Identifying Cell-Penetrating Peptides for Effectively Delivering Antimicrobial Molecules into Streptococcus suis

Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. Streptococcus suis, a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into S. suis cells: HIV-1 TAT efficiently penetrated S. suis cells with low toxicity against S. suis; (RXR)4XB had high penetration efficiency with inherent toxicity against S. suis; (KFF)3K showed lower penetration efficiency than HIV-1 TAT and (RXR)4XB; K8 failed to penetrate S. suis cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti-gyrA PNA) effectively inhibited the growth of S. suis. TAT-anti-gyrA PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of S. suis, which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating S. suis infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells.

To determine whether the CPPs K8, (KFF) 3 K, HIV-1 TAT, and (RXR) 4 XB listed in Table 1 can enter S. suis cells, we initially utilized super-resolution structured illumination microscopy (SR-SIM) to observe the intracellular localization of FITC-labeled CPPs in the S. suis cells.SR-SIM achieves higher spatial resolution than traditional microscopes through structured illumination patterns, allowing for the observation of smaller-scale bacterial structures and details.As shown in Figure 1, the S. suis cell membrane and FITC-labeled CPPs were observed with lasers at 640 nm (WGA, red) and 488 nm (FITC, green), respectively.Upon combined observation of WGA and FITC fluorescence signals, SIM images demonstrated that peptides HIV-1 TAT and (RXR) 4 XB at a concentration of 10 µM were able to efficiently cross the S. suis cell membrane and distribute within the cells (Merge).In contrast, (KFF) 3 K shows lower penetration efficiency than HIV-1 TAT and (RXR) 4 XB, while K8 fails to penetrate S. suis cells.However, we observed that (RXR) 4 XB appeared to possess a membrane-disruptive effect on S. suis, leading to a substantial accumulation of FITC-labeled (RXR) 4 XB within the cells.In contrast, HIV-1 TAT exhibited a more even intracellular distribution.suis cell membrane was counterstained with Alexa Fluor 633-WGA (wheat germ agglutinin) and observed with a laser at a wavelength of 640 nm (WGA, red), while the fluorescence signal of FITC was observed using a laser at a wavelength of 488 nm (FITC, green).

CPPs Uptake Efficiency Analysis by Flow Cytometry
To assess the uptake efficiency of K8, (KFF)3K, HIV-1 TAT, and (RXR)4XB in S. suis cells, we exposed S. suis serotype 2 strain SC070731 cells to 10 µM FITC-labeled CPPs and measured fluorescence using flow cytometry after one hour of treatment.A total of 50,000 events were collected, and over 95% of bacterial cells were acquired and analyzed by gating on side scatter (SSC) and forward scatter (FSC).As shown in Figure 2A, bacterial cells exhibited varying fluorescence intensities after treatment with different CPPs.The Figure 1.Representative images were used for analyzing the internalization efficiency of FITC-labeled CPPs in S. suis by SR-SIM.The negative control received an equivalent volume of water, while an equivalent concentration of FITC single molecules was also included as a control.The S. suis cell membrane was counterstained with Alexa Fluor 633-WGA (wheat germ agglutinin) and observed with a laser at a wavelength of 640 nm (WGA, red), while the fluorescence signal of FITC was observed using a laser at a wavelength of 488 nm (FITC, green).

CPPs Uptake Efficiency Analysis by Flow Cytometry
To assess the uptake efficiency of K8, (KFF) 3 K, HIV-1 TAT, and (RXR) 4 XB in S. suis cells, we exposed S. suis serotype 2 strain SC070731 cells to 10 µM FITC-labeled CPPs and measured fluorescence using flow cytometry after one hour of treatment.A total of 50,000 events were collected, and over 95% of bacterial cells were acquired and analyzed by gating on side scatter (SSC) and forward scatter (FSC).As shown in Figure 2A, bacterial cells exhibited varying fluorescence intensities after treatment with different CPPs.The baseline fluorescence threshold of bacterial cells was determined by the negative control (water) and set as the negative confidence area (quadrant 3).S. suis cells treated with 10 µM FITC molecule, K8, (KFF) 3 K, HIV-1 TAT, or (RXR) 4 XB mainly clustered in quadrant 4, accounting for 64.80%, 71.20%, 98.90%, 99.80%, and 99.90% of total cells, respectively (Figure 2A).As shown in Figure 2B, S. suis cells treated with (KFF) 3 K, HIV-1 TAT, or (RXR) 4 XB exhibited an unimodal distribution in the histogram.The peaks of (RXR) 4 XB, HIV-1 TAT, and (KFF) 3 K were shifted to the right compared to those of the negative control and FITC molecule.The peak of (RXR) 4 XB was positioned furthest to the right, suggesting the most vigorous fluorescence intensity, followed by HIV-1 TAT and (KFF) 3 K.In contrast, K8 exhibited the weakest fluorescence intensity.We analyzed the mean fluorescence intensity (MFI) of S. suis cells using FlowJo TM v10 software.Significant differences existed in the MFI of S. suis cells treated with 10 µM of different FITC-labeled CPPs.We found that (RXR) 4 XB exhibited the highest MFI, followed by HIV-1 TAT.In contrast, (KFF) 3 K showed a lower MFI than HIV-1 TAT, while K8 possessed the lowest MFI (Figure 2C).The results from flow cytometric analysis concurred with the observations from SR-SIM.These findings indicate that (RXR) 4 XB and HIV-1 TAT can efficiently penetrate S. suis cells.However, (KFF) 3 K displayed a lower penetration efficiency than (RXR) 4 XB and HIV-1 TAT, while K8 displayed a lowest penetration efficiency among them.Notably, the MFI of (RXR) 4 XB was significantly higher than that of HIV-1 TAT.This phenomenon might be associated with the potential cell membrane disruption effect of (RXR) 4 XB on S. suis cells observed by SR-SIM.To select suitable CPPs, we must balance their cell penetration efficiency and toxicity to target cells.Therefore, for CPPs with high penetration efficiency, further analysis of their toxicity to target cells is crucial.
contrast, (KFF)3K showed a lower MFI than HIV-1 TAT, while K8 possessed the lowest MFI (Figure 2C).The results from flow cytometric analysis concurred with the observations from SR-SIM.These findings indicate that (RXR)4XB and HIV-1 TAT can efficiently penetrate S. suis cells.However, (KFF)3K displayed a lower penetration efficiency than (RXR)4XB and HIV-1 TAT, while K8 displayed a lowest penetration efficiency among them.Notably, the MFI of (RXR)4XB was significantly higher than that of HIV-1 TAT.This phenomenon might be associated with the potential cell membrane disruption effect of (RXR)4XB on S. suis cells observed by SR-SIM.To select suitable CPPs, we must balance their cell penetration efficiency and toxicity to target cells.Therefore, for CPPs with high penetration efficiency, further analysis of their toxicity to target cells is crucial.

Toxicity Analysis of HIV-1 TAT and (RXR) 4 XB
We determined the minimum inhibitory concentrations (MICs) of HIV-1 TAT and (RXR) 4 XB, which exhibited high penetration efficiency in S. suis cells, using S. suis serotype 2 strain SC070731.We found that HIV-1 TAT did not induce significant growth inhibition in SC070731 (MIC > 128 µM; Figure 3A).However, (RXR) 4 XB resulted in growth inhibition (MIC of 4 µM; Figure 3B).We investigated the concentration-dependent bactericidal activity of HIV-1 TAT or (RXR) 4 XB by treating S. suis serotype 2 strain SC070731 with concentrations ranging from 8 to 64 µM.The results revealed no reduction in bacterial counts after HIV-1 TAT incubation within the 8 to 64 µM range.Conversely, a significant decrease in bacterial counts was observed after (RXR) 4 XB incubation within the 8 to 64 µM range (Table 2).Thus, (RXR) 4 XB possesses inherent toxicity against S. suis, whereas HIV-1 TAT is not.(MIC of 4 µM; Figure 3B).We investigated the concentration-dependent bactericidal activity of HIV-1 TAT or (RXR)4XB by treating S. suis serotype 2 strain SC070731 with concentrations ranging from 8 to 64 µM.The results revealed no reduction in bacterial counts after HIV-1 TAT incubation within the 8 to 64 µM range.Conversely, a significant decrease in bacterial counts was observed after (RXR)4XB incubation within the 8 to 64 µM range (Table 2).Thus, (RXR)4XB possesses inherent toxicity against S. suis, whereas HIV-1 TAT is not.CFU, colony-forming units.The count of surviving bacteria after treatment with CPPs was compared with that of water using an unpaired t-test.A summary of the p-values is provided, with an asterisk indicating a significant difference (p < 0.05) and 'ns' denoting no significant difference.

HIV-1 TAT-Coupled gyrA-Specific PNA Exhibits Bactericidal Activity
To assess the capability of HIV-1 TAT to deliver cargo molecules into S. suis, we utilized a widely used gyrA-specific antisense PNA as the delivered molecule.The gyrA-

HIV-1 TAT-Coupled gyrA-Specific PNA Exhibits Bactericidal Activity
To assess the capability of HIV-1 TAT to deliver cargo molecules into S. suis, we utilized a widely used gyrA-specific antisense PNA as the delivered molecule.The gyrAspecific antisense PNA fully complements the 12 nucleotides surrounding the gyrA mRNA start codon (Figure 4A), a sequence highly conserved among different serotypes of S. suis (Figure 4B).HIV-1 TAT is the carrier, conjugated to the 5 ′ end of the gyrA-specific antisense PNA via the linker 8-amino-3,6-dioxaoctanoic acid.
Antibiotics 2024, 13, x FOR PEER REVIEW 6 of 14 specific antisense PNA fully complements the 12 nucleotides surrounding the gyrA mRNA start codon (Figure 4A), a sequence highly conserved among different serotypes of S. suis (Figure 4B).HIV-1 TAT is the carrier, conjugated to the 5′ end of the gyrA-specific antisense PNA via the linker 8-amino-3,6-dioxaoctanoic acid.The effectiveness of HIV-1 TAT as a PNA carrier targeting S. suis was assessed by evaluating the bactericidal activity.The results revealed that free peptide or PNAs controls did not induce significant growth inhibition in S. suis serotype 2 strain SC070731 (MIC > 32 µM; Figure 5A).However, HIV-1 TAT-coupled gyrA-specific PNA (TAT-anti-gyrA PNA) led to efficient growth inhibition (MIC of 4 µM; Figure 5A).To clarify whether the observed growth inhibition of TAT-anti-gyrA PNA on S. suis serotype 2 strain SC070731 is bacteriostatic or bactericidal, we conducted a 4-h bactericidal assay using 1 × MIC and 2 × MIC concentrations against 1 × 10 5 CFU/mL of bacteria.As shown in Figure 5B, colony-forming units (CFU) counting on THA plates revealed that 4 µM and 8 µM of TAT-anti-gyrA PNA significantly reduced CFU counts, with a noticeable decrease The effectiveness of HIV-1 TAT as a PNA carrier targeting S. suis was assessed by evaluating the bactericidal activity.The results revealed that free peptide or PNAs controls did not induce significant growth inhibition in S. suis serotype 2 strain SC070731 (MIC > 32 µM; Figure 5A).However, HIV-1 TAT-coupled gyrA-specific PNA (TAT-anti-gyrA PNA) led to efficient growth inhibition (MIC of 4 µM; Figure 5A).To clarify whether the observed growth inhibition of TAT-anti-gyrA PNA on S. suis serotype 2 strain SC070731 is bacteriostatic or bactericidal, we conducted a 4-h bactericidal assay using 1 × MIC and 2 × MIC concentrations against 1 × 10 5 CFU/mL of bacteria.As shown in Figure 5B, colony-forming units (CFU) counting on THA plates revealed that 4 µM and 8 µM of TAT-anti-gyrA PNA significantly reduced CFU counts, with a noticeable decrease observed as early as 30 min post-treatment.As shown in Figure 5C, a significant reduction in bacterial numbers was observed for serotypes 2, 4, 5, 7, and 9 strains of S. suis exposed to TAT-anti-gyrA PNA after 2 h, indicating a bactericidal effect of TAT-anti-gyrA PNA on various serotypes of S. suis.

Discussion
ASOs, primarily comprising antisense PNAs and PMOs, are a type of synthetic noncytotoxic nucleic acid derivative known as antisense antimicrobial molecules [8,38].PNAs, constructed by attaching nucleobases to a modified polyamide backbone, are resistant to nucleases and proteases, maintaining high stability in complex biological environments, including human serum and cell extracts [13,39].Their uncharged nature

Discussion
ASOs, primarily comprising antisense PNAs and PMOs, are a type of synthetic noncytotoxic nucleic acid derivative known as antisense antimicrobial molecules [8,38].PNAs, constructed by attaching nucleobases to a modified polyamide backbone, are resistant to nucleases and proteases, maintaining high stability in complex biological environments, including human serum and cell extracts [13,39].Their uncharged nature contributes to their high affinity for mRNA [13].Upon delivery to the cellular target, they can leverage Watson-Crick nucleic acid base pairing to function as translation inhibitors by sterically hindering the ribosome-mRNA interaction, thereby silencing the expression of specific genes [8,13].However, due to the selective permeability of cell membranes, PNAs are usually unable to enter bacterial cells on their own.Therefore, PNAs are typically conjugated to CPPs to facilitate uptake into bacterial cells [8,40].Several studies have reported that the essential gyrA gene has been successfully targeted using PNAs delivered by CPPs such as K8, (KFF) 3 K, HIV-1 TAT, and (RXR) 4 XB, demonstrating antibacterial effects in both Gram-positive and Gram-negative organisms.(KFF) 3 K-coupled gyrA-specific PNAs have shown antimicrobial effects in S. pyogenes [18,19], S. aureus [41,42], and A. baumannii [43]; HIV-1 TAT and (RXR) 4 XB have been identified as valuable carriers for gyrA-specific PNAs in S. pyogenes [18,19] and S. pneumoniae [9]; K8-coupled gyrA-specific PNAs have shown antimicrobial effects in S. pyogenes but did not demonstrate bactericidal activity in S. pneumoniae [9,18].Various CPPs demonstrate different efficiencies in delivering PNAs, and the same CPPs may exhibit varying delivery efficiencies in different bacterial species.To achieve the optimal antibacterial effect of CPP-ASO conjugates, selecting suitable CPPs specific to the targeted bacterial species is crucial.
Previous research has indicated that arginine-rich polymers exhibit significantly enhanced cellular uptake compared to polymers of similar length containing lysine [44].This study demonstrated the efficient penetration of S. suis cells by arginine-rich CPPs HIV-1 TAT and (RXR) 4 XB.In contrast, (KFF) 3 K shows lower penetration efficiency than HIV-1 TAT and (RXR) 4 XB, while K8 fails to penetrate S. suis cells.Although (RXR) 4 XB demonstrates high efficiency in penetrating S. suis cells, it exerts considerable toxicity towards the bacteria.The length of peptides is a crucial factor influencing their cellular uptake capability.Mitchell et al. reported that the penetration efficiency of arginine-rich polymers with fewer than five amino acids is notably lower than that of peptides containing six or more amino acids [44].As the peptide length extends to 15 amino acids, penetration ability increases; however, cell toxicity rises concurrently [44].The toxicity exhibited by (RXR) 4 XB in S. suis may be associated with its length.CPPs as delivery vectors should ideally not exhibit toxicity towards target bacterial cells [40].The current primary research approach involves selecting various CPPs to conjugate with ASOs, evaluating their antibacterial efficacy by determining the MIC values of CPP-ASO conjugates, and assessing the ability of the chosen CPPs to deliver antimicrobial agents to specific bacterial species [18,20,45].While this method is effective in screening CPP-ASO conjugates with antibacterial effects, it requires the evaluation of a significant number of conjugates.It may even require the conjugation of peptides with ASOs targeting different genes, thereby significantly amplifying the complexity and cost of the screening process.Here, by combining SR-SIM, flow cytometry, and toxicity analysis assays, we determined that HIV-1 TAT efficiently penetrates S. suis cells with low toxicity.HIV-1 TAT-conjugated PNA specific for gyrA effectively inhibited the growth of S. suis.Our findings demonstrate that CPP-PNA conjugates can be promising new antimicrobial compounds against S. suis infections and offer convenient, intuitive, and cost-effective methods for screening suitable CPPs to deliver cargo molecules into targeted bacterial cells.
Another innovative synthetic ASO strategy is targeting non-essential genes, such as genes involved in antibiotic resistance and virulence [13].The strategy of targeting antibiotic resistance genes aims to decrease the expression of antibiotic resistance, thereby restoring susceptibility to an approved antibiotic that would be co-administered with the oligomer.Recently, it has been shown in various bacterial species that utilizing CPP-ASO conjugates to target antibiotic resistance genes can reinstate the susceptibility of resistant strains to standard antibiotics [46][47][48][49].Moreover, suppressing the expression of virulence genes is regarded as a promising alternative antibacterial therapy compared to conventional methods [50][51][52].This method aims to inhibit the expression of crucial virulence factors in pathogens, making them more susceptible to clearance by the host immune system.Furthermore, anti-virulence agents impose less selective pressure on the pathogen, thus decreasing the emergence of drug-resistant mutants compared to traditional antimicrobial drugs [50, [52][53][54].
In addition to ASOs, CPPs have great potential for delivering various cargo molecules, including peptides and foreign DNA, to bacterial cells.In a recent study, Islam et al. demonstrated the transfer of large plasmid DNA into Escherichia coli cells through CPPmediated delivery [55].This delivery method is recognized for safeguarding the integrity of nucleic acid and is considered suitable for transporting large nucleic acid molecules [56].Although electroporation and pheromone-induced natural transformation were used to introduce foreign DNA into S. suis cells [57,58], for some S. suis clinical isolates, it remains challenging to introduce foreign DNA into cells.The possibility of using CPPs to introduce foreign DNA into S. suis deserves further investigation.
In conclusion, our study has shown the potential of HIV-1 TAT as a delivery vehicle for antimicrobial molecules into S. suis cells and has demonstrated that using CPP-ASO conjugates represents a promising approach for combating S. suis infections.Future research should investigate more potential CPPs and target genes to identify antisense antimicrobial compounds with high antibacterial activity against S. suis.Additionally, our results underscore that SR-SIM and flow cytometry analysis serve as valuable tools for assessing the penetration efficiency of CPPs in S. suis, providing a convenient, intuitive, and cost-effective reference method for screening suitable CPPs with high penetration efficiency to deliver cargo molecules into bacteria.

Bacterial Strains and Culture Conditions
The following S. suis strains were used in the study: serotype 2 virulent strain SC070731, isolated from a pig with meningitis [59]; serotype 4 virulent strain ND90, isolated from a diseased pig [28]; serotype 5 strain GX169, isolated from a human patient and kindly provided by Dr. Han Zheng, Chinese Center for Disease Control and Prevention, China; serotype 7 virulent strain WUSS013, isolated from a diseased pig [30]; serotype 9 virulent strain GZ0565, isolated from a pig with meningitis [60].All strains were streaked on Todd-Hewitt agar (THA) containing 5% (v/v) sheep blood and cultured in Todd-Hewitt broth (THB) at 37 • C with 5% CO 2 .

CPPs Synthesis and Fluorescent Labeling
All peptides in this study, listed in Table 1, were synthesized and purified using highperformance liquid chromatography (HPLC purity ≥ 95%) provided by GenScript Inc. (Nanjing, China).A fluorescein moiety (FITC) was attached to the N-terminus of peptides via a 6-aminohexanoic acid spacer (GenScript Inc.), and all FITC-labeled peptides were dissolved in ultrapure water.

SR-SIM Analysis
S. suis serotype 2 strain SC070731 was streaked onto THA plates and incubated at 37 • C with 5% CO 2 for 12 h.A bacterial colony was picked and inoculated overnight in 2 mL of THB at 37 • C. The overnight culture was then diluted 100-fold in fresh THB and grown to an OD 600nm of 0.6 (approximately 3 × 10 8 CFU/mL).The obtained culture was washed twice with PBS and resuspended in PBS.Further, 900 µL of the bacterial solution was transferred into a 2 mL tube.Immediately, 100 µL of a 10× FITC-labeled CPPs working solution was added to a final concentration of 10 µM.Controls were prepared using an equivalent volume of water or a 10× FITC molecule solution.The reaction tubes were incubated for one hour at 37 • C and 10 rpm rotation in the dark.The bacterial cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and washed again before resuspending in PBS.They were then incubated with 5 µg/mL Alexa Fluor 633-WGA (wheat germ agglutinin) purchased from Thermo Fisher for 30 min at room temperature in the dark.The cells were then washed twice and resuspended in PBS.Subsequently, 3 µL of bacterial solution was applied onto a microscope slide and dried using an alcohol lamp.A droplet of ProLong TM Diamond antifade mountant (Thermo Fisher, Waltham, MA, USA) was added next.The microscope slide was then covered with a cover slip, and fluorescent cells were observed using SR-SIM (Nikon, Tokyo, Japan).The fluorescence emitted by the 640 nm laser was used to analyze the cell membrane stained with Alexa Fluor 633-WGA.In contrast, the fluorescence emitted by the 488 nm laser was used to analyze the distribution of FITC-labeled CPPs in bacterial cells.Each experiment has been performed on at least three independent biological replicates.

Flow Cytometry Analysis
As mentioned, the bacterial cells were treated with FITC-labeled CPPs and fixed with 4% paraformaldehyde.To analyze the cell membrane penetration ability of the CPPs, flow cytometry analysis was performed using a FACSVerse TM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) equipped with a 488 nm laser for FITC detection.Bacterial cells in suspension were analyzed at a 200-300 events/s flow rate, and 50,000 events were recorded for analysis.The acquisition and analysis were performed by gating on side scatter (SSC) and forward scatter (FSC).The mean fluorescence intensity of FITC was analyzed using FlowJo TM v10 software (BD Biosciences).Each experiment has been performed on at least three independent biological replicates.

Synthesis of PNAs and CPP-PNA Conjugates
PNAs and CPP-PNA conjugates, as listed in Table 1, were obtained from PANAGENE Inc. (Daejeon, Republic of Korea).The PNAs contained a sequence complementary to the mRNA of the target gene for the gyrase A subunit (gyrA), a cellular target for quinolone antibiotics [61].The peptide HIV-1 TAT was attached to gyrA-specific PNAs via the linker 8-amino-3,6-dioxaoctanoic acid.The quality and purity of these constructs were confirmed to be sufficient through HPLC analysis (purity > 99%) and mass spectrometry.The PNAs and CPP-PNA conjugates were dissolved in ultrapure water.The absorbance at 260 nm of these solutions was measured using a NanoDrop spectrophotometer, and the concentrations were then calculated using the extinction coefficient based on recently published methods [23,62].

MIC Determination
The broth microdilution method was employed to determine the MIC values, following a previous study and a recently published protocol with a few modifications [23,62].In brief, an overnight S. suis serotype 2 strain SC070731 culture was diluted 100-fold in fresh THB and grown to an OD 600nm of 0.6.Subsequently, the bacterial suspension was further diluted 2000-fold in fresh Mueller-Hinton broth (MHB) to a final concentration of approximately 1 × 10 5 CFU/mL.Then, 180 µL of the bacterial solution was transferred into a 96-well plate.Immediately, 20 µL of a 10× peptides, PNAs, or CPP-PNA conjugates working solution was added.As a negative control, an equivalent volume of water was added instead.The plate was then incubated at 37 • C with 5% CO 2 for 18 h, and the growth was periodically monitored by measuring the OD 595nm every hour using a BioTek ELx800 plate reader.The MIC value was determined as the lowest concentration inhibiting visible growth in the wells (OD 595nm < 0.1).

Figure 1 .
Figure 1.Representative images were used for analyzing the internalization efficiency of FITClabeled CPPs in S. suis by SR-SIM.The negative control received an equivalent volume of water, while an equivalent concentration of FITC single molecules was also included as a control.The S. suis cell membrane was counterstained with Alexa Fluor 633-WGA (wheat germ agglutinin) and observed with a laser at a wavelength of 640 nm (WGA, red), while the fluorescence signal of FITC was observed using a laser at a wavelength of 488 nm (FITC, green).

Figure 2 .
Figure 2. Cellular uptake of CPPs in S. suis was analyzed by flow cytometry.S. suis cells were exposed to 10 µM FITC-labeled CPPs, with fluorescence measured one hour post-treatment.The negative control (NC) received an equivalent volume of water, while an equivalent concentration of FITC single molecules was also included as a control.A total of 50,000 events were collected during

Figure 2 .
Figure 2. Cellular uptake of CPPs in S. suis was analyzed by flow cytometry.S. suis cells were exposed to 10 µM FITC-labeled CPPs, with fluorescence measured one hour post-treatment.The negative control (NC) received an equivalent volume of water, while an equivalent concentration of FITC single molecules was also included as a control.A total of 50,000 events were collected during the flow cytometry analysis.Dot plots (A) and histograms (B) were analyzed using FlowJo™ v10 software, with quadrant 3 representing the negative confidence region.SSC, or cell count, was plotted on the y-axis, and FITC fluorescence intensity was plotted on the x-axis.The MFI of FITC was analyzed using FlowJo™ v10 software (C).The unpaired t-test was used to compare the MFI of S. suis.* indicates p < 0.05, ** indicates p < 0.01.

Figure 3 .
Figure 3. Growth kinetics and MIC determination for S. suis serotype 2 strain SC070731 in various concentrations of CPPs.(A) HIV-1 TAT at concentrations ranging from 128 to 8 µM.(B) (RXR)4XB at concentrations ranging from 32 to 2 µM.An equivalent volume of water was included as a control.The MIC value is shown and was determined as the lowest concentration inhibiting visible growth in the wells (OD595nm < 0.1).

Figure 3 .
Figure 3. Growth kinetics and MIC determination for S. suis serotype 2 strain SC070731 in various concentrations of CPPs.(A) HIV-1 TAT at concentrations ranging from 128 to 8 µM.(B) (RXR) 4 XB at concentrations ranging from 32 to 2 µM.An equivalent volume of water was included as a control.The MIC value is shown and was determined as the lowest concentration inhibiting visible growth in the wells (OD 595nm < 0.1).

Figure 4 .
Figure 4. Schematic illustration of the PNA target region of gene gyrA.(A) Region of gyrA mRNA in S. suis, with the start codon (AUG) shown in bold type.For the location relative to the start site, 'A' of AUG is defined as +1 in this study.The Shine-Dalgarno and PNA target sequences are shaded in green and blue, respectively.Below, the PNA sequence is shown (blue box) with the conjugated CPP HIV-1 TAT for delivery into S. suis.(B) Multiple sequence alignments of the gene gyrA in different serotypes of S. suis, including serotype 2 strain SC070731, serotype 4 strain ND90, serotype 5 strain GX169, serotype 7 strain WUSS013, and serotype 9 strain GZ0565.A defined section (−26 to +60 nt), including the region around the PNA binding site (blue dashed border), is shown.

Figure 4 .
Figure 4. Schematic illustration of the PNA target region of gene gyrA.(A) Region of gyrA mRNA in S. suis, with the start codon (AUG) shown in bold type.For the location relative to the start site, 'A' of AUG is defined as +1 in this study.The Shine-Dalgarno and PNA target sequences are shaded in green and blue, respectively.Below, the PNA sequence is shown (blue box) with the conjugated CPP HIV-1 TAT for delivery into S. suis.(B) Multiple sequence alignments of the gene gyrA in different serotypes of S. suis, including serotype 2 strain SC070731, serotype 4 strain ND90, serotype 5 strain GX169, serotype 7 strain WUSS013, and serotype 9 strain GZ0565.A defined section (−26 to +60 nt), including the region around the PNA binding site (blue dashed border), is shown.

Antibiotics 2024 , 14 Figure 5 .
Figure 5.An antisense gyrA-specific PNA coupled to HIV-1 TAT (TAT-anti-gyrA PNA) exhibits antibacterial activity against S. suis strains.(A) Growth kinetics and MIC determination of SC070731 in various concentrations of free gyrA-specific PNA, free HIV-1 TAT, or TAT-anti-gyrA PNA at concentrations ranging from 32 to 2 µM.The MIC value is shown and was determined as the lowest concentration inhibiting visible growth in the wells (OD595nm < 0.1).(B) Bactericidal effects of TATanti-gyrA PNA were determined at 1 × MIC (4 µM) and 2 × MIC (8 µM) against S. suis serotype 2 strain SC070731 during a 4-h time course.After the indicated time points, aliquots of each condition were subjected to spot assays or CFU determination on THA plates to investigate the number of viable cells.(C) Concentration-dependent reduction in the bacterial counts following treatment of S. suis strains of various serotypes with TAT-anti-gyrA PNA for 2 h.The unpaired t-test was used to compare the number of viable bacterial cells.** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

Figure 5 .
Figure 5.An antisense gyrA-specific PNA coupled to HIV-1 TAT (TAT-anti-gyrA PNA) exhibits antibacterial activity against S. suis strains.(A) Growth kinetics and MIC determination of SC070731 in various concentrations of free gyrA-specific PNA, free HIV-1 TAT, or TAT-anti-gyrA PNA at concentrations ranging from 32 to 2 µM.The MIC value is shown and was determined as the lowest concentration inhibiting visible growth in the wells (OD 595nm < 0.1).(B) Bactericidal effects of TAT-anti-gyrA PNA were determined at 1 × MIC (4 µM) and 2 × MIC (8 µM) against S. suis serotype 2 strain SC070731 during a 4-h time course.After the indicated time points, aliquots of each condition were subjected to spot assays or CFU determination on THA plates to investigate the number of viable cells.(C) Concentration-dependent reduction in the bacterial counts following treatment of S. suis strains of various serotypes with TAT-anti-gyrA PNA for 2 h.The unpaired t-test was used to compare the number of viable bacterial cells.** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

Table 1 .
The list of CPPs, PNAs, CPP-PNA conjugates, and their MIC values for strain SC070731.
CFU, colony-forming units.The count of surviving bacteria after treatment with CPPs was compared with that of water using an unpaired t-test.A summary of the p-values is provided, with an asterisk indicating a significant difference (p < 0.05) and 'ns' denoting no significant difference.