ML364 exerts the broad-spectrum antivirulence effect by interfering with the bacterial quorum sensing system

Abstract

Antivirulence strategy has been developed as a nontraditional therapy which would engender a lower evolutionary pressure toward the development of antimicrobial resistance. However, the majority of the antivirulence agents currently in development could not meet clinical needs due to their narrow antibacterial spectrum and limited indications. Therefore, our main purpose is to develop broad-spectrum antivirulence agents that could target on both Gram-positive and Gram-negative pathogens. We discovered ML364, a novel scaffold compound, could inhibit the productions of both pyocyanin of Pseudomonas aeruginosa and staphyloxanthin of Staphylococcus aureus. Further transcriptome sequencing and enrichment analysis showed that the quorum sensing (QS) system of pathogens was mainly disrupted by ML364 treatment. To date, autoinducer-2 (AI-2) of the QS system is the only non-species-specific signaling molecule that responsible for the cross-talk between Gram-negative and Gram-positive species. And further investigation showed that ML364 treatment could significantly inhibit the sensing of AI-2 or its nonborated form DPD signaling in Vibrio campbellii MM32 and attenuate the biofilm formation across multi-species pathogens including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The results of molecular docking and MM/GBSA free energy prediction showed that ML364 might have higher affinity with the receptors of DPD/AI-2, when compared with DPD molecule. Finally, the in vivo study showed that ML364 could significantly improve the survival rates of systemically infected mice and attenuate bacterial loads in the organs of mice. Overall, ML364 might interfere with AI-2 quorum sensing system to exert broad-spectrum antivirulence effect both in vitro and in vivo.

Keywords: ML364; antimicrobial resistance; autoinducer-2; broad-spectrum antivirulence agent; quorum sensing.

Figure 1

The in vitro studies of ML364. (A) Growth curves of Pseudomonas aeruginosa PAO1 and carbapenem-resistant P. aeruginosa (CRPA) 16-2 treated with and without ML364. (B) Growth curves of S. aureus ATCC 29213 and methicillin-resistant Staphylococcus aureus (MRSA) 08-50 treated with and without ML364. (C) Images from SEM for P. aeruginosa PAO1 treated with or without 256 μg/ml ML364. (D) Images from SEM for S. aureus ATCC 29213 treated with or without 64 μg/ml ML364. (E) Production of pyocyanin by P. aeruginosa PAO1 in the presence or absence of ML364. (F) Production of staphyloxanthin by S. aureus ATCC 29213 in the presence or absence of ML364. (G) Quantitation of pyocyanin in P. aeruginosa PAO1 and CRPA 16-2 treated with and without 64–256 μg/ml ML364. (H) Quantitation of staphyloxanthin in S. aureus ATCC 29213 and MRSA 08-50 treated with and without 16–64 μg/ml ML364. Control represents the solvent treatment group. Data were calculated with one-way ANOVA and Bonferroni’s multiple comparisons, compared to those of the control group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 2

ML364 treatment attenuated virulence factor production by interfering with quorum sensing systems. Pseudomonas aeruginosa PAO1 was cultured for 6 h with or without 256 μg/ml ML364. (A) Volcano plots of differentially expressed genes in P. aeruginosa PAO1 treated with ML364. (B) KEGG enrichment analysis of differentially expressed genes in P. aeruginosa PAO1 treated with ML364. (C) Schematic diagram of the P. aeruginosa quorum-sensing hierarchy and biosynthesis system of pyocyanin. (D) Transcription levels of quorum sensing-related genes in P. aeruginosa PAO1 with ML364 treatment determined by real-time PCR. Data were analyzed via the Student’s t-test, compared to those of the PAO1 control group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 3

ML364 blocks DPD/AI-2 signaling response and inhibit biofilm formation of different species of bacteria. (A) The AI-2 and DPD signaling response of Vibrio Campbellii MM32 (LuxN⁻, LuxS⁻) treated with various concentrations of ML364. (B) The biofilm quantification of Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700721 and Staphylococcus aureus ATCC 29213 treated with ML364. Data were calculated with one-way ANOVA and Bonferroni’s multiple comparisons, compared to those of the control groups; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 4

The interactions and per-residue free energy decomposition between DPD or ML364 with DPD/AI-2 receptors. (A) The interactions between DPD or ML364 with LuxP protein of V. Campbellii. (B) The per-residue energy decomposition between LuxP with DPD or ML364. (C) The interactions between DPD or ML364 with PctA protein of Pseudomonas aeruginosa. (D) The per-residue energy decomposition between PctA with DPD or ML364. (E) The interactions between DPD or ML364 with TlpQ protein of P. aeruginosa. (F) The per-residue energy decomposition between TlpQ with DPD or ML364. Protein-ligand interactions are colored as following: blue solid line, hydrogen bond; dash line, hydrophobic interaction; cyan solid line, halogen bond.

Figure 5

The in vivo studies of ML364. (A) The chemical structure of ML364. (B) The drug administration procedure in systemically infected mice. (C) Mice were intraperitoneally infected with Pseudomonas aeruginosa PAO1, CRPA 16-2, Staphylococcus aureus ATCC 29213, and MRSA 08-50. A dose of 3 mg/kg ML364 was intraperitoneally injected at 0, 12, and 24 h post-infection. Survival rates of mice were monitored until 4 days post-infection. (D) Bacterial loads in organs (liver, spleen, and kidney) influenced by ML364 therapy in the CRPA 16-2 and MRSA 08-50 infection model counted at 26 h post-infection (n = 7 per group). ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 (Compared with the model group).