Diffusion of β-Lactam Antibiotics Through Oligomeric or Monomeric Porin Channels of Some Gram-Negative Bacteria

The penetration of anionic β-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12. Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce β-lactamase. These two bacteria were sensitive to the different β-lactams tested. The other four bacteria were resistant to all or to several β-lactams. A modified liposome swelling method was used for determining the rate of penetration of charged β-lactam antibiotics. Zwitterionic β-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged β-lactams are different for oligomeric and monomeric porins. Negatively charged β-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged β-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to β-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to β-lactams, and only the existence of a potential β-lactamase that can destroy the penetrating drug will cause resistance. Received: 28 January 2002 / Accepted: 4 May 2002     

In vitro assembly of the functional porin trimer from dissociated monomers in Pseudomonas aeruginosa

The molecular weights of monomeric and oligomeric forms of the newly identified porins, protein D2, of the outer membrane of Pseudomonas aeruginosa appeared to be 47,000 and 137,000, respectively, as determined by the light scattering technique. Presence of the trimeric aggregates of the homologous subunits in the intact outer membrane, the liposome membrane, and the non-ionic surfactant were confirmed through cross-linking experiments and immunoblotting techniques. The protein D2 monomers prepared in 0.1% of sodium dodecyl sulfate at 23 degrees C spontaneously reassembled into the trimeric aggregate when the surfactant dropped below critical concentration. The diffusion rates of saccharides and beta-lactam antibiotics through the liposome membranes reconstituted from the reassembled protein D2 trimers were indistinguishable from those of the native protein D2. This study shed some light on the porin trimer assembly as well as on the mechanism of carbapenem diffusion through the protein D2 pores.     

Bacterial porin and antibiotic susceptibility

In Gram negative bacteria, hydrophilic antibiotics such as beta-lactams and fluoroquinolons used the bacterial porin channel during their entry. The balance of the porin expression level and the molecular parameters which govern the molecule diffusion through the pore are important physiological points. Acquired in vivo beta-lactam resistance is often associated with porin loss, and recently clinical resistant strains synthetizing mutated porin have been described. These data highlight both the importance of the channel characteristics and the amino acid residues involved in the drug diffusion process. In addition, several mechanisms, including various repressors or activators as well as molecules inhibiting the pore synthesis or activity, argue for the complexity and plasticity of the bacterial control of porin function. All these aspects play a key role in both membrane permeability and efficiency of the antibiotic resistance process.     

Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.     

Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function.

FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting of 16 transmembrane segments with eight surface-exposed loops. Several conserved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of beta-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic beta-lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.     

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.     

Molecular and Structural Characterization of the HMP-AB Gene Encoding a Pore-Forming Protein from a Clinical Isolate of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.     

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.     

Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.     

Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response

The intensive antibiotic treatment of cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa has improved the survival rate and the clinical condition of Danish patients. Acquirement of resistance to anti-pseudomonal antibiotics is one of the main drawbacks of this therapeutic strategy and our results showed the development of resistance of P. aeruginosa to several antibiotics during 25 years of intensive antibiotic treatment. Our studies have been concentrating on the development of resistance to beta-lactam antibiotics. We have shown an association between the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression. Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P. aeruginosa CF isolates. We have also shown that the high alginate producing P. aeruginosa isolates, that characterize the chronic lung infection in CF patients, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates. We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce high levels of beta-lactamase. The beta-lactamase produced by the non-mucoid isolates might play a protective role in the biofilm, defending the mucoid isolates from the action of beta-lactam antibiotics and helping them to maintain their antibiotic susceptibility. We have also shown that beta-lactamase, which is a periplasmic enzyme, can be secreted extracellulary packed in membrane vesicles liberated by high beta-lactamase-producing P. aeruginosa. The continuos presence in the CF lungs of bacteria producing high basal levels of beta-lactamase (partial derepressed) induces a humoral immune response to beta-lactamase. We have shown that antibodies against the chromosomally encoded beta-lactamase (a beta ab) might be considered a marker of the development of resistance to beta-lactam antibiotics. We investigated the humoral immune response to beta-lactamase by quantifying a beta ab specific IgG and IgG subclass antibodies, by investigating the influence of the allotypes on the IgG subclass response and by measuring the avidity of the IgG a beta ab. We found that CF patients with good lung function had in the early stages of the chronic lung infection higher titers of a beta ab of good avidity than patients with poor lung function. Therefore, we raised the hypothesis that some of the a beta ab might have beta-lactamase neutralizing effect, playing a beta-lactamase inhibitor role and improving the effect of the treatment with beta-lactam antibiotics. Finally, we tested our hypothesis in the rat model of chronic lung infection by assessing the effect of a beta ab raised by vaccination with purified chromosomal beta-lactamase on the outcome of the treatment with ceftazidime of bacteria resistant to beta-lactam antibiotics. Our results showed that significantly lower bacterial load and better lung pathology were found in rats with neutralizing antibodies compared to non-immunized rats or rats without neutralizing antibodies. Our findings might be of potential importance for the improvement of the treatment with beta-lactam antibiotics of resistant P. aeruginosa hyperproducing chromosomal beta-lactamase that represent a threat especially for patients with CF and chronic lung infection.     

Molecular Cloning, Sequencing, and Expression of omp-40, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.     

Isolation and characterization of heat-modifiable proteins from the outer membrane of Porphyromonas asaccharolytica and Acinetobacter baumannii

Active porins were isolated and purified from the outer membranes of the gram-negative anaerobic rod Porphyromonas asaccharolytica and the aerobic coccobacillus Acinetobacter baumannii. The porins from both bacteria appear to be monomers when isolated and purified. Both porins exhibited decreased mobility on SDS-PAGE after boiling for 10 min in the sample buffer. After heating, their molecular weight is estimated at 43 kDa while without heating they run as proteins with a molecular weight of approximately 37 kDa. Due to their characteristic heat-modifiability, these proteins were named HMP (heat-modifiable protein)-P. asaccharolytica and HMP-A. baumannii. Amino acid analysis revealed both porins to be hydrophilic proteins. These proteins have been shown to be active in transporting sugars when incorporated into liposomes. The permeability of both porins for L-arabinose was less than that produced by the porin of Escherichia coli B. Permeability to high molecular weight disaccharides was lower than for small monosaccharides. Western blot analysis did not reveal any antigenic cross reaction between HMP-A. baumannii and the HMP-P. asaccharolytica. The results obtained in this study confirm that although these heat-modifiable proteins are pore forming proteins and have similar activity they differ in their antigenicity.     

Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.     

A common channel-forming motif in evolutionarily distant porins.

Four new crystal packings of Escherichia coli porins are presented (phosphoporin, maltoporin, and two crystal forms of matrix porin). These were determined by molecular replacement methods using a polyalanine trial model acquired from the refined coordinates of porin from Rhodobacter capsulatus. The successful molecular replacement shows that the dominant motif found in R. capsulatus porin (a 16-stranded antiparallel beta-barrel) also applies to the E. coli porins, despite the lack of significant amino acid sequence homology. A 30 degrees-40 degrees tilt of the beta-strands with respect to the membrane normal was derived from the intensity distributions in the X-ray diffraction patterns for each porin studied, stressing their similarity. In view of the evolutionary distance between enteric and photosynthetic bacteria, the antiparallel beta-barrel may have significance as a basic structural motif for the formation of bacterial membrane channel structures.     

Indicator method of determining the beta-lactamase-inhibiting activity of various compounds

An indicator for determination of beta-lactamase inhibitory activity of various compounds was developed. The method is based on the direct contact of beta-lactamase with the compounds tested. It excludes the use of test-bacteria and provides recording in the data on the day of the experiment. The indicator method enables the detection of the beta-lactamase inhibitory properties of both beta-lactamase inhibitors and beta-lactam antibiotics, not subjected to destruction by beta-lactamases. The method is likely to be fit for detection of atypical beta-lactams having beta-lactam groups in their molecules (bleomycin group). Antibiotics not belonging to the group of beta-lactams, such as gentamicin, sisomicin, lincomycin and fusidin showed no beta-lactamase inhibitory activity under the conditions of the indicator method. The use of the indicator method provided determination of the inhibitory activity with respect to penicillinase of Bac. licheniformis 749/C in 30 (8.5 per cent) out of 350 fermentation broths of actinomycetes.     

State of penicillin-binding proteins and requirements for their bactericidal interaction with beta-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum beta-lactams

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum beta-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum beta-lactam antibiotics and with constitutively overproduced chromosomal beta-lactamase in the periplasm. The binding of structurally different beta-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1.5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested beta-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced beta-lactamase did not prevent PBP-beta-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific beta-lactams.     

Diffusion of beta-lactam antibiotics into proteoliposomes reconstituted with outer membranes of isogenic imipenem-susceptible and -resistant strains of Enterobacter cloacae.

The influence of outer membrane (OM) permeability on carbapenem susceptibility was studied in strains of Enterobacter cloacae, a species in which carbapenem resistance depends upon the conjunction of overproduction of the chromosomal cephalosporinase and reduction of OM permeability. Relative trans-OM diffusion rates were measured using the liposome swelling assay. Proteoliposomes were reconstituted with OM from the members of an isogenic set of E. cloacae strains, selected in vivo or in vitro, which produced either porins F and D (wild-type), or F or D only, or neither. For all but one mutant, and compared with the wild-type strain, the respective increases in MICs and decreases in trans-OM diffusion of carbapenems were: nil and 13 to 18%; 4- to 32-fold and 33 to 50%; > or = 64-fold and > or = 90%. Our results suggest (i) that carbapenems (and other beta-lactam antibiotics) diffuse through porins F and D, but more rapidly through porin F, and (ii) that OM permeability is the critical factor in determining the level of MICs of carbapenems for cephalosporinase-overproducing strains of E. cloacae. The OM of one particular low-level carbapenem-resistant and porin F- and D-deficient mutant was at least five times more permeable to carbapenems than the similarly porin-deficient high-level resistant mutants. We infer from this observation the possible existence of an alternative carbapenem penetration pathway which could be associated with two as yet uncharacterized overproduced OM proteins of about 22 and 47 kDa.     

The translocation kinetics of antibiotics through porin OmpC: Insights from structure‐based solvation mapping using WaterMap

Poor permeability of the lipopolysaccharide‐based outer membrane of Gram‐negative bacteria is compensated by the existence of protein channels (porins) that selectively admit low molecular weight substrates, including many antibiotics. Improved understanding of the translocation mechanisms of porin substrates could help guide the design of antibiotics capable of achieving high intracellular exposure. Energy barriers to channel entry and exit govern antibiotic fluxes through porins. We have previously reported a hypothesis that the costs of transferring protein solvation to and from bulk medium underlie the barriers to protein‐ligand association and dissociation, respectively, concomitant with the gain and loss of protein‐ligand interactions during those processes. We have now applied this hypothesis to explain the published rates of entry (association) and exit (dissociation) of six antibiotics to/from reconstituted E. coli porin OmpC. WaterMap was used to estimate the total water transfer energies resulting from transient occupation by each antibiotic. Our results suggest that solvation within the porin cavity is highly energetically favorable, and the observed moderately fast entry rates of the antibiotics are consistent with replacement of protein‐water H‐bonds. The observed ultrafast exit kinetics is consistent with the lack of intrachannel solvation sites that convey unfavorable resolvation during antibiotic dissociation. These results are aligned with known general relationships between antibiotic efficacy and physicochemical properties, namely unusually low logP, reflecting an abundance of H‐bond partners. We conclude that antibiotics figuratively “melt” their way through porin solvation at a rate determined by the cost of exchanging protein‐solvent for protein‐antibiotic H‐bonds. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.