Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pyogenes

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane.


Introduction
Peptide nucleic acids (PNAs) are synthetic DNA analogues with a pseudo-peptide backbone consisting of N-(2-aminoethyl)glycine units that are linked by amide bonds.Nucleobases are bound to the polyamide backbone via methylene carbonyl linkages.PNAs are able to hybridize with high affinity to complementary DNA and RNA molecules in a sequence-specific and reversible manner.PNA binding is independent of the salt concentration and the molecules are chemically stable [1].Since PNAs are not recognized by proteases and nucleases, they exhibit a high serum stability [2].Strong specific binding in combination with a high stability make PNAs a suitable tool for gene silencing.Antimicrobial activity is achieved by targeting essential bacterial genes.Typical genes are involved in central metabolic pathways that are in many cases also classical targets of conventional antibiotics.Examples include gene coding for subunits of the RNA polymerase (rpoD) or gyrase (gyrA), cell wall synthesis enzymes (murA), or enzymes involved in the synthesis of fatty acids (acpP, fabI) [3][4][5][6][7][8][9].However, surface barriers, including membranes, cell walls, and capsules, are a hindrance to translocation of PNAs into bacteria.Uptake of PNAs into bacteria can be improved by coupling with cell penetrating peptides (CPPs).These molecules have been intensively investigated in the context of cargo delivery into eukaryotic cells [10].
The uptake of CPP-PNA into bacterial cells depends on surface characteristics and therefore differs between species, strains, and even serotypes.CPP-antisense PNAs exhibit antibacterial activity towards bacteria.Over recent years, antisense inhibition of Gram-negative bacteria has been investigated in detail [4].In contrast, the number of CPPs supporting PNA uptake into Gram-positive bacteria is low.CPPs facilitating bacterial PNA uptake have been identified for Listeria monocytogenes and Staphylococcus aureus [3,11].Recently, we identified CPPs efficiently promoting the uptake of antisense PNAs in Streptococcus pyogenes and Streptococcus pneumoniae.We identified three different CPPs that enhance the antibacterial effect of antisense PNAs in S. pyogenes-targeting gyrA: HIV-1 TAT, K8, and (RXR) 4 XB [12].HIV-1 TAT and (RXR) 4 XB were suitable CPPs for antisense PNA delivery in S. pneumoniae [13].
An improved uptake of CPP-antisense PNA conjugates will increase their antibacterial activity and allow the application of lower CPP-PNA concentrations.This may lead, for example, to a considerable cost reduction in gene expression silencing experiments.Addition of counter anions has been shown to facilitate translocation of arginine-rich CPPs across intact lipid bilayer membranes [14,15].One example that has already been studied for this approach is pyrenebutyrate (PyB), which is obtained from 4-(1-pyrenyl)-butyric acid [16,17].PyB is hydrophobic due to its aromatic ring system and has a negatively charged carboxylic group.An improved uptake of oligoarginine-cargo conjugates into various cell lines has been demonstrated using cell fluorometry and confocal microscopy and confirmed by bioactive cargo delivery experiments [16,17].Competition with various ionic species in complex solutions such as media and sera impedes the complex formation of arginine-rich peptides with PyB [17].Therefore, the application of this counterion is limited to in vitro experiments in the absence of complex solutions.
Overall, PyB has been shown to increase the translocation efficacy of arginine-rich CPPcargo conjugates in various cell lines, but translocation of CPP-coupled cargos has not yet been investigated in bacteria.In contrast to mammalian cells, bacteria produce polymeric surface structures that provide barriers to both CPPs and PyB, e.g., peptidoglycan-based cell walls or capsules composed of polysaccharides or proteins.Additionally, the plasma membrane of bacteria differs from the mammalian cell membrane in its transmembrane potential and lipid composition.Bacterial membranes contain a high amount of negatively charged phospholipids such as cardiolipin and phosphatidylglycerol [18], while mammalian membranes are composed of mostly neutral lipids such as cholesterol, phosphatidylcholine and sphingomyelin [19].
These structural differences between mammalian membranes and bacterial surfaces raise the question whether PyB is able to support translocation of peptides into bacterial cells.Here, we investigated the effect of PyB on CPP-coupled antisense PNA activity in the Gram-positive bacteria S. pyogenes and S. pneumoniae.PyB administered alone showed no toxic effect on the bacteria.Pre-incubation of S. pyogenes M49 strain 591 with PyB enhanced the antimicrobial activity of CPP-coupled antisense PNAs.In contrast, the effect of CPP-antisense PNAs in S. pneumoniae was not significantly influenced by PyB.Uptake of K8-FITC by S. pyogenes and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae increased in the presence of PyB.These results indicate that PyB enhances the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by supporting CPP translocation over the bacterial membrane.

PNA Synthesis
CPP-PNAs were synthesized on RinkAM resin using Fmoc chemistry followed by HPLC purification (Peps4LS GmbH, Heidelberg, Germany) [20].The ethylene glycol linker (Fmoc-8-amino-3,6-dioxaoctanoic acid) and the respective Fmoc-protected amino acids for the CPPs were coupled directly to the PNA part on the resin.Sequences of all CPP-PNAs used in this study are listed in Table 1.

Bacterial Killing Assay
S. pneumoniae serotype 4 strain TIGR4: after culturing, S. pneumoniae serotype 4 strain TIGR4 was diluted to approximately 10 5 CFU/mL in phosphate-buffered saline (PBS)/THY (95%/5%).S. pyogenes serotype M49 strain 591: An overnight culture of S. pyogenes serotype M49 strain 591 was diluted to approximately 10 5 CFU/mL in PBS/BHI (70%/30%).An amount of 400 µL of the respective bacterial suspension was transferred to a 2 mL polypropylene reaction tube.4-(1-pyrenyl)-butyric acid (Merck, Darmstadt, Germany) was dissolved to 1 M in DMSO.The solution was stored in the dark and further dilution steps were performed in PBS and 3% DMSO.An amount of 50 µL PyB in PBS/DMSO was added to the bacterial suspension at a final concentration of 50-400 µM and pre-incubated for 10 min at 37 • C and 7 rpm (Rotor SB3, Stuart, Staffordshire, UK).An amount of 50 µL PBS served as a mock control.Following pre-incubation, 50 µL of CPP-PNA solution was added to a final concentration of 5-10 µM or as indicated.An amount of 50 µL molecular grade H 2 O served as a mock control.The reaction tubes were incubated for another 6 h at 37 • C and 7 rpm.Killing kinetics were observed over 12 h.Samples were collected at time points of 2-12 h following addition of CPP-PNAs, and bacteria were plated on BHI solid medium following serial dilution.Agar plates were incubated for 24 h at 37 • C under a 5% CO 2 atmosphere and CFUs were counted.The bacterial count at the beginning of the experiment was 1-3 × 10 5 CFU/mL.Each experiment was conducted at least three times in independent biological replicates.

Fluorescence Peptide Uptake Assay
S. pyogenes, S. pneumoniae, E. coli, or K. pneumoniae, respectively, were pre-incubated with 100 µM PyB.Bacteria were then incubated with 10 µM K8-FITC (Eurogentec, Seraing, BE) for 30 min at 37 • C, washed in PBS, and lysed.For lysis, bacteria were incubated in lysis buffer (Quiagen, Hilden, Germany).S. pyogenes samples were supplemented with Mutanolysin (Merck, Darmstadt, Germany).Cell debris was removed by centrifugation.Afterwards, the fluorescence intensity of cell lysates was measured with a fluorescence microplate reader in arbitrary units at excitation/emission wavelengths of 488 nm/521 nm.Cell lysates of bacteria treated with H 2 O with or without pre-treatment with 100 µM PyB served as background control.

Statistical Analyses
All experiments were performed at least three times or as indicated in the respective figure legends by sample size (n).Statistical significance was determined using the tests indicated in the respective figure legends.Statistical analyses were performed using GraphPad Prism (version 7, GraphPad Software, Boston, MA, USA).

Effects of Pyrenebutyrate on the Activity of CPP-Coupled Antisense PNAs in S. pyogenes and S. pneumoniae
Recently, we identified peptide-coupled antisense PNAs specific for S. pyogenes and S. pneumoniae that exhibited antibacterial activity [12,13].Since hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to support translocation of peptides over membranes, we wanted to investigate whether PyB is able to support uptake of antisense CPP-PNAs into S. pyogenes and S. pneumoniae.PyB has only been studied in eukaryotes but not in bacteria.Therefore, the toxicity of PyB towards bacteria was determined in a bacterial killing assay [23].
A total of 5 × 10 5 CFU/mL S. pyogenes 591 or 8 × 10 5 CFU/mL S. pneumoniae TIGR4 were incubated with PyB concentrations from 50 µM to 200 µM or 50 µM to 400 µM, respectively, for 6 h and the bacterial count was determined.The concentrations tested are based on the doses of PyB applied in similar experiments in cell cultures [17].CFU/mL values following incubation with PyB were compared to an untreated control (Figure 1).No significant toxicity of PyB was observed towards S. pyogenes up to a concentration of 200 µM PyB (Figure 1A).S. pneumoniae was unaffected by PyB up to a concentration of 400 µM (Figure 1B).Since viable counts decreased slightly after treatment of S. pyogenes with 200 µM PyB, 100 µM PyB was used for treatment of S. pyogenes and 200 µM PyB was used for treatment of S. pneumoniae in the following experiments.To study the effects of PyB on the antimicrobial activity of CPP-PNA, bacteria were pre-incubated for 10 min with PyB.
The antisense-PNA target used in this study was the essential gene gyrA, which encodes the subunit A of the DNA gyrase.Peptides were coupled to PNAs via a flexible ethyleneglycol linker (8-amino-3, 6-dioxaoctanoic acid).The sequences of all peptideconjugated PNAs used in this study are listed in Table 1.Antisense PNAs were designed to be complementary to the start region of the target gene.Scrambled control PNAs (scrambled PNAs, scPNAs) are composed of the same base pairs as the antisense PNAs but cannot bind to the target mRNA because of their randomized order.All constructs have been used previously to study the antimicrobial effects of antisense PNAs in the respective streptococci [12,13].
Since previous studies with eukaryotic cells only determined the effects of PyB with arginine-rich CPPs, the numbers of positive amino acid residues of the carriers studied in this work are listed in Table 2 for comparison.The antisense-PNA target used in this study was the essential gene gyrA, which codes the subunit A of the DNA gyrase.Peptides were coupled to PNAs via a flexi ethyleneglycol linker (8-amino-3, 6-dioxaoctanoic acid).The sequences of all peptide-co jugated PNAs used in this study are listed in Table 1.Antisense PNAs were designed be complementary to the start region of the target gene.Scrambled control PNAs (scra bled PNAs, scPNAs) are composed of the same base pairs as the antisense PNAs but c not bind to the target mRNA because of their randomized order.All constructs have be used previously to study the antimicrobial effects of antisense PNAs in the respect streptococci [12,13].
Since previous studies with eukaryotic cells only determined the effects of PyB w arginine-rich CPPs, the numbers of positive amino acid residues of the carriers studied this work are listed in Table 2 for comparison.S. pyogenes M49 strain 591 was incubated for 6 h with CPP-antisense PNA conjuga with or without pre-treatment with 100 µM PyB for 10 min.A reduction in the bacter CFU/mL caused by 5 µM of different CPP-coupled anti-gyrA-PNA constructs compar to the untreated control was observed.Antisense PNAs coupled to three different CP were tested: (RXR)4XB, HIV-1 TAT (TAT), and Oligolysin (K8) (Table 1).The number  S. pyogenes M49 strain 591 was incubated for 6 h with CPP-antisense PNA conjugates with or without pre-treatment with 100 µM PyB for 10 min.A reduction in the bacterial CFU/mL caused by 5 µM of different CPP-coupled anti-gyrA-PNA constructs compared to the untreated control was observed.Antisense PNAs coupled to three different CPPs were tested: (RXR) 4 XB, HIV-1 TAT (TAT), and Oligolysin (K8) (Table 1).The number of CFUs in CPP-PNA-treated samples was significantly reduced in comparison to the untreated control sample (Figure 2A).Pre-treatment with PyB resulted in a greater reduction in bacterial counts.The effect increased as a function of the positive charge of the CPPs.Treatment with K8-anti-gyrA-PNA caused a log CFU reduction of 3.7 that was significantly enhanced to 4.8 following pre-treatment with PyB.
Incubation of S. pneumoniae TIGR4 for 6 h with 10 µM (RXR) 4 XB-and TAT-conjugated anti-gyrA-PNA (Table 1) was performed with or without pre-treatment with 200 µM PyB for 10 min (Figure 2B).As observed before [13], the CPP (RXR) 4 XB supports uptake of antisense PNAs more efficiently than TAT in S. pneumoniae.Pre-treatment with PyB did not significantly enhance the CFU reduction in this experiment.K8-coupled antisense PNA did not affect S. pneumoniae in previous experiments [13].Pre-treatment with PyB was not able to increase the efficacy of this CPP.We also tested Oligoarginine-(R9) and PenArg-coupled anti-gyrA-PNAs, which caused a log CFU reduction of 4 and 3.3, respectively.There was also no significant effect of PyB treatment on the antimicrobial efficacy of these constructs.
A slight, albeit non-significant, improvement in the antimicrobial effect of CPP-PNA on S. pneumoniae mediated by PyB depends on the proportion of positively charged amino acid residues of the carriers.In pneumococci, the activity of CPP-PNAs containing less than 75% arginine and lysine ((RXR) 4 XB and PenArg, Table 2) was not enhanced by PyB, whereas the conjugates TAT-anti-gyrA PNA (75%) and R9-anti-gyrA PNA (100%) (Table 2) showed slightly increased antibacterial activities in the presence of PyB.Overall, PyB did not significantly enhance the antimicrobial activity of CPP-PNA constructs in S. pneumoniae under the conditions used in this study.CFUs in CPP-PNA-treated samples was significantly reduced in comparison to the untreated control sample (Figure 2A).Pre-treatment with PyB resulted in a greater reduction in bacterial counts.The effect increased as a function of the positive charge of the CPPs.
Treatment with K8-anti-gyrA-PNA caused a log CFU reduction of 3.7 that was significantly enhanced to 4.8 following pre-treatment with PyB.Incubation of S. pneumoniae TIGR4 for 6 h with 10 µM (RXR)4XB-and TAT-conjugated anti-gyrA-PNA (Table 1) was performed with or without pre-treatment with 200 µM PyB for 10 min (Figure 2B).As observed before [13], the CPP (RXR)4XB supports uptake of antisense PNAs more efficiently than TAT in S. pneumoniae.Pre-treatment with PyB did not significantly enhance the CFU reduction in this experiment.K8-coupled antisense

Bactericidal Kinetics of CPP-Coupled Antisense PNAs in the Presence of PyB in S. pyogenes
In S. pyogenes, addition of PyB resulted in the significantly increased antimicrobial activity of K8-anti-gyrA PNA.Therefore, the kinetics of the antimicrobial activity of K8conjugated anti-gyrA PNA in the presence of PyB were studied in an in vitro time killing assay in S. pyogenes.Bacteria were treated with 2.5 or 5 µM CPP-antisense PNAs or scrambled control PNAs (Figure 3) with or without pre-treatment with 100 µM PyB.Samples were collected at 2, 4, 6, and 12 h following antisense treatment.CFUs per milliliter were determined by plating of serial dilutions and plotted over time.Untreated bacteria grew by 1-1.5 log CFU over the course of the experiment.Incubation with K8-anti-gyrA PNA resulted in a constant decrease in viable bacteria over time.However, S. pyogenes was not eradicated during the course of the assay (Figure 3A,B).In contrast, following pre-treatment with PyB, application of 5 µM K8-anti-gyrA PNA S. pyogenes CFUs steadily declined until clearance after 12 h (Figure 3B).Following treatment with 2.5 µM K8-conjugated scPNA, no reduction in bacterial counts was observed throughout the experiment.Upon treatment with 5 µM K8-conjugated scPNA, growth was reduced by 0.6 log CFU in comparison to the untreated control, indicating an inhibitory effect of the CPP under these conditions.Overall, pre-treatment with PyB enhanced the activity of K8-anti-gyrA PNA and allowed the eradication of S. pyogenes in vitro.
constructs in S. pneumoniae under the conditions used in this study.

Bactericidal Kinetics of CPP-Coupled Antisense PNAs in the Presence of PyB in S
In S. pyogenes, addition of PyB resulted in the significantly increased ant activity of K8-anti-gyrA PNA.Therefore, the kinetics of the antimicrobial activ conjugated anti-gyrA PNA in the presence of PyB were studied in an in vitro ti assay in S. pyogenes.Bacteria were treated with 2.5 or 5 µM CPP-antisense PNAs bled control PNAs (Figure 3) with or without pre-treatment with 100 µM PyB were collected at 2, 4, 6, and 12 h following antisense treatment.CFUs per mill determined by plating of serial dilutions and plotted over time.Untreated bac by 1-1.5 log CFU over the course of the experiment.Incubation with K8-anti-g resulted in a constant decrease in viable bacteria over time.However, S. pyogen eradicated during the course of the assay (Figure 3A,B).In contrast, following ment with PyB, application of 5 µM K8-anti-gyrA PNA S. pyogenes CFUs steadil until clearance after 12 h (Figure 3B).Following treatment with 2.5 µM K8-c scPNA, no reduction in bacterial counts was observed throughout the experim treatment with 5 µM K8-conjugated scPNA, growth was reduced by 0.6 log CF parison to the untreated control, indicating an inhibitory effect of the CPP un conditions.Overall, pre-treatment with PyB enhanced the activity of K8-anti-g and allowed the eradication of S. pyogenes in vitro.

PyB Supports Transport of K8-FITC Peptides into Bacteria
Since PyB enhanced the antibacterial effect of K8-anti-gyrA PNA in S. pyogenes, we hypothesized that uptake of K8 was facilitated in the presence of PyB.Therefore, we investigated uptake of K8-FITC into S. pyogenes with or without pre-treatment with 100 µM PyB for 10 min.Additionally, to study whether the uptake of K8-FITC is also facilitated by PyB in Gram-negative bacteria, we pre-incubated E. coli and K. pneumoniae with 100 µM PyB for 10 min before incubation with K8-FITC.Bacteria were incubated with 10 µM K8-FITC for 30 min, washed in PBS, and lysed.Subsequently, the fluorescence intensity of the bacterial cell lysates was measured with a fluorescence microplate reader in arbitrary units at excitation/emission wavelengths of 488 nm/521 nm.The fluorescence intensity of cell lysates from S. pyogenes incubated with K8-FITC increased 4.5-fold when the cells were pre-treated with PyB (Figure 4). Figure 3. Killing kinetics of CPP-anti-gyrA PNA treatment in S. pyogenes with or without pre-treat-ment with PyB.(A) Bacterial counts following treatment with 2.5 µM K8-anti-gyrA PNA or 2.5 µM K8-anti-gyrA scPNA, respectively.(B) Bacterial counts following treatment with 5 µM K8-anti-gyrA PNA or 5 µM K8-anti-gyrA scPNA, respectively.Data are presented as means with standard deviation.Sample size: n = 3.

PyB Supports Transport of K8-FITC Peptides into Bacteria
Since PyB enhanced the antibacterial effect of K8-anti-gyrA PNA in S. pyogenes, we hypothesized that uptake of K8 was facilitated in the presence of PyB.Therefore, we investigated uptake of K8-FITC into S. pyogenes with or without pre-treatment with 100 µM PyB for 10 min.Additionally, to study whether the uptake of K8-FITC is also facilitated by PyB in Gram-negative bacteria, we pre-incubated E. coli and K. pneumoniae with 100 µM PyB for 10 min before incubation with K8-FITC.Bacteria were incubated with 10 µM K8-FITC for 30 min, washed in PBS, and lysed.Subsequently, the fluorescence intensity of the bacterial cell lysates was measured with a fluorescence microplate reader in arbitrary units at excitation/emission wavelengths of 488 nm/521 nm.The fluorescence intensity of cell lysates from S. pyogenes incubated with K8-FITC increased 4.5-fold when the cells were pre-treated with PyB (Figure 4).It can therefore be concluded that pre-incubation of S. pyogenes with PyB increases the uptake of PNA conjugates depending on the carrier.The same effect was observed in E. coli and K. pneumoniae, with a fluorescence intensity that increased 6-fold and 5-fold, respectively (Figure 4).These data support the idea that counterions enhance the bacterial uptake of CPP-coupled cargos.

Discussion
Antisense molecules as potential therapeutic agents provide species specificity due to the free choice of the target gene and the sequence variability of a given gene between species.To date, no naturally occurring resistance of bacteria to antisense oligonucleotides has been observed.One advantage of antisense molecules is the possibility of easily modifying the antisense sequence of the molecule once the pathogen in question develops resistance.Antisense-PNAs are potentially interesting antisense compounds, which are currently under intensive investigation.One challenge to their successful application is the It can therefore be concluded that pre-incubation of S. pyogenes with PyB increases the uptake of PNA conjugates depending on the carrier.The same effect was observed in E. coli and K. pneumoniae, with a fluorescence intensity that increased 6-fold and 5-fold, respectively (Figure 4).These data support the idea that counterions enhance the bacterial uptake of CPP-coupled cargos.

Discussion
Antisense molecules as potential therapeutic agents provide species specificity due to the free choice of the target gene and the sequence variability of a given gene between species.To date, no naturally occurring resistance of bacteria to antisense oligonucleotides has been observed.One advantage of antisense molecules is the possibility of easily modifying the antisense sequence of the molecule once the pathogen in question develops resistance.Antisense-PNAs are potentially interesting antisense compounds, which are currently under intensive investigation.One challenge to their successful application is the limited translocation of PNAs into bacteria.CPPs are coupled to many cargos, including PNAs, to enhance their uptake into bacterial and eukaryotic cells [24,25].
The counterion PyB can be employed as a translocation catalyst, accelerating the direct translocation of arginine-rich CPPs in various cell lines [16,17].In artificial model membranes, the effects of PyB on R8 translocation were studied.PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8.PyB also increased the fluidity of the negatively charged membranes and induced membrane curvature [26].In this work, the effect of PyB on the antimicrobial activity of CPP-PNAs in bacteria was investigated.In the presence of PyB, uptake of K8-FITC conjugates into S. pyogenes, E. coli, and K. pneumoniae was strongly elevated, and the antibacterial effect of CPP-PNA on S. pneumoniae and S. pyogenes was increased.Thus, this work demonstrated that the enhancing impact of PyB on translocation is not limited to eukaryotes.PyB is also effective in bacteria, despite differences in membrane composition and charge and the presence of bacterial barriers such as the peptidoglycan layer, LPS, and capsules.The effective concentration of PyB in the tested streptococcal species was comparable to those used in cells [16,17].PyB alone had neither intrinsic effects on bacterial viability at the concentrations used nor did it increase the toxic effects of CPP-scPNA controls.
The strongest effect of PyB on the antimicrobial activity of CPP-PNAs was observed with K8-anti gyrA PNA in S. pyogenes.Until now, it was assumed that the guanidinium cations of arginine residues of CPPs are particularly important for improved uptake via counter anions [26][27][28].Oligolysine K8 is a cationic CPP without guanidinium moieties.Therefore, interactions of PyB with guanidinium cations of the CPPs do not appear to be essential to increase the translocation of CPPs into bacteria.It seems likely that the interaction of positively charged CPPs with negatively charged PyB is sufficient to increase the accumulation of CPPs on the membrane surface, thereby increasing the efficiency of CPP uptake.Furthermore, it has been shown that PyB enhances the direct translocation of oligoarginines through artificial membranes not only directly due to electrostatic interaction with the CPP, but also indirectly by increasing membrane fluidity and the formation of membrane curvatures [26].Eukaryotic cell and bacterial membranes are not homogenous in composition and charge, but some areas are more fluid than others.As a rule, the translocation rate of cationic CPPs is higher in fluid membrane domains [29][30][31].Incubation with PyB was shown to disperse phase separation and increase the fluidity of negatively charged artificial membranes, which might result in an enhanced translocation of cationic CPPs.Additionally, PyB caused formation of inward tubular structures in artificial membranes, which disappeared upon addition of oligoarginine.Formation of curvatures and recovery might enhance the uptake of CPPs [26].Whether one of these effects of PyB demonstrated with artificial membranes or the combination of these effects enhances uptake of positively charged oligopeptides in bacteria is not known.It has also not yet been tested whether PyB increases the uptake of oligolysine not only in S. pyogenes but also in eukaryotic cells.
The effect of PyB was stronger in S. pyogenes than in pneumococci.The efficiency of different carriers varies generally between the two streptococcal species, for example, K8 is a very efficient CPP in S. pyogenes but does not support translocation in pneumococci [13].Surface structures such as the capsule play a role in the uptake of carriers [6,[32][33][34] and might also influence the activity of the counterion.Pneumococci generally have a thick capsule, and particularly the capsule of S. pneumoniae TIGR4 is quite wide at 185 nm [35,36].In addition to thickness, the capsules of individual streptococcal species also differ in composition.S. pyogenes capsules are composed of hyaluronic acid (N-acetlyglucosamine and glucuronic acid), whereas pneumococcal capsules consist of 2-8 saccharides that are often substituted with O-acetyl, phosphoglycerol, or pyruvyl acetal residues [37][38][39][40].
PyB application is limited by the fact that CPP delivery is not efficiently supported in the presence of culture medium or serum.It has been speculated that ionic species in the medium compete with the carrier for interaction with PyB [17].Use of a counterion is consequently restricted to in vitro approaches, in which the experimental conditions can be adapted in a way that the CPP-PNA uptake can be increased.If the required concentration of CPP-PNA can be decreased, the expenses for in vitro studies of the antisense PNA function will be reduced.To enable the use of PyB in vivo, Mishra and coworkers tested an approach in which they covalently coupled PyB to a CPP-cargo molecule.Compared to CPP alone, the uptake of the PyB-CPP-cargo conjugates into cells was increased 4-5-fold and was shown to be independent of the medium [41].In future experiments, it could be tested whether linking PyB to CPP-PNA might result in a conjugate with an improved activity in bacteria.
The fluorescence intensity of cell lysates from S. pyogenes, E. coli, and K. pneumoniae incubated with K8-FITC increased 4-fold, 6-fold, and 5-fold, respectively, when the cells were pre-treated with PyB.A similar effect of PyB has been observed on the uptake of GABA-FITC-labelled R9 peptide in HeLa cells [16].In the same study, the impact of PyB on the relative uptake of labelled pTat 48-60 and Pen was much lower.In conclusion, the efficacy of the translocation-enhancing effect of PyB depends on the properties of the target cell as well as on the nature of the CPP used.Based on our results, it should be investigated whether PyB is able to enhance the uptake of CPP-PNAs in Gram-negative bacteria.

Conclusions
Overall, we demonstrated that PyB supports the antimicrobial effect of CPP-coupled antisense PNAs in S. pyogenes.The effect of PyB was not limited to arginine-rich CPPs, but PyB was able to enhance the activity of PNAs coupled to lysine-rich CPPs.Pretreatment of S. pyogenes, E. coli, and K. pneumoniae with PyB improved the cellular uptake of FITClabelled K8.Together with data from previous studies in eukaryotic cells and artificial membranes [14,16,17], these results indicate that PyB directly interacts with the bacterial membrane and thereby facilitates CPP uptake.In future studies, more potential enhancing counterions should be identified, which may promote membrane translocation in the presence of complex media and sera.Alternatively, covalent coupling of PyB to CPPs could be investigated as a measure to optimize PyB-supported uptake.

Figure 1 .
Figure 1.Toxicity of PyB to S. pyogenes and S. pneumoniae.(A) Treatment of S. pyogenes with 50-µM PyB for 6 h.(B) Treatment of S. pneumoniae with 50-400 µM PyB for 6 h.Data are presented mean values with standard deviation.Statistical significance was determined by a one-w ANOVA, multiple comparisons.Sample size: n = 4.

Figure 1 .
Figure 1.Toxicity of PyB to S. pyogenes and S. pneumoniae.(A) Treatment of S. pyogenes with 50-200 µM PyB for 6 h.(B) Treatment of S. pneumoniae with 50-400 µM PyB for 6 h.Data are presented as mean values with standard deviation.Statistical significance was determined by a one-way ANOVA, multiple comparisons.Sample size: n = 4.

Figure 2 .
Figure 2. Reduction in bacterial CFU/mL following treatment with CPP-antisense PNAs for 6 h with or without pre-treatment with PyB.(A) Bacterial counts following treatment of S. pyogenes with 5 µM CPP-anti-gyrA PNA or 5 µM CPP-anti-gyrA scPNAs.(B) Bacterial counts following treatment of S. pneumoniae with 10 µM CPP-anti-gyrA PNA or 10 µM CPP-anti-gyrA scPNAs.Data are presented as mean values with standard deviation.Statistical significance was determined by a oneway ANOVA, multiple comparisons.Differences between PNA conjugate samples and mock control (untreated) were expressed as p ≤ 0.05 (*), p ≤ 0.01 (**) or p ≤ 0.0001 (****).Differences between PNA conjugate samples in the presence of PyB and PNA conjugate samples without PyB were expressed as p ≤ 0.05 (#).Sample size: n = 5.

Figure 2 .
Figure 2. Reduction in bacterial CFU/mL following treatment with CPP-antisense PNAs for 6 h with or without pre-treatment with PyB.(A) Bacterial counts following treatment of S. pyogenes with 5 µM CPP-anti-gyrA PNA or 5 µM CPP-anti-gyrA scPNAs.(B) Bacterial counts following treatment of S. pneumoniae with 10 µM CPP-anti-gyrA PNA or 10 µM CPP-anti-gyrA scPNAs.Data are presented as mean values with standard deviation.Statistical significance was determined by a one-way ANOVA, multiple comparisons.Differences between PNA conjugate samples and mock control (untreated) were expressed as p ≤ 0.05 (*), p ≤ 0.01 (**) or p ≤ 0.0001 (****).Differences between PNA conjugate samples in the presence of PyB and PNA conjugate samples without PyB were expressed as p ≤ 0.05 (#).Sample size: n = 5.

Table 2 .
Positive amino acid residues per CPP.

Table 2 .
Positive amino acid residues per CPP.