Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection

Abstract

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that has antimicrobial activities against a broad spectrum of Gram-positive pathogens. Here, we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes. Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes. Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens, and accelerated rates of wound healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

Fig. 1.. GmPcide PS757 treatment caused nucleoid and cell wall abnormalities in S. pyogenes cells.

(A) S. pyogenes HSC5 cells treated under both sublethal (0.4 μM) and bactericidal (20 μM) concentrations of PS757 exhibited nucleoid abnormality with altered nucleoid structure that was less dense and very filamentous (white arrows). S. pyogenes HSC5 cells treated under bactericidal (20 μM) concentration of PS757 were observed with small dense globular structures at periphery of bacterial cell wall (red arrows), suggesting that PS757 produced cell wall abnormalities. (B) S. pyogenes HSC5 cells treated under bactericidal (20 μM) concentration of PS757 were observed with irregular and heterogeneous cell wall structures, indicating that PS757 induced cell wall damages. DMSO, dimethyl sulfoxide.

Fig. 2.. GmPcide PS757 was active against S. pyogenes biofilm.

(A and B) Bacterial growth (A) and biofilm formation (B) of S. pyogenes HSC5 strain were measured in microplate assays for 24 hours using BHI medium, which identified 4, 7, and 24 hours as the time points for three different phases of S. pyogenes biofilm formation, including biofilm initiation, biofilm development, and fully mature biofilm. (C) At 4 hours, PS757 treatment at the concentrations of 0.7 and 1.0 μM prevented S. pyogenes HSC5 bacterial growth and biofilm formation in the initiation phase, respectively. (D) At 7 hours, PS757 treatment at the concentrations of 2.0 and 5.0 μM ceased S. pyogenes HSC5 bacterial growth and biofilm formation in the maturing phase, respectively. (E) At 24 hours, PS757 treatment at the bactericidal concentration of 20 μM eradicated mature S. pyogenes HSC5 biofilm. OD₆₀₀, optical density at 600; A595, absorbance at 595.

Fig. 3.. GmPcide PS757 was effective in treating S. pyogenes SSTI in mice.

(A) Timeline of the 3-day infection and treatment protocol by using PS757 to treat S. pyogenes SSTI in mice. (B) PS757 treatment alleviated acute weight loss caused by S. pyogenes SSTI in mice in 3 days. (C and D) PS757 treatment reduced ulcer formation at day 3 of S. pyogenes SSTI in mice (P < 0.0001). (E) PS757 treatment attenuated bacterial burden at day 3 of S. pyogenes SSTI in mice (P < 0.0001). (F to H) Generation of host pro-inflammatory inflammation cytokines, TNFα [(E) P ≤ 0.05] and IL-6 [(F) P ≤ 0.05], but not IL-1β (H), were reduced in the PS757-treated group at day 3 of S. pyogenes SSTI in mice. (I) Immunofluorescence microscopy characterizations of thigh tissue samples from both vehicle-treated and PS757-treated mice demonstrated the accumulation of streptococcal and neutrophil cells and intensive interaction in between at the infection site, as well as elevated amounts of these cell types in the vehicle-treated mice group. Statistics were performed with Mann-Whitney U test. P ≤ 0.05 is considered as statistically significant. *P ≤ 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns indicates not significant. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 4.. GmPcide PS757 treatment promoted ulcer healing and bacterial clearance in S. pyogenes SSTI in mice.

(A) Timeline of the 12-day infection and treatment protocol by using PS757 to treat S. pyogenes SSTI in mice. (B and C) PS757 treatment promoted ulcer healing in S. pyogenes SSTI in mice during 12 days of infection (P < 0.001). (D) PS757-treated mice were observed with quicker eschars slough-off from the infected skin than the untreated group in 12-day S. pyogenes SSTI in mice. (E) PS757 treatment promoted the clearance of bacterial infection at day 12 in S. pyogenes SSTI in mice (P < 0.0001). Statistics were performed with Mann-Whitney U test. P ≤ 0.05 is considered as statistically significant. ***P < 0.001 and ****P < 0.0001.

Fig. 5.. GmPcide PS757 treatment of S. pyogenes resulted in altered transcriptome featuring the inhibition of two major virulence factors (emm5 and speB).

(A) PCA was conducted for comparing the transcriptomes of S. pyogenes HSC5 (obtained from RNA-seq) between two conditions under the treatments of 0.4 μM PS757 or DMSO vehicle. Score plot (PC2 ~ PC1) of the first (PC1) and second (PC2) principal components revealed the transcriptome differences between these two conditions, with clear separation observed along the PC1 axis (accounting for 48.2% of total variance between specimens). (B) A volcano plot comparing the PS757-treated versus vehicle (DMSO)-treated was used for identifying DEGs in RNA-seq analysis. DEGs with log₂(FC) > 0.5 and P < 0.05, including down- and up-regulated genes, were indicated as blue and red dots, respectively. A more stringent criteria, upper limits of the 99% confidence intervals (CIs) for log₂(FC) and −log(P) among DEGs, was further applied to identify the most significant DEGs, with down- and up-regulated genes indicated as bigger blue and red dots, respectively. (C to E) Seven genes were identified as the most down-regulated group of genes by the two more stringent criteria [(C) and (D)]. Among which, gene emm5, a major and multifunctional virulence factor of S. pyogenes, was identified with the highest inhibition among all genes by PS757 treatment (D), followed by validation by RT-qPCR test (E). (F) Another major S. pyogenes virulence factor, speB, was also identified with down-regulated transcription induced by PS757 treatment, which was validated by RT-qPCR and protease activity assays. Statistics were performed with Mann-Whitney U test. P ≤ 0.05 is considered as statistically significant. **P < 0.01 and ***P < 0.001.