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.
An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to -lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with -lactam molecules. Determination of -lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem Ͼ Ͼ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to -lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to -lactam antibiotics.Providencia stuartii is an opportunistic pathogen involved in community-acquired as well as hospital-acquired infectious diseases. Clinical strains of P. stuartii are mostly isolated from urinary tract infections of patients with long-term indwelling urinary catheters and, in fewer cases, from respiratory and skin infections (1, 2). P. stuartii is reported as one of the most resistant species in the family of Enterobacteriaceae (3). P. stuartii strains show high levels of resistance to the majority of antibiotic classes but were found to remain susceptible to carbapenems (3, 4). P. stuartii produces a chromosomally encoded cephalosporinase, AmpC, which causes the natural resistance to aminopenicillins and narrow-spectrum cephalosporins (5). The productions of different extended-spectrum -lactamases (6, 7, 8, 9, 10, 11) and metallo--lactamases (12, 13, 14) have been reported in association with resistance to carbapenems in Providencia spp. Other enzymatic mechanisms of antibiotic resistance identified in P. stuartii include acetyl aminotransferases targeting aminoglycoside antibiotics and an integronencoded erythromycin esterase involved in the resistance to macrolides (15, 16). Moreover, mutations of the gyrA gene leading to a modification of target site were described in resistance to fluoroquinolones (17). In contrast, little is known about the involvement of membrane proteins in antibiotic resistance of P. stuartii. One study suggested a role of outer membrane porins in antibiotic permeability for Proteus, Morganella, and Providencia strains by selection of mutants resistant to cefoxitin, a cephalosporin of the second generation (18). However, the authors sugg...
Biofilms are organized communities of bacterial cells that are responsible for the majority of human chronic bacterial infections. Providencia stuartii is a Gram-negative biofilm-forming bacterium involved in high incidence of urinary tract infections in catheterized patients. Yet, the structuration of these biofilms, and their resistance to environmental insults remain poorly understood. Here, we report on planktonic cell growth and biofilm formation by P. stuartii, in conditions that mimic its most common pathophysiological habitat in humans, i.e. the urinary tract. We observed that, in the planktonic state, P. stuartii forms floating communities of cells, prior to attachment to a surface and subsequent adoption of the biofilm phenotype. P. stuartii planktonic and biofilm cells are remarkably resistant to calcium, magnesium and to high concentrations of urea, and show the ability to grow over a wide range of pHs. Experiments conducted on a P. stuartii strain knocked-out for the Omp-Pst2 porin sheds light on the role it plays in the early stages of growth, as well as in the adaptation to high concentration of urea and to varying pH.
The emergence of Gram-negative "superbugs" exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram-negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure-translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC- E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC-expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k(on) ≈ 2 × 10(4) M(-1) s(-1)). Zwitterionic compounds with highly polar groups attached to the penem-2 ring, including panipenem, imipenem and doripenem exhibit faster k(on) (>10(4) M(-1) s(-1)), while meropenem and biapenem with fewer exposed polar groups exhibit slower k(on) (∼5 × 10(3) M(-1) s(-1)). Tebipenem pivoxil and razupenem exhibit ∼13-fold slower k(on) (∼1.5 × 10(3) M(-1) s(-1)) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure-kinetic relationships of carbapenem translocation are governed by H-bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups).
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