Center for Genomics Research, Karolinska Institute, Berzelius väg 37, 171 77, Stockholm, Sweden1
Center for Biomolecular Recognition, Panum Institute, Blegdamsvej 3c, DK 2200 Copenhagen N., and Pantheco A/S, Fruebjergvej 3, DK 2100 Copenhagen Ø., Denmark2
Author for correspondence: Liam Good. Tel: +46 8 728 6697. Fax: +46 8 323950. e-mail: liam.good{at}cgr.ki.se
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ABSTRACT |
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Keywords: peptide nucleic acid, bacteria, lipopolysaccharide
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; PNA, peptide nucleic acid
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INTRODUCTION |
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PNA is a DNA mimic with nucleobases attached to a pseudopeptide backbone (Nielsen et al., 1991 ) (Fig. 1a
). PNA hybridizes to complementary DNA, RNA or PNA oligomer sequences through Watson and Crick base pairing and helix formation (Egholm et al., 1993
; Wittung et al., 1994
; Jensen et al., 1997
). The peptide backbone provides improved hybridization affinity and specificity properties (Egholm et al., 1993
; Jensen et al., 1997
), resistance to enzymic degradation (Demidov et al., 1994
) and access to a variety of chemical modifications (Nielsen & Haaima, 1997
; Püschl et al., 1998
). For antisense applications, target-bound PNA can hinder the activities of DNA and RNA polymerases, reverse transcriptase, telomerase and the ribosome (Hanvey et al., 1992
; Knudsen & Nielsen, 1996
; Good & Nielsen, 1998a
). Also, in vitro assays for telomerase activity and mitochondrial genome replication show that PNAs are often effective in applications where other (modified) oligonucleotides perform poorly (Norton et al., 1996
; Taylor et al., 1997
).
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To study factors that limit PNA antisense effects apart from nonspecific inhibition, we designed a PNA to target the lac repressor mRNA. Limited repressor expression was seen as induced ß-galactosidase (LacZ) activity in E. coli cells and taken as an indicator of antisense effect. The anti-lac repressor PNA was used together with mutant E. coli strains and chemicals known to disrupt cell wall components. The results show that the outer-membrane LPS layer is a major limit to PNA cell entry and antisense effects in E. coli.
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METHODS |
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Bacterial growth conditions.
Growth on solid media was initiated with an inoculum of 104 cells ml-1 in an overlay of agar media, and growth in liquid media was initiated with an inoculum of 104 cells ml-1 in 200 µl cultures in siliconized polypropylene microdilution tubes. The cultures were grown in 10% LuriaBertani medium at 37 °C for 18 h. The chemicals listed in Table 2 were included in liquid cultures over the nanomolar concentration range. PNAs and the chemicals listed in Table 2
were added to cultures prior to overnight growth.
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Nitrocefin outer cell barrier permeability assay.
Outer-membrane permeability was determined essentially as described by Angus (1982) . E. coli cells transformed with pBR322 were cultured to mid-exponential phase, recovered by centrifugation at 3000 g and rinsed with 5 mM HEPES (7·4). Reactions were in 96-well microtitre plates containing 0·1 OD550 units of cells in 5 mM HEPES (7·4), 5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 20 µg nitrocefin ml-1. Nitrocefin cleavage was monitored by measuring A500.
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RESULTS AND DISCUSSION |
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LPS layer and limited antisense effects
The outer cell membrane of Gram-negative bacteria is coated with an LPS layer that stringently limits the influx of foreign molecules (Nikaido, 1994 ; Hancock, 1997
), and this barrier also may restrict PNA. In our previous experiments, efficient inhibition in vitro was observed with PNA concentrations in the nanomolar range, whereas growth inhibition in cells required micromolar concentrations, dilute growth medium and the cell-wall-deficient strain AS19 (Good & Nielsen, 1998a
, b
). These results suggest that cell uptake, rather than limited intracellular activities, is the major limit against the use of antisense PNAs in bacteria. Therefore, we aimed to determine whether PNA susceptibility correlates with outer-barrier permeability and whether PNA antisense effects can be improved by compromising the integrity of the LPS. It was hoped that the results would clarify the need for improved uptake properties in the next generation of antisense PNAs.
Wild-type and permeable mutant E. coli strains were tested for susceptibility to antisense PNA #1709. Among the strains listed in Table 1, AS19 and to a lesser extent D22 showed increased induction of lacZ by PNA #1709; both of these susceptible strains are LPS defective. Strain AS19 has a severely depleted LPS layer (Zorzopulos et al., 1989
) and is more susceptible to antibiotics (Sekiguchi & Iida, 1967
). Also, this strain is unusually susceptible to PNAs targeted to the rRNA and reporter gene mRNAs (Good & Nielsen, 1998b
). Strain D22 is defective in the lipid A component of its LPS and more susceptible to antibiotics (Normark et al., 1969
). Therefore, LPS defects in E. coli appear to confer unusual PNA susceptibility.
To establish a more direct link between LPS integrity and PNA susceptibility for strains AS19 and D22, we tested outer-barrier permeability to nitrocefin, a chromogenic cephaloporin antibiotic. Nitrocefin is normally excluded from cells, but nitrocefin molecules that do permeate the outer membrane are cleaved by periplasmic ß-lactamase and undergo a change in absorbance maximum from 385 nm to 490 (Angus et al., 1982 ). E. coli strains were transformed with pBR322, selected for ampicillin resistance and subjected to the nitrocefin permeability assay (Table 1
). To block respiration and eliminate the potential for active efflux of nitrocefin, CCCP was included at 5 µM. The results showed high outer cell barrier permeability for strain AS19 and to a lesser extent for strain D22 (Table 1
, Fig. 2
). These strains also showed high PNA susceptibilities (Table 1
). The link between PNA susceptibility and LPS permeability suggests that the integrity of the LPS layer is a major determinant of PNA entry into E. coli.
To further investigate the importance of the LPS layer in the susceptibility of E. coli to PNA, we asked whether PNA antisense effects in wild-type E. coli strain K-12 can be improved by using chemicals that compromise LPS integrity. The anti-lac repressor PNA #1709 was used in combination with a range of compounds that permeabilize E. coli and can increase the uptake or potency of standard antibiotics (Table 2). Several of these agents enhanced PNA potency; the most effective was polymyxin B nonapeptide, which enhanced lacZ induction by approximately sixfold when present at low nanomolar concentrations (Fig. 2
). An important factor in the effectiveness of polymyxin B nonapeptide in this application may be its ability to permeabilize the outer membrane with relatively low toxicity to E. coli (Chunhong et al., 1998
). Antibiotics that block peptidoglycan formation did not enhance PNA effects (Table 2
). Synergism between polymyxin B nonapeptide and antisense PNAs is unlikely to be useful in practical applications, but it is important as further evidence that the LPS is a major barrier to PNA uptake.
Multidrug resistance translocase activity and PNA susceptibility
Intracellular accumulation of PNAs and antisense effects also could be limited by multidrug resistance translocases, which can clear a diverse range of foreign agents from cells, including peptide antibiotics (Shafer et al., 1998 ). Several drug translocases are associated with multidrug-resistance phenotypes in bacteria, and these efflux pumps display surprising substrate flexibility. Therefore, we investigated whether the best-known E. coli drug pumps could limit PNA-mediated effects. The level of lacZ induction by PNA #1709 was determined in E. coli strains with altered drug efflux activities. Strains with defective copies of the acrAB or emrA transporter genes did not differ from the wild-type in susceptibility to PNAs (Table 3
). Also, plasmids bearing cloned copies of the acrA, B and emrB transporter genes were introduced by transformation to complement the defective endogenous genes in strains N43 and SJ261, and active copies of these genes did not alter PNA susceptibilies (Table 3
).
The acr and emr drug translocase genes are among the best-characterized E. coli drug pumps, but other translocases also could affect PNA susceptibility. A range of translocases are implicated in drug resistance and there may be as many as 18 ABC-type transporter homologues within the E. coli genome (Blattner et al., 1997 ). Accordingly, drug translocase involvement in PNA susceptibility was tested in a more general way using the inhibitors verapamil and reserpine, which can reduce drug efflux in E. coli and other types of cells (Beja & Bibi, 1996
; Ng et al., 1994
). Neither of these compounds altered the PNA susceptibility of E. coli K-12. Therefore, the activity of drug efflux translocases does not appear to alter the susceptibility of E. coli to PNAs.
Conclusions
Induction of lacZ by an anti-lacI PNA provides a positive assay for antisense effects. In this study the assay was used to learn which cell-barrier components limit PNA cell entry and antisense effects in E. coli. The results, together with our previous work, provide three lines of evidence indicating that the LPS layer is a major barrier against PNA uptake. First, the LPS mutant strains AS19 and D22 are more susceptible to PNA than wild-type. Second, mutant E. coli strains that are more susceptible to PNA also are more permeable to the antibiotic nitrocefin. Third, chemical agents that compromise LPS integrity can also increase cellular PNA susceptibility. Fortunately, for practical applications, there are opportunities to modify PNAs to better permeate the outer-membrane LPS layer. Also, the activity of multidrug resistance translocases did not significantly alter the susceptibility of E. coli to PNA. This resistance against active efflux, along with evidence for PNA stability in biological fluids, suggests that PNAs that are able to gain access into bacteria are likely to remain active within cells. Therefore, efforts to improve the technology can focus on achieving improved uptake. Finally, the observation that an antisense PNA can up-regulate, as well as down-regulate, gene expression supports the idea that antisense PNAs can provide sequence specific tools for the analysis of microbial gene expression and function.
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ACKNOWLEDGEMENTS |
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Received 2 May 2000;
revised 21 June 2000;
accepted 4 July 2000.