strain M4 under all conditions studied. These included the genes encoding the enzymes involved in branched-chain amino acid biosynthesis and two genes (patA and patB) with sequences suggestive of ABC transporter proteins. Expression of the patA and patB genes was induced by ciprofloxacin in both strains, but in strain M4 it only reached the levels observed in strain M22 after long incubation with high concentrations of ciprofloxacin. The altered expression profile observed with strain M22 suggested that the mutation or mutations acquired during resistance selection bring the cell into a state in which the expression of critical genes is preemptively altered to correct for the potential effects of ciprofloxacin on gene expression in the parent strain.
Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. β-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel β-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different β-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Δ1-36 rPBP5). The soluble protein, Δ1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH2-terminal protein sequence was verified by amino acid sequencing. Using β-lactams with different R1 side chains, we show that azlocillin has greater affinity for Δ1-36 rPBP5 than piperacillin and ampicillin (apparent Ki = 7 ± 0.3 μM, compared to 36 ± 3 and 51 ± 10 μM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k2 = 15 ± 4 M−1 s−1). Meropenem demonstrates an affinity for Δ1-36 rPBP5 comparable to that of ampicillin (apparent Ki = 51 ± 15 μM) but is slower at acylating (apparent k2 = 0.14 ± 0.02 M−1 s−1). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a β-lactam, and suggests that the specific side chain interactions of β-lactams could be modified to improve inactivation of resistant PBPs.
We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all β-lactams. Affinity for penicillin generally correlated with β-lactam MICs for the mutants, but these associations were not strictly proportional.
Polyclonal rabbit antibodies against SHV-1 and CMY-2 β-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC β-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC β-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E. coli possessing TEM-1, SHV-1, K-1, or OXA-1 β-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC β-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC β-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 β-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV β-lactamases, and the results were confirmed with PCR amplification of blaAmpC and blaSHV genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC β-lactamases. The ELISA format can facilitate the identification of AmpC and SHV β-lactamases and can be used to quantify relative amounts of β-lactamase enzymes in clinical and laboratory isolates.
New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10−7). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.
An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of Enterococcus faecium. Its MIC at which 90% of the isolates tested were inhibited (MIC90) for methicillin-resistant Staphylococcus aureus (MRSA) was 4 μg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant Streptococcus pneumoniae (MIC90 = 2 μg/ml). It was active against ceftazidime-susceptible strains of Pseudomonas aeruginosa and against Enterobacteriaceae except Proteus vulgaris and some isolates producing extended-spectrum β-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2′ of MRSA, and its stability towards β-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.