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. 2022 Dec 21;10(6):e0049722.
doi: 10.1128/spectrum.00497-22. Epub 2022 Nov 2.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae

Gina Barkowsky #  1 , Corina Abt #  1 , Irina Pöhner  1 , Adam Bieda  1 , Sven Hammerschmidt  2 , Anette Jacob  3   4 , Bernd Kreikemeyer  1 , Nadja Patenge  1
Affiliations

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae

Gina Barkowsky et al. Microbiol Spectr. .

Abstract

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

Keywords: Streptococcus pneumoniae; antimicrobial activity; antimicrobial therapy; antisense molecules; cell penetrating peptide; peptide nucleic acid; pneumococcus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Concentration-dependent reduction of the pneumococcal CFU/mL following treatment of Streptococcus pneumoniae TIGR4 with cell-penetrating peptide (CPP)-antisense peptide nucleic acids (PNAs) for 6 h. scPNA, scrambled PNA controls. (A) Treatment with TAT-anti-gyrA PNAs. (B) Treatment with TAT-anti-rpoB PNAs. (C) Treatment with (RXR)4XB-anti-gyrA PNAs. (D) Treatment with (RXR)4XB-anti-rpoB PNAs. Data are presented as means and standard deviation. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons. Differences between PNA conjugate samples and mock control (untreated) are shown as *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.0001. Sample size: n = 5.
FIG 2
FIG 2
Killing kinetics of CPP-anti-gyrA PNA treatment in S. pneumoniae strain TIGR4. (A) Bacterial counts following treatment with 10 μM TAT-anti-gyrA PNA or 10 μM TAT-anti-gyrA scPNAs. (B) Bacterial counts following treatment with 10 μM (RXR)4XB-anti-gyrA PNAs or 10 μM (RXR)4XB-anti-gyrA scPNAs. Data are presented as means and standard deviation. Sample size: n = 3.
FIG 3
FIG 3
Relative expression of target genes and virulence genes in S. pneumoniae strain TIGR4 following treatment with CPP-antisense PNAs. The 5S rRNA gene served as internal control. Relative expression was calculated using the threshold cycle (2–ΔΔCT) method. (A) Treatment with 7.5 μM TAT-anti-gyrA PNAs and 7.5 μM (RXR)4XB-anti-gyrA PNAs. Relative expression of gyrA. (B) Treatment with 10 μM TAT-anti-rpoB PNAs and 10 μM (RXR)4XB-anti-rpoB PNAs. Relative expression of rpoB. (C) Treatment with 10 μM TAT-anti-rpoB PNAs and 10 μM (RXR)4XB-anti-rpoB PNAs. Relative expression of ply. (D) Treatment with 10 μM TAT-anti-rpoB PNAs and 10 μM (RXR)4XB-anti-rpoB PNAs. Relative expression of pspA. Data are presented as means and standard deviation. Statistical significance was determined by one-way ANOVA with multiple comparisons. Differences between PNA conjugate samples and the mock control (untreated) are shown as *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.0001. Sample size: n = 3.
FIG 4
FIG 4
Antimicrobial and cytotoxic effects of (RXR)4XB-anti-gyrA PNA in a cell-based infection system. (A) Detroit 562 cells infected with S. pneumoniae TIGR4 and subsequently treated with 1 μg levofloxacin, 20 μM (RXR)4XB-anti-gyrA PNA, or 20 μM (RXR)4XB-anti-gyrA scPNA, respectively. Sample size: n = 3. Statistical significance was determined by one-way ANOVA with multiple comparisons. Differences between PNA conjugate samples and mock control (untreated) are shown as *, P ≤ 0.05. (B) Detroit 562 cells were lysed and treated with 20 μM (RXR)4XB-PNA or 20 μM TAT-PNA, respectively. Biomass was stained with crystal violet, bound crystal violet was extracted, and the optical density of the solution was measured at 600 nm. Sample size: n = 4. Statistical significance was determined by one-way ANOVA with multiple comparisons. Differences between samples and maximal cell lysis are shown as **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 5
FIG 5
Survival of Galleria mellonella larvae treated with 10 nmol CPP-PNAs following infection with S. pneumoniae TIGR4. (A) G. mellonella larvae infected with S. pneumoniae TIGR4 and subsequently treated with 10 or 1 μg levofloxacin. Sample size: n = 40 larvae per group. (B) G. mellonella larvae infected with S. pneumoniae TIGR4 and subsequently treated with TAT-anti-gyrA PNA or TAT-anti-gyrA scPNA, respectively. Sample size: n = 60 larvae per group. (C) G. mellonella larvae infected with S. pneumoniae TIGR4 and subsequently treated with (RXR)4XB-anti-gyrA PNA or (RXR)4XB-anti-gyrA scPNA, respectively. Sample size: n = 60 larvae per group. Statistical significance was determined using the log-rank test. Differences between curves are shown as *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001.
FIG 6
FIG 6
Bacterial load of G. mellonella larvae following infection with S. pneumoniae TIGR4. G. mellonella larvae infected with S. pneumoniae TIGR4 and subsequently treated with either levofloxacin or (RXR)4XB-PNAs. After 24 h, larvae were homogenized. Bacterial load was determined by serial dilution, plating on selective medium, and CFU calculation. Sample size: n = 4 larvae per group in three independent experiments. Data are presented as means and standard deviation. Statistical significance was determined by one-way ANOVA with multiple comparisons. Differences between treated samples and mock control (NaCl) are shown as *, P ≤ 0.05.
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