Inhibition of Escherichia coli Growth by Vitamin B12–Peptide Nucleic

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Inhibition of Escherichia coli Growth by Vitamin B12−Peptide Nucleic Acid Conjugates ́ nicki,†,‡ Zofia Dąbrowska,† Monika Wojciechowska,† Aleksandra J. Wierzba,§ Marcin Row Ksenia Maximova,† Dorota Gryko,*,§ and Joanna Trylska*,† †

Centre of New Technologies and ‡College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland § Institute of Organic Chemistry, Polish Academy of Sciences, M. Kasprzaka 44/52, 01-224 Warsaw, Poland

ACS Omega 2019.4:819-824. Downloaded from pubs.acs.org by 5.189.201.61 on 01/14/19. For personal use only.

S Supporting Information *

ABSTRACT: The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B12−PNA conjugates. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA−vitamin B12 conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B12 and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF)3K. We found that vitamin B12−PNA conjugates inhibit E. coli growth at a concentration of 5 μM, similar as (KFF)3K−PNA. We also showed that vitamin B12−PNA conjugates are stable in the presence of biological media. This study provides the foundation for improving and developing PNAs conjugated to vitamin B12 as antibacterials.

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INTRODUCTION The misuse of antimicrobials has decreased the efficacy of many commonly used antibiotics. However, the increase of bacterial resistance to antibiotics has not resulted in sufficient increase in the discovery of new antimicrobials.1 This fact necessitates pursuing alternative therapies to treat drugresistant bacterial infections.2 An alternative therapeutic approach that has shown promise in recent years is silencing essential genes by antisense oligonucleotides.3 Antisense antibacterials are short (about 10−20 nucleotides) synthetic DNA analogues that inhibit gene expression at the mRNA level in a sequence-specific manner.4 Chemically modified antisense oligomers, such as peptide nucleic acids5 (PNAs, Figure 1), possess favorable physicochemical properties. These include improved target specificity, binding affinity, chemical and enzymatic stability conferred by their pseudopeptide backbone, as well as low toxicity to host tissues.6 PNAs have been tested as antimicrobial agents in the past decade in a variety of bacterial species.7−9 The mRNA of several essential genes has been targeted by PNA antisense interference to achieve inhibition of bacterial growth, including the gene for the gyrase A subunit (gyrA),10 the gene coding the acyl carrier protein (acpP),11 and the rpoD gene coding RNA polymerase.12,13 In a different approach, bacterial protein synthesis has been inhibited by PNAs specific for the 16S14 or 23S rRNA.15 However, one significant limitation of antisense PNAs is that their cellular uptake is restricted by bacterial cell membranes.16 This fact precludes successful use of PNA in © 2019 American Chemical Society

targeting bacterial pathogens. To overcome the delivery problem, PNA has been covalently conjugated to various cell-penetrating peptides including TAT,17 (RXR)4XB (X6aminohexanoic acid, Bβ-alanine),13 and many others.18 Even though all are cationic and amphipathic, none of them significantly improved the cell delivery of PNA when compared to the most popular cell-penetrating peptide(KFF)3K.19 Therefore, a new more efficient carrier to deliver PNAs to bacterial cells would be of great value. One promising approach is to use a nonpeptidic natural carriervitamin B12 (Figure 2a) to transport PNA to bacterial cells. We have previously shown that vitamin B12 delivers PNA to Gram-negative bacteria.20 Vitamin B12 is a natural organometallic molecule and essential nutrient for both mammalian and many bacterial cells.21 Although only few bacterial species produce vitamin B12, most aerobic bacteria do require vitamin B12 to grow.22 Furthermore, the mechanisms of vitamin B12 uptake in bacteria are quite well characterized,23 especially in Escherichia coli.24,25 All these features make vitamin B12 an attractive and promiscuous candidate as a PNA carrier. However, to exploit the natural uptake properties of vitamin B12, the molecule has to be modified and conjugated to PNA oligomers. To successfully use the vitamin B12 pathway in the transport of PNA, the conjugate must still be recognized by Received: November 10, 2018 Accepted: December 26, 2018 Published: January 10, 2019 819

DOI: 10.1021/acsomega.8b03139 ACS Omega 2019, 4, 819−824

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antibacterial potential of vitamin B12−PNA conjugates, we used PNA complementary to the mRNA transcript of the acpP gene, encoding acyl carrier protein, involved in fatty acid biosynthesis. The acpP gene is a well-described target for antisense antibacterial oligonucleotides.27,28 We used a 10base-pair anti-acpP PNA designed by Good et al. (2001)28 with the following sequence: Nterm-ctcatactct-Cterm (Figure 2b). The antibacterial activity of this PNA sequence was verified by determination of its minimal inhibitory concentration (MIC) on E. coli cells.28,29 Moreover, the site-specific activity of this anti-acpP PNA sequence was also confirmed by the real-time quantitative PCR.30 For our work, the anti-acpP PNA was coupled to vitamin B12 by either a noncleavable carbamate and triazol or a cleavable −(SS)− linker (Figure 2b). To evaluate the sequence specificity of the anti-acpP PNA, we designed and synthesized scrambled sequences (PNAscr; Nterm-ctctcacattCterm) in which the overall base composition remained the same, but the sequences were not complementary to the selected mRNA target (Figure 2b). For comparison, we also synthesized PNA conjugates (anti-acpP and scrambled PNA sequences) with the most commonly used cell-penetrating peptide (KFF)3K at the N-terminus. This cell-penetrating peptide has been successfully used to achieve PNA uptake in a variety of bacterial species, so it was used as a reference carrier. Inhibition of E. coli Growth by (KFF) 3 K−PNA Conjugates. First, we examined the biostability of vitamin B12−PNA and (KFF)3K−PNA conjugates in vitro in the bacterial Scarlett and Turner medium and Mueller Hinton Broth (MHB), which is of major importance to reach the desired inhibitory effect. The resulting high-performance liquid chromatography (HPLC) chromatograms did not show any detectable differences before and after incubation. Therefore, all conjugates were considered stable in the presence of biological media. Next, we evaluated the potential of the antisense PNA to inhibit bacterial growth by targeting the acpP gene. We cultured bacteria in MHB in the presence of anti-acpP PNA coupled to the (KFF)3K peptide. We observed a concentration-dependent reduction of E. coli growth after overnight incubation. The MIC of (KFF)3K−PNA was 5 μM (Figure 3). At this concentration, the visible growth of E. coli was completely inhibited. To control for the specificity of interactions, we used PNAscr coupled to (KFF)3K. A comparison with (KFF)3K−PNAscr revealed that growth reduction caused by treatment with (KFF)3K−PNA was sequence-specific (Figure 3). No growth inhibition was

Figure 1. Schematic model of PNA and RNA oligomers showing the PNA structure and its complementary base-pairing scheme with RNA.

the vitamin B12 receptors, and the PNA has to interact with its target and cause the desired inhibitory effect. In this work, we have investigated the antibacterial activity of a PNA oligomer conjugated to vitamin B12. The PNA oligomer sequence was targeting the essential acpP gene involved in the fatty acid synthesis pathway in E. coli. Under growth conditions promoting vitamin B12 uptake by E. coli, the antisense PNA inhibited bacterial growth, confirming that vitamin B12 delivers PNA to E. coli cells.

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RESULTS AND DISCUSSION Previously, we investigated the ability of the natural noncationic carrier vitamin B12 to transport PNA oligomers into E. coli cells.20,26 Encouraged by the observation that conjugation of vitamin B12 transports PNA to E. coli cells, we now tested the antibacterial potential of such conjugates using a PNA sequence with antibacterial properties, namely, the PNA aimed to target the mRNA transcript of an essential gene. Such PNA, targeting the expression of an essential protein, should inhibit bacterial growth provided that it is delivered to the cell interior and bound to the target at required concentrations. To test the

Figure 2. (a) Cyanocobalaminthe form of vitamin B12 used in this study and (b) structures, naming, and PNA sequences (marked in red) of vitamin B12−PNA conjugates. The carrier is attached to the N-terminus of PNA, and a lysine is attached at the PNA C-terminus. 820

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To obtain favorable conditions for E. coli to uptake vitamin B12 from the environment, we further used the Scarlett and Turner medium.33 In this broth, ethanolamine is a sole source of nitrogen. Fermentation of ethanolamine by an ammonialyase enzyme to ammonia, acetate, and ethanol requires vitamin B12 as a coenzyme.33−35 Because E. coli is not able to produce vitamin B12, it has to take this nutrient from the environment. However, in such medium, E. coli requires much longer lag phase of growth (up to 48 h) to adapt to the growth conditions. In this period, the individual bacteria mature and not yet able to divide; therefore, the conditions of experiments had to be changed and prolonged to 144 h (details are given in Methods). E. coli Growth Supplied with Vitamin B12 Derivatives. First, we verified if E. coli is able to grow in the Scarlett and Turner medium with the derivatives of vitamin B12 used in this study. We grew bacteria in the presence of natural vitamin B12, B12−(CH2)6−N3 (Figure S2),36 and B12−SS−Py (Figure S3).37 The shortest lag phase of E. coli growth (∼24 h) was when nonmodified vitamin B12 was added (Figure 4). The time

Figure 3. Concentration-dependent growth inhibition of E. coli with the (KFF)3K−PNA conjugate and no growth inhibition with the vitamin B12−PNA conjugates and (KFF)3K−PNAscr in the MHB medium. Error bars represent standard deviation of the mean from three independent experiments. For (KFF)3K−PNA, the differences between 0 and 5, 10, and 20 μM are statistically significant with P < 0.01. For other compounds, the differences between 0 μM and the respective concentrations are not significant with P > 0.05.

achieved by adding (KFF)3K−PNAscr up to the concentration of 20 μM. Furthermore, application of either the free (KFF)3K peptide or PNA sequence without the carrier (KFF)3K peptide, at a concentration up to 20 μM, did not reduce bacterial growth (Figure S1). Therefore, we concluded that the observed inhibitory effect was dependent on the PNA sequence delivered in a conjugate. Inhibition of E. coli Growth by Vitamin B12 Conjugates with Anti-acpP PNA. Under the same conditions as were used for the (KFF)3K−PNA conjugates, we next investigated the ability of vitamin B12 derivatives conjugated to the anti-acpP PNA (Figure 2b) to inhibit E. coli growth. In the compound B12−(CH2)6−PNA, vitamin B12 was conjugated to PNA via a triazole ring, which is generally stable in the bacterial cytoplasm. However, in the second conjugate (B12− SS−PNA, Figure 2b), vitamin B12 was connected to PNA via the disulfide bond that can be reduced by glutathione (GSH), an antioxidant widely distributed in bacteria. For both conjugates, contrary to (KFF)3K−PNA, we did not observe any significant inhibitory effect on E. coli after overnight incubation with a concentration up to 20 μM (Figure 3). We speculate that the MHB medium, used for this study, creates unfavorable environmental conditions for the uptake of vitamin B12 by E. coli cells, thus reducing vitamin B12 ability to deliver PNA. MHB is a nonselective and nondifferential medium used for cultivation of a wide variety of microorganisms. Thus, MHB is a nutritionally rich medium containing, for example, beef infusion and casamino acids as a source of energy and nutrients. The uptake of vitamin B12 in E. coli starts with specific association and binding of vitamin B12 to the outer membrane receptor protein BtuB whose expression is regulated by the btuB riboswitch.24,31 The btuB riboswitch RNA interacts with vitamin B12, as well as its derivatives, leading to RNA structural changes and downregulating the expression of the BtuB transporter to control the cellular levels of B12 vitamers. Thus, at high enough vitamin B12 concentrations, further transport of vitamin B12 in E. coli is precluded.32 Therefore, it seems that in the MHB broth, vitamin B12 uptake was not sufficient to achieve required amounts of PNA inside the cells, presumably because of saturation of the receptors involved in vitamin B12 uptake. As a consequence, we did not observe inhibition of E. coli growth.

Figure 4. Growth of E. coli in the Scarlett and Turner medium with addition of different vitamin B12 derivatives. The prolonged lag phase is visible. Error bars represent standard deviations of the mean from two independent experiments. The differences between all compounds at 144 h are not statistically significant with P > 0.05.

of lag phase with supplementation of vitamin B12−SS−Py was 48 h, and the longest maturing time (∼120 h, Figure 4) that E. coli needed was when the vitamin B12−(CH2)6−N3 derivative was added to the medium. This suggests that E. coli can transport the above modified vitamin B12 from the medium and use it as a nitrogen source, however with different efficiencies. We also confirmed that bacteria do not grow in this medium without addition of any vitamin B12 (data not shown). We next tested the potential of vitamin B12−PNA conjugates to reduce E. coli growth in the conditions that force bacteria to uptake vitamin B12. Because the MIC for (KFF)3K−PNA in the MHB medium was 5 μM, we decided to use this concentration of vitamin B12−PNA conjugates. Both B12− (CH2)6−PNA and B12−SS−PNA inhibited E. coli growth at 5 μM concentrations (Figure 5). The optical density of cultures remained unchanged during the entire experiment, which means that the growth of bacteria was completely inhibited. This again confirms that vitamin B12 carries PNA to the interior of E. coli cells. Similarly, a combination of (KFF)3K− PNA and vitamin B12 in one test tube showed an inhibitory effect at 5 μM concentration of PNA (Figure 5). As controls, we used PNAscr attached to vitamin B12 via −(CH2)6− (Figure 2b) and to (KFF)3K. Analogously, to keep 821

DOI: 10.1021/acsomega.8b03139 ACS Omega 2019, 4, 819−824

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possible induction of resistance,41,42 lack of bacterial specificity of a peptide-mediated delivery system, and incomplete understanding of their uptake mechanism. It was also shown that (KFF)3K may activate an inflammatory response8 and has hemolytic activity at low concentration of 40 μg/mL (for comparison, the antibiotic polymyxin B is hemolytic at concentrations over 1500 μg/mL).19 At least some of the disadvantages of the cationic peptides could be in principle avoided by using vitamin B12. Contrary to (KFF)3K, vitamin B12 is a natural compound. It is not harmful for humans, cannot be overdosed, and does not induce resistance in bacteria. Moreover, vitamin B12 can be chemically modified just in few steps with high synthetic efficacy; the conjugation procedure is simple and high yielding.20,26 Furthermore, some derivatives of vitamin B12 (e.g., derivatives of cobyric acid and cobinamide) are specifically recognized by bacteria, and not by mammalian cells, so they can be applied directly to bacterial cells.43,44 Undeniably, the natural uptake properties of vitamin B12 in different bacteria still need to be further researched; however, all the above should make vitamin B12, at least in principle, a better transporter of antisense PNAs in terms of further medical use. In this study, we demonstrated that vitamin B12 could be considered as a suitable vehicle to be conjugated with PNA to achieve its uptake to E. coli cells in laboratory conditions. We have shown that vitamin B12−PNA conjugates inhibit the growth of E. coli at 5 μM concentration and are stable in the presence of bacterial media up to 168 h. Modifications of vitamin B12 structure influence the use of such vitamin B12 derivatives as essential coenzymes in E. coli. The use of vitamin B12 as a carrier could be thus extended to other antisense oligonucleotides such as 2′OMe RNA45 and locked nucleic acid.

Figure 5. Growth inhibition of E. coli after incubation with vitamin B 12 −PNA anti-acpP conjugates. Controlsconjugates with PNAscrshow no inhibitory effect. Error bars represent the mean ± SD from two independent experiments. Statistically significant differences at 144 h (with P < 0.01) were found for comparison of ((KFF)3K−PNAscr + B12) and ((KFF)3K−PNA + B12), as well as for B12−(CH2)6−PNAscr and other B12−PNA conjugates. In addition, the difference between ((KFF)3K−PNAscr + B12) and B12−(CH2)6− PNAscr is also significant with P < 0.01.

the test conditions identical, (KFF)3K−PNAscr had to be supplemented with vitamin B12 (5 μM). Neither B12−(CH2)6− PNAscr nor (KFF)3K−PNAscr had any inhibitory effect on E. coli growth (Figure 5), confirming the sequence-specific antisense effect of PNA on the expression of the essential acpP gene. Characteristic for the growth of E. coli in this medium is the prolonged lag phase (Figure 5), which is similar to the experiments without adding PNA (Figure 4). The duration of the lag phase during treatment with B12−(CH2)6− PNAscr (Figure 5) is comparable to the lag phase after supplementation with B12−(CH2) 6−N3 (Figure 4). In addition, the maturation time for E. coli incubated with (KFF)3K−PNAscr + B12 (∼48 h, Figure 5) is identical as after incubation with nonmodified vitamin B12 (Figure 4). This suggests adaptation to environmental conditions and lack of inhibitory effect of PNA. We also conclude that conjugation of PNA to vitamin B12 did not affect its antisense properties in E. coli. However, chemical modification of the vitamin B12 structuresuch as attaching spacers (e.g., NH2−(CH2)6− N3)altered the possibility of using vitamin B12 derivatives as essential coenzymes in E. coli. Efficient delivery of PNAs to bacterial cells is a major obstacle, which significantly prevents their clinical application.38 The use of cationic cell-penetrating peptides, such as (KFF)3K, is still the most extensively reported strategy for the delivery of PNA into bacteria.18 Moreover, several studies demonstrated a significant efficacy of PNA conjugated to cellpenetrating peptides in animal infection models.39,40 However, cell-penetrating peptides have many adverse properties such as

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METHODS Reagents and Conditions. Commercial reagents and solvents were used as received. Fmoc-/Bhoc-protected PNA monomers and Fmoc-XAL PEG-PS resin were obtained from Panagene and Merck, respectively. Rink-amide resin (TentaGel S RAM resin) for peptide synthesis and two Nα-Fmocprotected L-amino acids (Fmoc-Phe-OH, Fmoc-Cys(Trt)− OH) were obtained from Sigma-Aldrich. One Nα-Fmocprotected L-amino acid (Fmoc-Lys(Boc)−OH) was purchased from Novabiochem. All reactions and product purities were monitored using reverse-phase (RP)-HPLC. Preparative chromatography was performed using C18 reversed-phase silica gel 90 Å (Sigma-Aldrich) with 0.1% trifluoroacetic acid solutions in redistilled water and HPLC grade with acetonitrile as eluents. HPLC measurement conditions were as follows: columnEurospher II 100-5 C18, 250 mm × 4.6 mm with a

Table 1. HPLC Methods and Retention Times (tR) of the Synthesized PNA Conjugatesa molecular mass [g/mol] b

conjugate name

HPLC tR [min]

HPLC method

calculated

detectedc

B12−(CH2)6−PNA B12−(CH2)6−PNAscr B12−SS−PNAd (KFF)3K−PNAe (KFF)3K−PNAscre

14.9 14.6 17.4 15.6 15.0

0−50%/30 min 10−40%30 min 0−50%/30 min 15−50%/30 min 15−50%/30 min

4343.46 4368.5 4236.90 4577.22 4306.57

4344.24 4369.00 4237.50 4577.26 4306.50

a

B12 stands for vitamin B12. For PNA sequences, see the main text. bThe product was analyzed by analytical RP-HPLC. cdata obtained from QTOF Premier. dMS MALDI-TOF m/z [M − CN]+ calculated and detected. eHPLC and mass spectra in ref 29. 822

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precolumn, or Kromasil C18, 5 μm, 250 mm × 4.0 mm; pressure10 MPa; flow rate1 mL/min; room temperature; detectionUV/vis at wavelengths (λ) of 361 and 267 nm. Preparation of Vitamin B12 Derivatives at the 5′ Position. The NH2−(CH2)6−N3 linker and the vitamin B12 derivative B12−(CH2)6−N3 (Figure S2) were synthesized according to the previously described procedure.36,46 The vitamin B12 derivative B12−SS−Py (Figure S3) was prepared as described in the literature.37 Syntheses of PNA Oligomers, (KFF)3K−N3 and PNA Conjugates with Vitamin B12 and (KFF)3K. PNA oligomers were synthesized manually using Fmoc chemistry as described previously.20,47 Azido-peptide was synthesized by manual solidphase peptide synthesis (SPPS) using the standard Fmoc/t-Bu chemistry according to the known procedure.20,47 The (KFF)3K−PNA and B12−(CH2)6−PNA conjugates were synthesized using copper-catalyzed azide−alkyne cycloaddition according to the procedures described in the literature studies.46,47 The B12−SS−PNA conjugate was prepared by the method reported in the literature.37 All PNAs contained a C-terminal lysine to enhance solubility. The products were purified by RP-HPLC (Table 1). HPLC chromatograms and mass spectra are shown in Section S1 (Figures S4−S6). According to HPLC analyses, the reactions proceeded with >90% conversion. The HPLC analysis of the vitamin B12−PNA and (KFF)3K−PNA products gave m/z values in accordance with their calculated molecular masses. The purity, determined by RP-HPLC (267 and 361 nm), was ≥90% for all the conjugates. To monitor the stability, the vitamin B12−PNA and (KFF)3K−PNA conjugates were added at 50 μM concentrations to Scarlett and Turner medium33 and cation-adjusted MHB.48 After 24 h (in MHB) or 168 h (in the Scarlett and Turner medium) of incubation at 37 °C with shaking, the RPHPLC analyses were performed. Testing of Growth Inhibition. The E. coli K-12 MG1655 strain49 was used. To prepare inocula, bacteria were grown overnight in LB at 37 °C with shaking. To monitor growth inhibition, bacteria were grown in either cation-adjusted MHB (growth medium commonly used for antibiotic susceptibility testing) or the Scarlett and Turner medium at 37 °C with shaking. Overnight cultures of E. coli K-12 MG1655 were diluted 1:99 in appropriate medium and allowed to grow to exponential growth phase. Then bacteria were diluted to ∼1 × 107 CFU/mL in fresh medium. All experiments were performed in sterile glass test tubes. The vitamin B12−PNA and (KFF)3K−PNA conjugates diluted in sterile H2O were added to a final PNA concentration of 5 μM per test tube. Growth rates were determined immediately after adding PNA to bacteria (t0 = 0 h), after 24 h in experiments with the MHB medium (t1 = 24 h) or in 24 h intervals for 144 h in the experiments with the Scarlett and Turner medium (t1; t2 = 48 h; t3 = 72 h; t4 = 96 h; t5 = 120 h; t6 = 144 h). The turbidity of the samples was determined at 600 nm. Experiments were performed in at least two independent biological replicates. For the evaluation of statistical significance, the two-way analysis of variance test following multiple comparisons was used. A probability value of P ≤ 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03139. OD600 after incubation of E. coli with either free PNA or (KFF)3K, structure of vitamin B12−(CH2)6−N3 and of B12−SS−Py, and HPLC chromatograms and mass spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +48223432051 (D.G.). *E-mail: [email protected]. Phone: +48225543683. Fax: +48225540801 (J.T.). ORCID

Dorota Gryko: 0000-0002-5197-4222 Joanna Trylska: 0000-0002-1464-5323 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from the National Science Centre, Poland (SYMFONIA DEC-2014/12/W/ST5/00589) and CeNT BST funds. JT and MR acknowledge support from the Polish-U.S. Fulbright Commission.

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REFERENCES

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DOI: 10.1021/acsomega.8b03139 ACS Omega 2019, 4, 819−824