Whole-Cell-Based Assay To Evaluate Structure Permeation

Article pubs.acs.org/journal/aidcbc

Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD Ramkumar Iyer, Mark A. Sylvester, Camilo Velez-Vega, Ruben Tommasi, Thomas F. Durand-Reville, and Alita A. Miller* Entasis Therapeutics, Inc., 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States S Supporting Information *

ABSTRACT: The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure−permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin−permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria. KEYWORDS: antibiotic, porin, OprD, permeation, Pseudomonas aeruginosa, carbapenem

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with Gram-negative activity have been established from the study of currently marketed antibiotics,8,9 there are no outer membrane “permeation rules” or chemical descriptors that inform rational design of compound uptake during the lead optimization phase. The differences in the composition and permeability of the outer membranes of various Gram-negative bacterial species further complicate this problem.7 Antibiotic entry across the outer membrane is thought to occur through two possible routes: (i) a hydrophilic pathway, exemplified by passage through outer membrane porin channels, or (ii) a hydrophobic pathway that involves entry through the LPS leaflet in a porin-independent manner.10 The aminoglycosides, quinolones (to some extent11), azithromycin, and polymyxin12 are the only examples described thus far as capable of using the hydrophobic pathway (self-promoted uptake mechanism involving the disruption of LPS-Mg2+ cross bridges12) in Escherichia coli13 and Pseudomona aeruginosa.14 While a number of antibiotic classes, such as the β-lactams,

ntibiotic resistance is rapidly becoming a real threat to global public health and economic development1 with ever rising potential for a post-antibiotic era, where common bacterial infections will be difficult if not impossible to treat.2 Of particular concern are infections caused by multi-drugresistant (MDR) Gram-negative bacteria, for which novel therapies are seriously lacking. The discovery of new antibiotics with activity against MDR Gram-negative pathogens is stymied both by the lack of attractive starting points from highthroughput target-based screens and high drop-out rates during lead optimization on the path to optimizing whole-cell antibacterial activity.3 This problem is exacerbated in Gramnegative bacteria both by the relative impermeability of their outer membranes and by the existence of multidrug efflux pumps with broad substrate specificity which limit the intracellular accumulation of small antibacterial molecules.4−6 The passive barrier function of the outer membrane is due to a unique glycolipid called lipopolysaccharide (LPS) in the outer leaflet of the membrane and the sieving function of outer membrane porin (OMP) protein channels.7 While general physicochemical characteristics that are unique to molecules © 2017 American Chemical Society

Received: November 22, 2016 Published: February 3, 2017 310

DOI: 10.1021/acsinfecdis.6b00197 ACS Infect. Dis. 2017, 3, 310−319

ACS Infectious Diseases

Article

Table 1. Effect of oprD Inactivation and Overexpression on Antibacterial Activity of Carbapenems and Comparator Antibiotics in P. aeruginosa MIC, μg/mLa inactivated OprD

a

overexpressed OprD

antibiotic

wild-type

ΔoprD

fold increase

pPSV35_empty

pPSV35_oprD

fold decrease

meropenem biapenem doripenem Imipenem faropenem carbenicillin aztreonam tetracycline

0.5 0.25 0.5 1 >32 128 4 16

4 4 2 8 >32 64 4 32

8× 16× 4× 8× nd 0.5× 1× 2×

0.5 0.25 0.25 1 >32 32 1 32

0.13 0.06 0.06 0.25 >32 32 2 32

4× 4× 4× 4× nd 1× 0.5× 1×

Values shown are the mean of at least two independent experiments; nd = not determined.

have been shown to cross the Gram-negative outer membrane via porin passage,15−18 the lack of generally applicable experimental approaches allowing a systematic investigation of structure porin−permeation relationships (SPPR) for small molecules is a major obstacle to discovery of new classes of antimicrobial compounds, especially for highly impermeable pathogens such as P. aeruginosa. Unlike E. coli, which utilizes several general diffusion porins with larger openings,6 the chromosome of P. aeruginosa encodes genes for at least 72 different porins that are divided into the OprD (small molecule uptake), OprM (efflux), and TonB-interacting families of gated porins.19 The OprD family is functionally specialized to take up specific nutrients (e.g., amino acids, dipeptides, tricarboxylates, sugars) and includes 19 members that take up cationic or anionic substrates.20 The most highly characterized member in this family, OprD, is one of the most highly expressed.19 It is involved in the uptake of basic amino acids such as arginine in P. aeruginosa21 and is upregulated in response to carbon and nitrogen sources such as arginine, glutamate, and alanine.22 Although studying the permeation properties of a specific porin directly in P. aeruginosa is desirable, the existence of multiple porins with potentially redundant functions in this organism makes this approach challenging. Historically, the characteristics of individual porins have been studied using liposome swelling6,23 and electrophysiological measurements involving purified proteins.4,24 The characterization of general diffusion porins from enteric bacteria such as E. coli OmpF and OmpC using electrophysiological methods has shown they possess relatively large (2.5−4 nanosiemens) conductances25,26 in bilayers and low substrate specificity in that they allow passage of different classes of antibiotics. In addition, these approaches have characterized the interaction of general diffusion porins with fluoroquinolones, zwitterionic penicillins,27,28 β-lactams,15,29 and the carbapenems,30,31 leading to the hypothesis that the ability of an antibiotic to optimally permeate through these porins requires displacement of organized water molecules in the porin constriction zone during entry and, in the case of zwitterionic molecules, favorable orientation along the charge axis of the constriction.29,31 In contrast, far fewer studies of this nature have examined the interactions between small, hydrophilic antibiotics and narrow channel porins, such as Pseudomonas OprD, presumably due to its much lower singlechannel conductance (20 picosiemens, even at 1 M KCl32) and greater substrate specificity.33

The following describes a sensitive, whole-cell-based approach called titrable outer membrane permeability assay system (TOMAS), which is an optimization of our previously described method34 that allows for the characterization of the molecular drivers of SPPR for individual porins. Here, we engineered an E. coli strain (K12ΔFCA) to have low intrinsic porin-dependent outer membrane permeability by deleting the three major porins ompA, ompC, and ompF. Controlled expression of P. aeruginosa porins in this strain resulted in a broad range of susceptibility to small antibacterial molecules traversing the porin of interest with a greater range of sensitivity. We validated the approach with carbapenem passage through the P. aeruginosa porin OprD for several reasons. First, it is well-established that meropenem, the most frequently prescribed member of this class, permeates P. aeruginosa through OprD.34−37 Moreover, meropenem resistance in P. aeruginosa clinical isolates is frequently mediated by mutations or deletions of oprD that lead to lower levels of expression or loss of function.38−40 Finally, the carbapenem class of antibiotics are known to use the OprD porin to varying extents.41,42 Although the current study is focused on OprD to establish proof of concept for the approach, our ultimate goal is to use TOMAS to define porin permeation characteristics of small molecule substrates for multiple porins and subsequently incorporate these learnings into early stage medicinal chemistry design in support of Gram-negative antibiotic discovery.

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RESULTS AND DISCUSSION Relative Effect of Inactivation versus Overexpression of OprD on Carbapenem Uptake in P. aeruginosa. Consistent with other reports,34,38,41 we found that the minimal inhibitory concentration (MIC) of meropenem against wildtype P. aeruginosa (PAO1) to be ∼0.5 μg/mL, whereas P. aeruginosa PAO1 lacking oprD was 8-fold less sensitive (Table 1). Other carbapenems tested (biapenem, doripenem, and imipenem) also showed MIC increases ranging from 4- to 16fold, whereas the other classes of antibiotics tested were unaffected by the absence of OprD (Table 1). Overexpression of OprD in PAO1 (as confirmed by Western blot, Supplemental Figure 1) caused at best 4-fold increases in sensitization (Table 1), suggesting that levels of OprD are not rate-limiting for these carbapenems in this wild-type background. Because of the relatively modest effects of deletion and overexpression of a single type of porin in P. aeruginosa, presumably due to the complexity of porin physiology as described above, we sought to develop a more robust, whole311



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Figure 1. Use of a titrable outer membrane permeability assay system to study small molecule permeation through the P. aeruginosa porin OprD. (A) SDS-PAGE analysis of purified E. coli K12ΔFCA outer membranes expressing P. aeruginosa OprD (lanes 2−5) or the 6 amino acid L7 loop deletion [Δ312−317 (ΔGSGAGG), lanes 7−10)]39 as a function of L-arabinose induction. Lanes 1 and 6 = SeeBlue Plus2 ladder. Densitometric analysis of the bands corresponding to OprD (indicated with arrows) showed an increase in protein expression with added L-arabinose. Relative band intensities for lanes 2−5: 0, 200 000, 342 000, 430 000. Lanes 7−10: 0, 246000, 269000, 331000. (B) Schematic representation of TOMAS, where the P. aeruginosa porin OprD is expressed from a plasmid (pB22) under the control of the arabinose promoter (PBAD) in the K12ΔFCA strain that allows for sensitive and uniform population-wide expression.

Table 2. MICs of Commercial Carbapenems and Faropenem in TOMAS

a

MIC values shown are the mean of at least two independent experiments.

of permeation of several carbapenems.34 The limitations of this particular strain were (i) the requirement for high concentrations of the inducer (several hundred μM) to effect phenotypic changes and (ii) the lack of uniform, populationwide induction due to the all-or-none nature of the inducer response,44 both of which reduced the sensitivity of the assay. Here, we employed an arabinose-titrable K12 E. coli that has been previously shown to have uniform induction across the

cell-based assay to characterize the ability of individual porins to mediate small molecule uptake. Development and Validation of TOMAS. Previously, we described the use of an E. coli B strain (BL21_omp8)43 deleted for its native complement of porins (ompF, ompC, lamB, and ompA), which expressed selected P. aeruginosa porins under the control of an L-arabinose-inducible (pBAD) expression system to provide preliminary information about the molecular drivers 312



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Figure 2. Comparison of two experimental paradigms for OprD-mediated uptake of commercially available carbapenems and faropenem. (A) While overexpression of OprD in E. coli BL21_omp8 selectively sensitizes the cells to meropenem, the E. coli K12ΔFCA strain provides a larger dynamic range of OprD-dependent antibacterial activity. The relative change in meropenem antibacterial activity upon addition of the inducer arabinose when normalized to the MIC in the absence of induction is plotted on the Y-axis. L-Arabinose concentrations are shown on the X-axis. Error bars represent standard errors of mean from 3 to 5 experiments. (B) Arabinose-dependent OprD overexpression in K12ΔFCA results in lowered MICs for commercially available carbapenems [meropenem (MEM), imipenem (IMI), doripenem (DOR), biapenem (BPM)] and faropenem (FAR). (C) Change in meropenem MIC in the presence of increasing amounts of ΔL7OprD in K12ΔFCA (ΔL7OprD-MEM) was much less compared to wildtype OprD (MEM). Error bars are standard errors of mean from 4 experiments for MEM, DOR, and IMI. FAR and BIA means are from two independent determinations.

population.45 Specifically, unlike BL21, the K12 strain expresses the high capacity inner membrane arabinose transporter (AraE) under control of a constitutive, arabinose-independent promoter.46 Thus, all of the cells in the population have comparable ability to take up arabinose, which helps avoid the all-or-none pitfall that is associated with the use of the arabinose (PBAD) promoter for protein expression in other E. coli strains. We deleted the three major endogenous porins ompF, ompC, and ompA, to create the triple porin knockout background K12ΔFCA. Although the loss of the three major porins impairs the growth of the strain in minimal medium (M9 glucose), presumably due to the decrease in outer membrane permeability, its growth rate is indistinguishable from the wild-type in rich media (Supplemental Figure 2). In addition to permeation, active efflux by TolC-dependent pumps is a major variable in determining antibacterial activity in E. coli,47 which was recently demonstrated in an E. coli strain overexpressing a large, nonselective porin in either wild-type or efflux-compromised backgrounds to differentiate between barrier- and efflux-limited antibiotics.16 In contrast to this hyperporinated strain background, we found that inactivation of efflux (via insertional inactivation of the gene that encodes the major RND pump, acrB48) in the porin-deficient K12ΔFCA resulted in fitness defects (as evidenced by poor overnight growth densities), which were severe enough to render it unsuitable for TOMAS. We therefore focused the current study on OprD substrates for which efflux competence had minimal to no effect on antibacterial activity in E. coli (Supplemental Table 1). We evaluated the expression of the P. aeruginosa OprD in this K12ΔFCA E. coli strain. Exposure of cells to increasing levels of the inducer, L-arabinose, resulted in the dose-dependent expression of OprD, as shown in Figure 1A. The inducible and titrable nature of the system led us to name the approach TOMAS (Figure 1B). The porin-deficient K12ΔFCA strain, either untransformed or bearing the pB22 vector alone, was 16- to 32-fold more resistant to the carbapenems and 8-fold more resistant to the single penem (faropenem) tested, as compared to the wild-type (porin-replete) background (Table 2). Induction of K12ΔFCA/pB22_oprD with L-arabinose resulted in a dosedependent sensitization of the cells to these compounds. The

meropenem MIC at the highest inducer concentration tested decreased 16-fold, from 2 to 0.125 μg/mL (Table 2). In contrast, in the absence of induction, no difference in susceptibility to meropenem or any control antibiotics was observed in K12ΔFCA/pB22_oprD plasmid (data not shown). To compare relative compound permeation, the MICs at each of the inducer concentrations were divided by the MIC without arabinose and plotted as fold change in MIC as a function of arabinose concentration. Normalizing the MIC data in this manner enables us to cancel out target potencies of the compounds being compared, thereby allowing the evaluation of OprD permeation in isolation. Figure 2A shows that the dynamic range of the TOMAS strain MIC shift is greater than that of the E. coli B strain used previously.34 The effect of OprD overexpression on MICs was specific to the carbapenems and faropenem because arabinose induction had little to no effect on the MICs of antibiotics from other classes (Supplemental Table 2). To further demonstrate the OprD specificity of the observed phenotypes, we expressed a recently described meropenem-resistant variant of OprD in our K12ΔFCA strain (Figure 2C). This mutant has a 6 amino acid deletion in loop L7 (ΔGSGAGG corresponding to residues 312−317) that has been shown to impede entry of meropenem while preserving wild-type levels of arginine uptake.39 Although addition of Larabinose causes a dose-dependent induction of the L7 loop mutant of OprD in the outer membrane fraction (Figure 1A) at levels comparable to those of the wild-type porin, significantly smaller shifts in meropenem MICs were observed under these conditions as compared to wild-type OprD (Figure 2C). Addition of arabinose to the K12ΔFCA strain containing the empty vector led to equally minimal decreases in MIC values. In addition, growth rates of cells induced for OprD expression (even at the highest arabinose tested) in the absence of antibiotic were comparable to uninduced cells (data not shown). Taken together, these results show that the addition of arabinose and consequent OprD expression did not cause a general increase in outer membrane permeability or nonspecific sensitization to antibiotics. Nature, Number, and Position of Charges in the Carbapenem Scaffold Affects the Level of OprD Dependence in TOMAS. OprD selectively takes up basic amino acids such as arginine, lysine, and the structurally related 313



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Table 3. OprD Dependence of a Set of Carbapenem Analogues in TOMAS and P. aeruginosa (Values Shown Are the Mean of at Least Two Independent Experiments)

Calculated as the ratio of the MIC in the absence of arabinose divided by the MIC in the presence of 120 μM arabinose condition. bCalculated as the ratio of the MIC for PAO1ΔoprD divided by the MIC for PAO1 wild-type. cAs described in Isabella et al.34

a

antibiotic class of carbapenems,21,22 pointing to a role for positive charge in determining transit. Earlier work from our group involved making two modifications around the carbapenem core to evaluate the uptake of these analogues through the OprD porin.34 We used TOMAS to extend and refine these results, beginning with an evaluation of the differences in OprD dependence for a set of commercially available carbapenems. While imipenem, doripenem, and biapenem MICs decreased as a function of OprD concentration, by 8- to 32-fold, faropenem showed only a 4-fold decrease (Figure 2B and Table 2). This difference could be attributed to the lack of positive charge on the side chain as all these compounds share a very similar core scaffold. While it is well-known that charged molecules permeate Gram-negative bacteria more efficiently that their neutral analogues,8 we designed and synthesized nine additional carbapenem analogues to further explore the chemical features that contribute to effective translocation through OprD. All the carbapenems in this set have the ability to traverse the native E. coli porins as the K12ΔFCA strain is at least an order of magnitude less susceptible to them than the parent K12 (Table 3). Expressing the OprD porin in K12ΔFCA led to differing levels of sensitization of the TOMAS strain (Table 3, column 5). In comparison to meropenem, removing the positive charge (compound A), removing the tertiary amide side chain (compound B), or introducing a negative charge (cmpd734)

lowered the OprD-dependent permeation of the molecule, pointing to a critical role for charge in recognition during transit of the carbapenems (Table 3). Following the common dogma of “more charges = better Gram-negative permeation”, we prepared several carbapenems with an additional positive charge. While the second charge in compounds F and G maintained or improved permeation through OprD compared to meropenem, despite the increase in molecular weight, this was not the case for compounds C and D which only showed 8× decreases in MIC. Greater OprD dependence could be restored for compound C by adding a negative charge via a carboxylate group in a specific location and specific stereochemistry (compound E). Finally, a surprising result was observed while comparing compounds B and H. Simply increasing the ring size by one methylene group (pyrrolidine to piperidine) had a striking influence on the OprD dependence (from 4×to 8× to 16−32× MIC decrease in TOMAS). Taken together, these results show that small changes in chemical structure can have dramatic effects on OprD-mediated permeation. While it has been established that smaller and more polar compounds tend to have better Gram-negative outer membrane permeation,8 this by itself seems insufficient to optimize OprD passage. Whereas some analogues demonstrated reduced OprD-mediated permeation relative to meropenem (compounds A, B, and cmpd7), others were 314



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with the lowest rmsf was harmonically tethered to the aforementioned COM during the following picosecond. In general, translocation of these compounds through OprD followed general mechanistic guidelines that were previously proposed.50 Namely, only two routes were observed for these molecules, the first one requiring the displacement of the L7 loop (Supplemental Figure 3A,B) and the second one the rupture of the salt bridge between residues ARG131 and ASP295 (Supplemental Figure 3C), corresponding to paths A and B described in Samanta et al.,50 respectively. Moreover, our simulations show that the compounds’ dipole moment achieves its highest magnitude at the so-called constriction region between the loops and the barrel wall, and that the dipole moment tends to reorient upon encountering the strong electric field of this region, presumably to minimize the free energy cost associated with its passage across OprD. The latter observation is also in line with earlier research55,56 which suggests that proper alignment of the compounds’ dipole moment vector may facilitate translocation through these channels. Overall, diverse interactions are observed in our simulations between each compound and the arginine ladder lining the OprD channel (through their carboxylate groups), as well as with the negatively charged residues and hydrogen bond acceptors around the constriction region (Supplemental Figure 3C). These results lay the groundwork for future extensive sampling which should identify any dominant compoundspecific interactions, estimate the translocation barriers, and measure the relative probability of translocation through paths A and B, all of which should help explain the differences in MIC shifts observed experimentally for these three compounds.

significantly improved in OprD passage (compounds F and H). For this small set of compounds, pKa, log D, physicochemical properties, or other simple 2D descriptors also were not predictive of OprD permeation in TOMAS. While there is a requirement for at least one positive charge on the side chain for permeation through OprD, the number, nature, and exact position of the charges together with the right array of hydrogen bond donors and acceptors additionally seem to be critical for OprD-mediated uptake. Comparison of OprD MIC Shifts in P. aeruginosa versus TOMAS. We compared the above results to the relative MICs of the carbapenem analogues in wild-type PAO1 versus the isogenic PAO1 ΔoprD mutant. The PAO1 wild-type was much less susceptible to the nine carbapenem analogues than E. coli K12 wild-type (Table 3), and deletion of oprD in PAO1 further reduced this susceptibility. The MIC decreases upon induction of OprD in TOMAS correlated with the MIC increases in PAO1 when oprD was deleted with the exception of three compounds (doripenem, compound E, and compound H) (Table 1 and Table 3). The general alignment of the data between the two phenotypes validates our approach and is not surprising given the unusual dependence of the carbapenems on OprD for permeation into P. aeruginosa, a phenotype which has not been observed for any other antibiotic class to date. Deletion of oprD in PAO1 had less of an effect on carbapenem activity than overexpression of OprD in TOMAS, possibly due to the existence of additional routes of entry for these compounds in P. aeruginosa. In addition, although the K12 E. coli TOMAS strain allows the isolation and quantification of porin-mediated small molecule permeation across the outer membrane, species-specific differences in the nature of the target(s) and/or barriers to compound entry or accumulation are factors that also might contribute to differences in these phenotypes, reinforcing the utility of TOMAS to study porin permeation in isolation. Molecular Dynamic Simulations. Molecular dynamics (MD) simulation is an atomistic simulation technique that provides information about dynamical processes at atomic resolution.49 Preliminary MD simulations were performed in order to further understand the observations described above, with the ultimate goal of establishing a practical and iterative model for porin permeation that may ultimately apply to different porins and various chemical series. Previous MD simulations to study substrate translocation through OprD have revealed key residues lining the channel which play a role in passage34 as well as the potential for two alternative paths through OprD.50 Accordingly, we used MD simulations to model the passage of three compounds, meropenem, cmpd7, and compound G across OprD. Specifically, we employed a variation of steered MD called fluctuation guided pulling (FGP),51 which was implemented by means of Gromacs 5.1.252 and Plumed 2.1.553 software. The permeation process was simulated using the crystal structure of OprD (PDB: 3SY754) in explicit solvent. Multiple, independent FGP runs of 10 ns were carried out for each of the three compounds (n = 6, 10, and 10 for meropenem, cmpd7, and compound G, respectively). For each simulation, the molecule was forced to translocate through OprD by gradually shortening the distance between the center of mass (COM) of four opposing carbonyl oxygens at the periplasmic vestibule region and the compound’s atom with lowest root-mean-square fluctuation (rmsf). The rmsf of all ligand atoms was determined at every picosecond, and the one

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CONCLUSIONS Antibiotic discovery for Gram-negative agents has traditionally characterized the molecular drivers of compound permeation and/or accumulation by comparing MICs of wild-type versus mutant strains that are either isogenic, that is, lack specific outer membrane components, or that have been nonspecifically permeabilized. Historically, the contribution of individual porins in small molecule conduction is evaluated by comparing the MICs of porin-replete wild-type and the corresponding porin deletion strain. Individually engineered porin deletions often lead to only modest decreases in MICs17 due to the presence of other porins either constitutively expressed or induced in response to the lack of the deleted porin gene and/ or non-porin-mediated entry mechanisms. In addition, the relevant chemical series must possess whole-cell activity against the organism of interest, a frequent conundrum for compounds with good biochemical target potency but poor outer membrane permeability. The main advantage of the TOMAS approach is that it can specifically answer the question as to what extent a compound of interest is able to use the porin under consideration, that is, independent of any variables that would influence porin activity in the host organism.22,57,58 Any differences in target potency between analogues of interest are accounted for by the fact that the output of the assay is normalized to the antibacterial activity in the uninduced K12ΔFCA strain. There are caveats inherent to TOMAS. First, as with any growth-based measurement, only compounds with antibacterial activity against E. coli K12 can be studied in this assay. Fortunately, this strain is among the most antibiotic-susceptible laboratory strains of Gram-negative bacteria and therefore is an attractive choice for the optimization of compounds which have 315



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no or weak activity against less-permeable organisms such as P. aeruginosa or Acinetobacter baumannii. Second, the method only addresses porin-mediated permeation across the outer membrane; it does not address the additional challenge of crossing the cytoplasmic membrane for those compounds with cytoplasmic targets.59 Finally, in the present version of TOMAS, we have not formally characterized the effect of porin overexpression on efflux competence in K12ΔFCA. Although we were able to insertionally inactivate the gene that encodes the major RND pump (acrB)48 in K12ΔFCA, the resulting strain was severely attenuated in fitness when heterologously expressing porins and therefore not amenable to testing in TOMAS. We note that the antibacterial activities of the carbapenems used in the present study were generally unaffected in the ΔacrB K12 E. coli strain, suggesting a minimal role for efflux for this set of compounds. In addition, the K12ΔFCA strain, which is missing the three major porins, is considerably more resistant to the carbapenem analogues as compared to the porin-replete K12 wild-type parent, consistent with a much greater contribution from porin-mediated outer membrane permeation than efflux to antibacterial activity. Finally, the antibacterial activities of several control antibiotics which are known efflux substrates in E. coli were also unaffected by the increase in outer membrane permeability specific to the overexpression of OprD (Supplemental Tables 1 and 2). This is in contrast to the aforementioned hyperporinated strain of E. coli, where efflux competence clearly affected activity of certain classes of antibiotics.16 Our main goal in developing TOMAS was to be able to selectively and sensitively “tune” expression of specific porins of interest (like OprD) in an attempt to relate antibacterial activity to efficiency of entry through the porin of interest. Thus, eliminating the contribution to outer membrane permeability by the three major E. coli porins and the use of a strain providing a sensitive inducer response were critical to meet this objective. In contrast, Krishnamoorthy et al.16 overexpressed a very large, nonselective porin in the outer membrane of E. coli to deliberately and nonspecifically increase outer membrane permeability, essentially eliminating permeation as the rate-limiting step in antibacterial activity, to differentiate between barrier- versus efflux-limited antibacterial activities. Efforts are currently underway to make TOMAS more universally applicable in drug discovery by generating an efflux-deficient version of the method, such as inactivating tolC (in lieu of acrB) or addition of an efflux pump inhibitor (e.g., 1naphthylmethylpiperazine60) to the assay. We believe that a fundamental understanding of how the chemical attributes of small molecules influence permeation across the bacterial outer membrane requires a systematic deconvolution of the contribution of each component to permeation. To begin to achieve this goal, we developed a whole-cell antibacterial activity-based assay to define structure porin−permeation relationships (SPPR) for the passage of novel antibiotics through individual porins of interest and demonstrated the use of this assay for novel carbapenem permeation through the P. aeruginosa porin OprD. The results of this study serve to establish proof of concept for the assay by providing refinement of the SPPR of carbapenems through OprD. It is important to emphasize that, given the clinical prevalence of OprD functional mutants, our ultimate goal is not to “design in” OprD-mediated uptake of small molecules into P. aeruginosa. Rather, we intend to use TOMAS to interrogate the SPPR of other major porins whose ability to transport antibiotics have yet to be well-defined, not only in P. aeruginosa

but also in other formidable pathogens. We hypothesize that elucidation of the structural determinants of small molecule transport through multiple porins may ultimately aid in the rational design of novel antibacterial compounds able to enter cells through multiple porins, which may concomitantly lower uptake-mediated resistance liabilities.

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METHODS Reagents and Bacterial Strains. L-Arabinose, rifampin, chloramphenicol, erythromycin, novobiocin, ampicillin, meropenem, biapenem, imipenem, and faropenem were purchased from Sigma Chemical Company (St. Louis, MO). Dehydrated Mueller-Hinton cation-adjusted broth (MHIIB) powder was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Most antibiotic stocks were in distilled water, with the exception of chloramphenicol, erythromycin, novobiocin, and rifampin, which were dissolved in DMSO. Strains of E. coli K12 (BW27783; CGSC# 12119), E. coli B (BL21_omp8), P. aeruginosa (PAO1-wt, PAO1-ΔoprD34) were grown in Mueller-Hinton cation-adjusted agar (MHIIA) or in MHIIB (BD cat. no. 297311). Ampicillin was used at 40 μg/mL (K12) or 100 μg/mL (BL21_omp8) for E. coli, and gentamicin was at 30 μg/mL for P. aeruginosa where appropriate. DNA oligonucleotides were from MilliporeSigma (St. Louis, MO). Plasmids pB2220,54 and pPSV3561 have been described before. Construction of the Triple Porin Deletion E. coli K12ΔFCA strain and the ΔL7 OprD Expression Construct. E. coli K12 strain (BW27783; CGSC#12119) and the individual outer membrane porin deletions (ompF746(del)::kan; CGSC# 8925, ompC768(del)::kan); CGSC# 9781 and ompA772(del)::kan; CGSC# 8942) were purchased from the Coli Genetic Stock Center (CGSC, Yale University, New Haven, CT). E. coli B (BL21_omp8) was a gift from the van den Berg laboratory (Newcastle University, United Kingdom). The E. coli K12 TOMAS triple porin deletion strain was constructed by transferring individual ompF, ompC, and ompA deletions by P1 transduction62 using the corresponding kanamycin marked knockouts as donor strains. The kanamycin cassette was resolved between each successive transduction using the FLP vector, pCP20.63 The final strain (K12ΔFCA) was confirmed to lack the three major outer membrane porins by PCR and whole genome sequencing. The L7 loop mutation (Δ312−317; ΔGSGAGG) was generated using forward and reverse primers RI290 (5′GACTCGATTTTCCTCGCCAACTC-3′) and RI291 (5′GTTGCGGCCGAAGCCGAT-3′), respectively. The plasmid pB22_oprD was amplified using the Q5 DNA polymerase and the primers above to create the final plasmid pB22_L7oprD with the desired 18 bp nucleotide deletion using the Q5 sitedirected mutagenesis kit (New England Biolabs, Ipswich, MA) as per manufacturer recommendations. The product was transformed into NEB-10β E. coli cells (New England Biolabs, Ipswich, MA). Plasmid was isolated from the transformants and sequenced to verify the presence of the desired deletion in oprD and transformed into the K12ΔFCA strain for use in TOMAS. Porin Overexpression. K12ΔFCA was transformed with the empty pB22 vector54 and pB22 containing P. aeruginosa oprD34 to obtain strains K12ΔFCA/empty and K12ΔFCA/ oprD, respectively. The native signal peptide sequence of OprD was replaced by the corresponding sequence of an E. coli native outer membrane protein (TamA) as described earlier20 to facilitate expression in E. coli. The E. coli B strain (BL21_omp8) was transformed with either empty pB22 plasmid or the version 316



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Antibacterial Activity Assays. Cultures of K12ΔFCA/ pB22_empty, K12ΔFCA/pB22_oprD, BL21/pB22_empty, or BL21/pB22_oprD were grown up overnight in MHIIB (35 °C; 240 rpm; 1 mL) to stationary phase in 15 mL polypropylene tubes containing 40 μg/mL (K12) or 100 μg/mL ampicillin (BL21_omp8). Appropriate concentrations of L-arabinose, typically, 0, 1, 3, 10, 30, 90, and 120 μM for the K12ΔFCA set or 0, 50, 100, 200, 300, 500, and 1500 μM for the BL21 set, were added to these cultures. Antibacterial assays were run in MHIIB, employing a standard 2-fold dilution series of the compounds being tested in water or DMSO. Carbapenem and penem stocks were in water, and chloramphenicol and rifampin were dissolved in DMSO. One microliter of the compound dilution series was spotted in wells of sterile, 96-well polystyrene microplates. Fifty microliters of fresh MHIIB was then added to the plates. Inoculum was prepared by diluting 6− 7 μL of the appropriately induced overnight culture to 25 mL of MHIIB containing 2× the corresponding and final desired concentration of L-arabinose. Fifty microliters of this inoculum was aliquoted to the wells of the MIC plates above to obtain the desired final L-arabinose and antibiotic concentration range. At the end of 18−20 h at 35 °C, the plates were gently mixed in an Eppendorf MixMate (Hauppauge, NY) for 30 s and scanned in a SpectraMax (Molecular Devices, Sunnyvale, CA) at 600 nm. The MIC was read as concentration of the drug in the first well that showed no visible growth (typically, an OD600 nm value of ≤0.07). Growth curves were generated in MHIIB at 35 °C with shaking (220 rpm). At various times, aliquots were removed from growing cultures, diluted in fresh MHIIB medium, and plated to measure colony forming units/mL.

containing P. aeruginosa oprD to obtain strains BL21/empty and BL21/oprD, respectively. Transformants were selected on plates containing 100 μg/mL ampicillin. Porin induction was controlled by the addition of L-arabinose to the growth medium. OprD overexpression in P. aeruginosa was from the pPSV35 vector, and induction was achieved by addition of 100 μM IPTG as described previously.34,61 Outer Membrane Purification and SDS-PAGE Analysis. Fifty milliliter cultures of the K12ΔFCA strains (wtOprD-expressing and L7OprD-expressing) or the P. aeruginosa strains (wt, ΔoprD, and OprD-overexpressing) were grown in 50 mL of MHIIB overnight at 35 °C (240 rpm). OprD (wt and L7 mutant) induction in K12ΔFCA was by addition of 3, 30, and 120 μM L-arabinose, whereas OprD overexpression in P. aeruginosa from pPSV35_oprD was achieved by addition of 100 μM IPTG. Induction was overnight (typically, 16 h), and corresponding uninduced cultures were also included. The cells were pelleted down at 5000×g rpm for 10 min at 4 °C, resuspended in 10 mM HEPES, pH 7.5, and then subjected to one or two rounds of lysis through an Aminco french press cell at 16 000 psi. The resulting lysate was centrifuged at 5000×g for 20 min at 4 °C to remove unlysed cells and cell debris, after which the supernatant was subjected to ultracentrifugation to pellet total bacterial membranes (62 500g, 1 h, 4 °C). The membrane pellet was resuspended in 1 mL of 1% N-lauroyl sarcosine (Sarkosyl) and incubated at 35 °C for 35 min to selectively solubilize inner membranes. The extract was diluted to 25 mL in 10 mM HEPES, pH 7.5, and centrifuged at 62 500g (1 h, 4 °C). The resulting outer membrane pellet was resuspended in 0.2−0.5 mL of 10 mM HEPES, pH 7.5. Total protein was estimated using the Pierce BCA protein assay kit (ThermoFisher, Waltham, MA). K12ΔFCA protein samples were diluted in sample buffer (Jule, Milford, CT) containing reducing agent (50 mM dithiothreitol) and denatured at 95 °C for 10 min. Four to five micrograms of total protein was loaded per lane into wells of a 16% Tris-glycine SDS polyacrylamide gel (Jule, Milford, CT). The protein bands were visualized by staining the gel with InstantBlue protein stain (MilliporeSigma, St. Louis, MO). Western Blot Analysis of ΔoprD and OprD-Overexpressing P. aeruginosa Strains. Purified outer membranes of the P. aeruginosa strains were analyzed as follows. Ten micrograms of total protein was denatured in reducing agent (DTT) containing SDS-PAGE sample buffer at 70 °C for 10 min as per manufacturers’ instructions, of which a total of 3 μg was loaded per lane into wells of a 12% TruPage precast gel (MilliporeSigma, St. Louis, MO) and electrophoresed using TEA-tricine-SDS buffer (MilliporeSigma, St. Louis, MO) per manufacturer recommendations. The separated proteins were transferred onto a nitrocellulose filter using a Western blot procedure using the iBLOT dry blotting system (ThermoFisher, Waltham, MA). The filter was blocked using a proteinfree blocking buffer (Azure Biosystems, Dublin, CA). Washes were with a protein-free wash buffer (Azure Biosystems, Dublin, CA). The primary anti-OprD Ab was rabbit polyclonal antiserum terminal bleed (1 h at room temperature followed by incubation overnight at 4 °C, used at a dilution of 1:15000) and the secondary antibody (alkaline-phosphatase conjugated goat anti-rabbit; 1 h at room temperature) was from Rockland Antibodies & Assays (Limerick, PA). The blot was developed using the BCIP(R)/NBT-purple liquid substrate system (MilliporeSigma, St. Louis, MO) per manufacturer instructions.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00197. Supplementary Tables 1 and 2 and Figures 1−3, screen shots of MD simulations for three carbapenem analogues, supplementary methods, and synthesis of carbapenem analogues (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alita A. Miller: 0000-0002-7110-0493 Author Contributions

R.I. generated reagents, designed experiments, generated and analyzed data, and co-wrote the manuscript; M.A.S. generated and characterized carbapenem analogues; C.V.V. performed MD simulations and analysis; R.T. contributed to experimental design and data interpretation; T.D.-R. led the chemistry efforts and wrote sections of the manuscript; A.A.M. led the biology efforts and co-wrote the manuscript. Notes

The authors declare the following competing financial interest(s): All authors are current employees of Entasis Therapeutics and are shareholders in the company. 317



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ACKNOWLEDGMENTS We would like to thank A.J. Campbell for his intellectual contribution to compound design; Rongfeng Chen, Banggen Wang, Longyu Xu, and Yongjie Li at Pharmaron Inc. for chemical synthesis support; and Adam Shapiro and Sarah McLeod for technical advice and critical reading of the manuscript.

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ABBREVIATIONS MHIIB, Mueller-Hinton cation-adjusted broth; MIC, minimum inhibitory concentration; SPPR, structure porin permeation relationship; MD, molecular dynamics; MWM, molecular weight marker; IPTG, isopropyl β-D-1-thiogalactopyranoside; DMSO, dimethylsulfoxide

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Whole-Cell-Based Assay To Evaluate Structure Permeation

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