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

Abstract

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.

Keywords: Streptococcus suis; antisense oligonucleotides; cell-penetrating peptides; peptide nucleic acids.

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).

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

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 (OD_595nm < 0.1).

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 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.