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Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture

AVI BioPharma, Inc., Corvallis, Oregon,¹ Department of Microbiology, Oregon State University, Corvallis, Oregon²

Received 30 September 2005/ Returned for modification 28 November 2005/ Accepted 3 June 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is ß-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC₅₀]) were 3.6, 10.8, 9.5, and 7.5 µM, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 µM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC₅₀ of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 µM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antisense antibiotics are short (about 10- to 20-base), synthetic analogues of DNA that inhibit gene expression in a sequence-specific manner (4, 12). There are about a half-dozen structurally distinct types of antisense oligomers, including peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs). Each type uses the naturally occurring DNA bases but differs in linkage between the bases. Modified linkages prevent degradation by nucleases while maintaining the architecture required for complementary base pairing.

Antisense PMOs inhibit expression of specific genes in pure cultures of Escherichia coli (3, 5). Inhibition is sequence specific and occurs by an antisense mechanism. However, entry of PMOs into E. coli is apparently limited by the outer membrane (5), which excludes solutes greater than about 600 Da (13). The molecular mass of an 11-base PMO is 4 kDa (5). Nevertheless, small amounts of PMO evidently gain entry, because modest levels of inhibition of gene expression are detectable (3).

Recently, a PMO targeted to an essential gene (acpP) of E. coli was shown to reduce infection in mouse peritonitis (6). AcpP is the scaffold on which fatty acids are synthesized, is essential for lipid biosynthesis (19), and has been targeted frequently by antisense oligomers to inhibit growth of bacteria in vitro and in vivo (3, 6, 7, 17). It is noteworthy that the AcpP PMO reduced viable bacterial cells in mouse peritonitis to a greater extent than in pure cultures of E. coli. Nevertheless, the reduction of infection seemed modest, and improvements in efficacy may be necessary for clinical development.

Efficacy of antisense oligomers has been improved dramatically by attaching membrane-penetrating peptides. Good and colleagues attached a short, membrane-penetrating peptide (KFFKFFKFFKC) to various PNAs, including a PNA complementary to acpP (7). The AcpP peptide-PNA reduced viability of E. coli by 4 orders of magnitude. The same peptide was attached to a PMO targeted to the Lac repressor (lacI). The LacI peptide-PMO induced expression of ß-galactosidase, whereas the same PMO without attached peptide did not induce ß-galactosidase (5). Results similar to those shown for PMOs in vivo have been demonstrated recently for peptide-PNAs and further show the efficacy of attaching membrane-penetrating peptides to antisense oligomers (17).

In this report, four different cell-penetrating peptides were attached to PMOs that are complementary to either a reporter gene for firefly luciferase or acpP. Three of the peptides, RFFRFFRFFXB, RXXRXXRXXB, and KFFKFFKFFKC, are similar and preserve the periodic spacing of cationic and hydrophobic residues that are important for membrane penetration (17). In RXXRXXRXXB, F is replaced with X (6-amino-hexanoic acid), which preserves the hydrophobic character but may be less sensitive to proteolysis. RTRTRFLRRTXB is a linear variation of a synthetic cyclic peptide that is nontoxic to bacteria and acts synergistically with antibiotics (18). The efficacy and specificity of the peptide-PMOs were tested in various in vitro systems and compared to PMOs without attached peptide.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria. E. coli W3110 (ATCC 27325) and E2348/69 (a gift from James Kaper, University of Maryland) and Salmonella enterica serovar Typhimurium TA1535 (ATCC 29629) were grown in LB broth at 37°C with aeration. E. coli LMG194 (pBAD-acpPmut4) (Invitrogen, Carlsbad, CA) and E. coli W3110 (pSE380myc-luc) (5) were grown in LB broth plus 100 µg/ml ampicillin at 37°C with aeration.

Peptide-phosphorodiamidate morpholino oligomers. PMOs were synthesized and purified at AVI BioPharma, Inc. (Corvallis, OR), as described previously (6, 15, 16). Three PMOs were used in the experiments and have been characterized and described previously (3): (i) Luc, 5'-ACGTTGAGGG; (ii) AcpP, 5'-CTTCGATAGTG; and (iii) Scr, 5'-TCTCAGATGGT. Each of the first two PMOs is complementary to the sequence immediately 3' of the start codon of its targeted mRNA, beginning at bases 5 and 6 of the coding regions for the luciferase reporter gene (5) and acpP, respectively.

Peptides were synthesized by 9-fluorenylmethoxy carbonyl (Fmoc) chemistry on solid-phase resin, purified, and characterized at AVI BioPharma, Inc., as follows. Wang resin (Novabiochem, San Diego, CA) was loaded with Fmoc-beta-alanine using diisopropylcarbidiimide and 4-dimethylaminopyridine in dichloromethane and N,N-dimethylformamide. This was followed by coupling of N--Fmoc-protected amino acids (Novabiochem, San Diego, CA) through activation with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in N-methylpyrrolidinone (NMP) in the presence of N,N-diisopropylethylamine (DIEA). Fmoc deprotection was performed with 20% piperidine-NMP. Arginine side chains were protected with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, and threonine side chains were protected with O-t-butyl. Lysine side chains were protected with the 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)isovaleryl (ivDde) protecting group when the peptide was to be used for conjugation but with tert-butoxycarbonyl when the peptide was to be used in control experiments. The N terminus of the completed peptidyl resin was acetylated with 0.2 M acetic anhydride and 0.4 M DIEA in NMP.

For the arginine-containing peptides, cleavage from the synthesis resin and side chain deprotection were carried out simultaneously by treating the peptidyl resin with a solution of 2.5% H₂O, 2.5% triisopropylsilane, and 95% trifluoroacetic acid, and isolation was achieved by precipitation from a 10-fold excess of diethyl ether. Strong-cation-exchange high-performance liquid chromatography (SCX HPLC) utilizing Source 15S resin (GE Healthcare, Piscataway, NJ) was used for purification, followed by reverse-phase desalt employing Amberchrom CG300M resin (Rohm & Haas, Philadelphia, PA). Desalted peptides werelyophilized and analyzed for identity and purity by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and SCX HPLC. The lysine-containing control peptide was treated in the same fashion.

To prepare lysine-containing peptides meant for conjugation, cleavage was performed with 5% H₂O in trifluoroacetic acid, followed by separation from the resin by filtration, evaporation, and partitioning between 10% isopropanol-chloroform, followed by washing with water to remove excess trifluoroacetic acid and isolation by removal of solvent. The crude, lysine-protected peptides were used directly in the conjugation.

PMOs were synthesized with a piperazine ring at the 5' terminus, which supplies a secondary amine for amide bond formation with the peptide (10, 11). All peptides included a non--amino acid at the C terminus to preclude racemization during the conjugation reaction. For the arginine peptides, a C-terminal peptide-benzotriazolyl ester was prepared by dissolving the peptide-acid (15 µmol), HBTU (14.25 µmol), and 1-hydroxybenzotriazole (15 µmol) in 200 µl NMP and adding DIEA (22.5 µmol). Immediately after the addition of DIEA, the peptide solution was added to 1 ml of a 12 mM solution of PMO in dimethyl sulfoxide. After 3 h at 30°C, the reaction mixture was diluted with a fourfold excess of water. The crude conjugate was purified first through a CM-Sepharose weak-cation-exchange column (Sigma, Inc., St. Louis, MO) to remove unconjugated PMO and then through a reverse-phase column (HLB column; Waters, Inc., Milford, MA). The conjugate was lyophilized and analyzed by MALDI-TOF MS and SCX HPLC.

For the lysine-containing peptides, 3 molar equivalents of peptide, together with equimolar amounts of HBTU and 1-hydroxybenzotriazole, were dissolved in NMP at a concentration of 100 mg peptide per ml NMP. When dissolution was complete, 3 equivalents of DIEA were added, and the solution was added to a stirring solution of the PMO in dimethyl sulfoxide at a concentration of 200 mg/ml. The reaction mixture was stirred at 35°C for 3 h. The ivDde protecting groups were removed from the lysine residues by adding 60 molar equivalents (relative to ivDde) of hydrazine as a 20% (vol/vol) solution in N,N-dimethylformamide and stirring at room temperature. After 2 h, the reaction was stopped and the mixture was prepared for purification by diluting with a fourfold volumetric excess of water and adjusting the pH to 6.5 with dilute phosphoric acid.

Purification of the deprotected (KFF)₃KXB-PMO conjugate was performed using Source 15S resin, followed by elution with a linear increasing salt gradient. The eluate was fractionated and analyzed by HPLC and MALDI-TOF MS. The pooled fractions were desalted by passage through Amberchrom CG300M resin, elution of the product with acetonitrile water, and lyophilization. The product was analyzed as described above.

Merodiploid. A mutated allele (acpP10) of acpP was synthesized, purified, and characterized at Blue Heron Biotechnology, Inc. (Bothell, WA). acpP10 was independently sequenced at Central Services Laboratory, Center for Gene Research and Biotechnology, Oregon State University (Corvallis, OR). Counting from the first base of the start codon, bases 6, 9, 12, and 15 were changed from C to T, T to C, C to T, and A to G, respectively. Three tandem stop codons in each reading frame were included at the 3' end. FatI and SpeI sites were included at the 5' and 3' ends, respectively. The sequence of acpP10 is5'-CATATGAGTACCATTGAG . . . . . . GTAAGTGAATAAGGATCC-3', where all bases between the indicated sequences are wild-type acpP from E. coli K-12, bold indicates the start codon, italics indicate mutated wobble bases, and underlining indicates stop codons. acpP10 was restricted with FatI and SalI and ligated to the 4-kbp NcoI-SalI restriction fragment of pBAD/myc-His/lacZ (Invitrogen, Carlsbad, CA). The resulting plasmid (pBAD-acpPmut4) was transformed into E. coli LMG194 [lacX74 galE thi rpsL phoA (PvuII) ara-714 leu::Tn10]. Standard cloning and transformation procedures were used (1).

Overnight cultures of E. coli LMG194 (pBAD-acpPmut4) were diluted 2 x 10^–2 into LB broth plus 100 µg/ml ampicillin and 200 µM arabinose. Aliquots of the culture were treated with 20 µM AcpP RFFRFFRFFXB-PMO. Growth was monitored by optical density at 600 nm (OD₆₀₀).

Bacterial growth in pure culture. Single-colony overnight cultures were diluted 2 x 10^–2 in LB broth (with antibiotics where indicated), which gave a starting inoculum of 4 x 10⁷ CFU/ml. Stocks of PMO, peptide-PMO, and peptide were added immediately to the indicated final concentrations. Three 100-µl aliquots of each culture were transferred to 96-well, low-binding microtiter plates and grown at 37°C on an orbital shaker (225 rpm). OD₆₀₀ readings were measured at approximately 1-h intervals. Where indicated, a sample of one of the triplicate cultures was diluted and plated in triplicate on LB agar plates and incubated overnight at 37°C. Each experiment was repeated three times, and the values shown in the figures are means.

Cell-free protein expression. Cell-free protein translation was done as described previously (3) using a cytoplasmic extract from E. coli (Promega, Madison, WI) programmed with gel-purified mRNA for luciferase. Luciferase enzyme activity was measured as described previously (5).

Tissue culture. Caco-2 cells were plated in 96-well culture dishes (Falcon, Franklin Lakes, NJ) at a concentration of 1.5 x 10⁴ cells/ml in a volume of 100 µl and grown at 37°C for 48 h in Dulbecco's minimal essential medium F12 (HyClone, Logan, Utah) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) in 95% air-5% CO₂. An overnight culture of E. coli E2348/69 (enteropathogenic E. coli [EPEC]) was diluted 4 x 10^–4 for fixed-concentration experiments (10 µM peptide-PMO) or 5 x 10^–6 for dose-response experiments into Dulbecco's minimal essential medium F12 with 10% fetal bovine serum and transferred to wells containing Caco-2 cells. The starting bacterial inoculum was 8 x 10⁵ CFU/ml in fixed-concentration experiments or 1 x 10⁴ CFU/ml in dose-response experiments. PMO, peptide-PMO, and peptide were immediately added, and the cultures were incubated at 37°C in 95% air-5% CO₂ for 24 h. After 24 h, cultures were examined and photographed with a Nikon inverted light microscope under x100 magnification. Culture supernatant was removed and stored on ice. Caco-2 cells were removed by treatment with trypsin (2.5 mg/ml in EDTA, 15 min, 37°C) and combined with the culture supernatant on ice. Control experiments showed that the same trypsin treatment of EPEC had no effect on bacterial viability. An aliquot of the Caco-2 cells prior to being combined with the culture supernatant was measured for viable cells by hemacytometer using trypan blue exclusion. The combined culture supernatant was diluted and plated on LB agar to measure viable bacterial cells.

IC₅₀. The concentration of antibiotic that reduced CFU/ml by 50% after 8 h of growth compared to an untreated control (50% inhibitory concentration [IC₅₀]) was calculated by nonlinear regression analysis using GraphPad Prism software (GraphPad Software, San Diego, CA).

Statistical analysis. Where indicated, mean values of triplicate data from each experiment were transformed logarithmically using InStat statistical software (GraphPad Software). Means of two or three separate experiments were calculated, and differences in treatment group means were analyzed with an unpaired t test.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of luciferase in E. coli. Four peptide-PMOs were tested for their abilities to inhibit expression of firefly luciferase in E. coli. Each peptide-PMO was synthesized with the same 10-base PMO (Luc). The PMO is complementary in base sequence to the region immediately 3' of the start codon for the reporter gene sequence. Covalently bound to the 5' end is one of four different peptides: RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, or KFFKFFKFFKXB (where X is 6-aminohexanoic acid and B is ß-alanine). Each Luc peptide-PMO was added (20 µM) to growing cultures of E. coli (pSE380myc-luc) that express luciferase. Controls included a culture without treatment and cultures treated separately with 20 µM peptide, PMO without attached peptide, each peptide coupled to a scrambled sequence PMO, or a mixture of peptide plus PMO. Growth was monitored by OD₆₀₀ for 8 h, and then each culture was analyzed for luciferase activity. The results (Fig. 1) show that each peptide-PMO inhibited luciferase activity. The Luc RTRTRFLRRTXB-PMO, Luc RXXRXXRXXB-PMO, and Luc KFFKFFKFFKXB-PMO each inhibited luciferase expression about 80%, whereas Luc RFFRFFRFFXB-PMO inhibited 23%. None of the controls inhibited, including the mixture of peptide plus PMO. There was no difference in growth of any of the cultures as measured by optical density or viable cell count (data not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 1. Luciferase expression in E. coli W3110 (pSE380myc-luc) treated with peptide-PMO. Growing cultures of E. coli W3110 (pSE380myc-luc) were treated separately with 20 µM Luc peptide-PMO (filled bars) or a Luc PMO (checkered bars) complementary to the region around the start codon of a reporter gene for firefly luciferase. Controls included untreated cultures (open bars) and cultures treated with 20 µM peptide (horizontally lined bars), a mixture of 20 µM peptide plus 20 µM Luc PMO (stippled bars), or 20 µM scrambled base sequence peptide-PMO (diagonally lined bars). Abbreviations: RLU, relative light units; RFF, RFFRFFRFFXB; RTR, RTRTRFLRRTXB; RXX, RXXRXXRXXB; KFF, KFFKFFKFFKXB. Error bars indicate standard errors of the means (n = 2 or 3).

View larger version (44K): [in this window] [in a new window]   FIG. 2. Cell-free translation reactions. Each peptide-PMO (RFF-Luc, RTR-Luc, and KFF-Luc) was added (100 nM) separately to cell-free translation reaction mixtures that were programmed to express firefly luciferase. Controls included reaction mixtures with 100 nM of PMO (Luc), scrambled base sequence PMO (Scr), scrambled base sequence PMO-peptide conjugates (RFF-, RTR-, and RXX-Scr), each peptide (RFF, RTR, and RXX), and mixtures of peptide plus PMO (RFF+Luc, RTR+Luc, and RXX+Luc). After 1 h at 37°C, luciferase activity was measured with a luminometer. Abbreviations are defined in the legend for Fig. 1. Error bars indicate standard errors of the means (n = 2). No, untreated cultures.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Dose-response in cell-free translation reactions. Various concentrations (Conc.) of Luc RTRTRFLRRTXB-PMO (triangles) or Luc PMO (squares) were added to bacterial cell-free translation reaction mixtures programmed to express firefly luciferase. Luciferase activity was measured in each reaction and is plotted as a function of log concentration. Error bars indicate standard deviations (n = 3). RLU, relative light units.

View larger version (22K): [in this window] [in a new window]   FIG. 4. (A) OD600 of growing cultures of E. coli W3110. Cultures were treated with 20 µM AcpP RFFRFFRFFXB-PMO (triangles), AcpP RTRTRFLRRTXB-PMO (inverted triangles), AcpP RXXRXXRXXB-PMO (diamonds), or KFFKFFKFFKXB-PMO (circles). The untreated culture is also indicated (squares). Control cultures (not shown) were treated with 20 µM peptide (RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, or KFFKFFKFFKXB), PMO, a mixture of 20 µM peptide plus 20 µM PMO, or 20 µM scrambled base sequence peptide-PMO, and all grew the same as the untreated culture. (B) Viable bacterial cells were measured in cultures after 8 h of growth. Untreated cultures (open bars) and cultures treated with peptide-PMO (filled bars), peptide (horizontally striped bars), PMO (checkered bars), a mixture of 20 µM each peptide plus PMO (stippled bars), or scrambled base sequence peptide-PMO (diagonally striped bars) are shown. Abbreviations are defined in the legend for Fig. 1. Error bars indicate standard errors of the means (n = 3).

View larger version (13K): [in this window] [in a new window]   FIG. 5. Dose-response of peptide-PMOs in E. coli W3110. Various concentrations of AcpP RFFRFFRFFXB-PMO (A), AcpP RTRTRFLRRTXB-PMO (B), AcpP KFFKFFKFFKXB-PMO (C), or ampicillin (Amp) (D) were mixed with growing cultures of nonpathogenic E. coli W3110 and grown aerobically for 8 h at 37°C. Samples of each culture were diluted and plated to measure viable cells. Other abbreviations are defined in the legend for Fig. 1. (E) The inhibition of growth (CFU/ml) was calculated as a percentage of the untreated culture, plotted as a function of log concentration of AcpP RFFRFFRFFXB-PMO (squares), AcpP RTRTRFLRRTXB-PMO (circles), AcpP KFFKFFKFFKXB-PMO (inverted triangles), or ampicillin (triangles), and analyzed by nonlinear regression (lines). Error bars indicate standard deviations (n = 2, except AcpP RFFRFFRFFXB-PMO [n = 4] and AcpP KFFKFFKFFKXB-PMO [n = 3]).

Inhibition of growth of pathogens. AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, and KFFKFFKXB-PMO were also tested against two pathogens: E. coli E2348/69 (EPEC) and S. enterica serovar Typhimurium TA1535. Remarkably, AcpP RFFRFFRXB-PMO and AcpP RTRTRFLRRTXB-PMO inhibited each strain to greater extents (Fig. 6) than the K-12 nonpathogenic strain W3110 (Fig. 4). AcpP KFFKFFKFFKXB-PMO inhibited the pathogenic E. coli strain to about the same extent as the nonpathogenic strain and inhibited S. enterica serovar Typhimurium to about the same extent as the other two AcpP peptide-PMOs.

View larger version (32K): [in this window] [in a new window]   FIG. 6. Growth of two different pathogens treated with peptide-PMOs. Growing cultures of (A) E. coli E2348/69 and (B) S. enterica serovar Typhimurium TA1535 were untreated (No) or treated with 20 µM AcpP RFFRFFRFFXB-PMO (RFF-PMO), 20 µM RFFRFFRFFXB peptide (RFF), a mixture of 20 µM each RFFRFFRFFXB peptide plus AcpP PMO (RFF+PMO), 20 µM scrambled RFFRFFRFFXB-PMO (Scr RFF-PMO), 20 µM AcpP RTRTRFLRRTXB-PMO (RTR-PMO), 20 µM RTRTRFLRRTXB peptide (RTR), a mixture of 20 µM each RTRTRFLRRTXB peptide plus AcpP PMO (RTR+PMO), 20 µM scrambled RTRTRFLRRTXB-PMO (Scr RTR-PMO), 20 µM AcpP PMO (PMO), 20 µM AcpP KFFKFFKFFKXB-PMO (KFF-PMO), or 20 µM scrambled KFFKFFKFFKXB-PMO (Scr KFF-PMO). Viable cells (CFU/ml) were measured after 8 h of growth. Error bars indicate standard errors of the means (n = 3).

Merodiploid rescue. The sequence-specific effect of the AcpP RFFRFFRFFXB-PMO was tested by constructing an acpP merodiploid. An arabinose-inducible expression plasmid was constructed with an allele of acpP (pBAD-acpPmut4) that has four point mutations at each of the wobble bases within the region targeted by the antisense peptide-PMO. The mutations cause a mismatch in base pairing with the peptide-PMO but do not change the protein sequence of the acyl carrier protein. A growing culture of E. coli (pBAD-acpPmut4) was divided, and half was induced with arabinose. The induced half was divided again, and AcpP RFFRFFRFFXB-PMO was immediately added (20 µM) to one half, while the other induced half was not treated with peptide-PMO. Growth was monitored by OD₆₀₀. The results show nearly identical growth curves for cultures induced with arabinose and either treated or not treated with AcpP RFFRFFRFFXB-PMO (Fig. 7). As expected, growth was reduced in the uninduced culture treated with the peptide-PMO.

View larger version (8K): [in this window] [in a new window]   FIG. 7. Growth of acpP merodiploid. Cultures of E. coli LMG194 (pBAD-acpPmut4) were induced with arabinose and not treated with AcpP RFFRFFRFFXB-PMO (squares), not induced with arabinose and treated with 20 µM AcpP RFFRFFRFFXB-PMO (X's), or induced with arabinose and treated with 20 µM AcpP RFFRFFRFFXB-PMO (triangles). Growth was monitored by OD600. Error bars indicate standard deviations (n = 3).

View larger version (23K): [in this window] [in a new window]   FIG. 8. Caco-2 cultures. Caco-2 cultures were infected with E. coli E2348/69 (EPEC) and treated with 10 µM AcpP RFFRFFRFFXB-PMO (RFF-PMO), scrambled base sequence RFFRFFRFFXBF-PMO (Scr RFF-PMO), AcpP PMO (PMO), RFFRFFRFFXB peptide (RFF), or a mixture of 10 µM each RFFRFFRFFXB peptide and AcpP PMO (RFF+PMO). Cultures that were uninfected or infected but untreated (No) are also shown. After 24 h, viable Caco-2 (open bars) and EPEC (checkered bars) were measured. Error bars indicate standard errors of the means (n = 3).

These results were confirmed by microscopic examination of the cultures, which showed that AcpP RFFRFFRFFXB-PMO-treated cultures appeared the same as uninfected cultures (Fig. 9). All infected controls appeared the same, independent of treatment, and showed few Caco-2 cells but massive amounts of bacteria, which made the cultures appear turbid. The darker color of the infected controls results from the turbidity and a change in the pH indicator from red to yellow, which indicates that the control cultures were acidified by the bacteria. Results obtained using AcpP RTRTRFLRRTXB-PMO were similar to those obtained using AcpP RFFRFFRFFXB-PMO (data not shown).

View larger version (56K): [in this window] [in a new window]   FIG. 9. Light micrographs of Caco-2 cultures. (A) Uninfected Caco-2 culture. (B) Caco-2 culture 24 h postinfection (untreated). (C) Caco-2 culture 24 h postinfection and treated with AcpP RFFRFFRFFXB-PMO. Magnification, x100.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results show that attaching RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFKXB to PMOs increased the antisense effects of the PMO by orders of magnitude. Attaching RXXRXXRXXB was generally less effective than attaching the other peptides, and the effects were more dependent on the strain used for testing. These effects were shown in three different systems: pure culture, cell-free protein synthesis reactions, and infected tissue cultures.

The effect of peptide-PMOs on viability is not limited to E. coli. Growth of S. enterica serovar Typhimurium was also inhibited to similar extents. It will be informative to design and test peptide-PMOs targeted to genes in other bacteria, especially antibiotic-resistant gram-positive bacteria. Efficacy of another type of antisense oligomer, PNA, has been demonstrated against both E. coli and the gram-positive bacterium Staphylococcus aureus (7, 10).

Increased efficacy of peptide-PMOs compared to PMOs may be a combination of two different effects of the peptide. One effect is the increased penetration of the bacterial cell. Mixtures of peptide and PMO had no effect on gene expression in pure cultures. This shows that the peptides tested did not generally permeabilize the bacterial cell to solutes. Instead, the peptides enabled attached PMOs to traverse the outer membrane and enter the bacterial cell. Second, peptide-PMOs were more effective than the corresponding PMOs in cell-free reactions. This suggests that some of the effect of conjugate may be attributed to enhanced inhibition of translation. Currently it is not known if the peptide remains attached once the conjugate enters a bacterial cell.

The relative efficacies of peptide-PMOs differed from strain to strain. One possible explanation for this is that the peptide may be hydrolyzed to different extents in different strains, either before or after entering the cell. Clearly, hydrolysis of the peptide moiety outside the cell would reduce efficacy. However, it is not clear if the same would be true if hydrolysis occurred in the cytoplasm. It seems possible that after entering the cytoplasm, peptide-PMO might reverse course and traverse back through the cytoplasmic membrane. If this were the case, then hydrolysis of the peptide moiety in the cytoplasm might act to trap the PMO in the cytoplasm, effectively increasing its intracellular concentration and antisense activity.

The results of dose-response experiments with E. coli W3110 show that the IC₅₀ of AcpP RFFRFFRFFXB-PMO was about half that of ampicillin and about one-third that of AcpP RTRTRFLRRTXB-PMO or AcpP KFFKFFKFFKXB-PMO. However, the pattern of inhibition of the peptide-PMOs appeared different from that of ampicillin. Higher concentrations of ampicillin resulted in increased inhibition, whereas inhibition by the peptide-PMOs did not increase nearly as much above 20 to 40 µM. This may suggest that entry of peptide-PMOs into the cell, at either the outer membrane or the plasma membrane, is rate limiting. This would not be surprising, considering that ampicillin is a small solute that readily diffuses through the porins of the outer membrane and immediately arrives at its site of action in the periplasm. Presumably, peptide-PMOs traverse the outer membrane through the lipid bilayer and then either are transported across the plasma membrane by an unknown transport protein or penetrate through the lipid bilayer. The leveling of the dose-response curves of peptide-PMOs is characteristic of a saturable mechanism, which may reflect a limited rate of entry into the cell. It seems less likely that the cytoplasmic concentration of peptide-PMOs has saturated the mRNA of acpP, because AcpP is one of the most abundant proteins in E. coli (2, 8) and the concentration of AcpP can be significantly reduced without affecting growth (8, 14). The mechanism of entry of peptide-PMO and its intracellular concentration remain important goals in our development of this remarkable experimental antibiotic.

The reductions of viability caused by AcpP peptide-PMOs appear similar to that shown for AcpP PNA coupled to a similar peptide, KFFKFFKFFK. Good et al. reported no apparent growth using 1 µM AcpP KFFKFFKFFK-PNA (7). Tan et al. reported no apparent growth of E. coli in vitro using 40 µM AcpP KFFKFFKFFK-PNA and an IC₅₀ of approximately 4 µM (17). However, it is difficult to compare results because of differences in starting inoculum and growth broth. Good et al. used a starting inoculum of 1 x 10⁵ CFU/ml in Mueller-Hinton broth, Tan et al. used 10⁵ CFU/ml in 1/10-diluted LB broth, and we used 4 x 10⁷ CFU/ml in LB broth. We observed no apparent growth of E. coli W3110 after 24 h in 4 µM AcpP RFFRFFRFFXB-PMO, as measured by either optical density or viable cell counts, using a starting inoculum of 5 x 10⁵ CFU/ml in Mueller-Hinton broth, which are the starting inoculum and broth prescribed for determining MIC under standard clinical laboratory methods (9). This result indicates that AcpP RFFRFFRFFXB-PMO has an MIC at most equal to or less than 4 µM, which is approximately the same as the IC₅₀ measured in LB broth with a starting inoculum of 4 x 10⁷ CFU/ml.

Induction of expression of the mutated acpP allele fully rescued the acpP merodiploid from the lethal effect of the AcpP peptide-PMO. This result shows that inhibition of growth by the peptide-PMO is sequence specific and not a result of toxicity. Although results using scrambled base sequence controls suggest sequence specificity of the peptide-PMO, the results using the merodiploid are more definitive.

AcpP peptide-PMOs cured or nearly cured infected cultures of Caco-2 cells, whereas treatment with scrambled base sequence peptide-PMO, PMO, or a mixture of peptide and PMO had no effect on bacterial infection. In addition, viability of Caco-2 cells was apparently unaffected by treatment with AcpP peptide-PMOs. Caco-2 cells were selected because they are derived from the intestinal epithelium, which is the site of infection of the enteropathogenic strain of E. coli that was used in our experiments. Similar results have been reported using an AcpP PNA conjugated to the peptide KFFKFFKFFK(7).

Although the IC₅₀ were similar in tissue culture and pure culture, the reduction in bacterial viability was orders of magnitude greater in tissue culture than in pure culture. Good et al. (7) also reported a significant increase in efficacy of peptide-PNA in tissue culture medium compared to bacterial growth medium. The reason for this difference is unknown but may result from large differences in starting inocula (1 x 10⁴ CFU/ml and 4 x 10⁷ CFU/ml for tissue culture and pure culture, respectively). Alternative possibilities include differences in bacterial growth rates in the two types of culture or an unidentified substance produced in tissue culture that counteracts a bacterial protease. These results show that peptide-PMOs are not toxic to Caco-2 cells at the concentrations tested and are more effective in environments that more closely mimic animal models than in pure cultures of bacteria. The reduction in CFU below the initial inoculum in pure culture and in infected tissue culture shows that the peptide-PMOs tested are bactericidal.

Previous work has shown that PMOs are more effective in animal infections than in pure cultures (6). We are optimistic that peptide-PMOs will perform similarly. Further improvements are likely, considering that few essential gene targets and membrane-penetrating peptides have been tested yet with antisense antibiotics.

	ACKNOWLEDGMENTS

 
We thank the entire chemistry department at AVI BioPharma and especially Candace Lovejoy for making and purifying the PMOs and peptide-PMOs and Dave Stein for reading the manuscript.

This work was funded by AVI BioPharma, Inc. Bruce L. Geller is an employee of both Oregon State University and AVI BioPharma, Inc.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1845. Fax: (541) 737-0496. E-mail: gellerb{at}orst.edu.

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