AgrC biotinylation inhibits Staphylococcus aureus infection

Abstract

Staphylococcus aureus (S. aureus) is a leading cause of nosocomial infections, particularly among antibiotic-resistant strains. S. aureus virulence is governed by the accessory gene regulator (Agr) quorum sensing (QS) system, which relies on AgrC, a two-component histidine kinase, to detect secreted auto-inducing peptides (AIPs). Emerging evidence highlights the potential of inhibiting the interaction between AgrC and AIPs as a promising therapeutic strategy. Given the limited clinic methods in inhibiting AgrC, we hereby report a novel method utilizing TurboID, an engineered biotin ligase, to inhibit Agr C on S. aureus via its biotinylation. To achieve this goal, a fusion protein named TurboID-AgrD[Formula: see text] (Agr-ID) was designed to include an AgrC binding domain (AgrID[Formula: see text]) and a catalytic domain (TurboID) for AgrC biotinylation. By incubating with Alexa Fluor 647-conjugated streptavidin, the biotinylated AgrC on S. aureus was successfully visualized through fluorescence microscopy with 100x objective. We further confirmed the specific biotinylation of AgrC using Western Blotting, and biotinylated AgrC resulted in inhibiting the growth of S. aureus strains, including S. aureus 25923, S. aureus 43300, and S. aureus 6538 (MRSA). The downstream biological effect of AgrC biotinylation exhibited decreased virulence protein generation as monitored by the lower presence of apoptotic HEK 293T cells after incubating with S. aureus cell lysates and supernatant. The impaired colonizing features from biotinylated S. aureus 6538 were investigated by calculating the decreased ratio of cell death versus live HeLa cells. By further investigating the efficiency of the immune clearance of biotinylated S. aureus by mouse macrophages, we observed the enhanced uptake of S. aureus by murine macrophages in vivo. Overall, our work reveals that the biotinylation of AgrC can inhibit the growth and toxicity of S. aureus while simultaneously promoting the clearance of biotinylated S. aureus via macrophage phagocytosis.

Fig 1. AgrC biotinylation by Agr-ID in Staphylococcus aureus (S. aureus). (A) Schematic model for the AgrC in situ biotinylation. The control group lacks biotin treatment. TurboID group represents none specific biotinylation without AgrC binding domain. Agr-ID is the experimental group targeting the biotinylation of AgrC. (B) Purity of Agr-ID analysis by SDS -10% PAGE and Coomassie blue staining. (C) Visualization of the biotinylated S. aureus by co-staining with Streptavdin-647. The percentage of biotinylated S. aureus was qualified by calculating the ratio of Streptavidin₊ S. aureus 25923 versus total DAPI₊ S. aureus 25923. N=5 independent biological replicates. (D-E) Pull-down assay for detecting the AgrC in situ biotinylation. Flag-agrC was transformed and overexpressed into S. aureus. The average fluorescent intensity of biotinylated AgrC was measured in all S. aureus pulled down by anti-Flag beads. N=5 independent biological replicates. (F-G) Pull-down assay for detecting the AgrC protein biotinylation using Western Blotting. Biotinylated proteins were pulled down by streptavidin-beads and analyzed by Western Blotting for detecting FLAG-AgrC. N=3 independent biological replicates. Data are presented as mean ± SEM. Two-sided P values were determined by unpaired student t-test. Data are presented as mean ± SEM. Two-sided P values were determined by a one-way ANOVA with Tukey’s multiple comparisons test.

Fig 2. AgrC biotinylation by Agr-ID inhibited the growth of S. aureus. (A) Schematic model for testing the growth of biotinylated S. aureus in LB medium and LB agar plate. (B-D) Growth inhibition in biotinylated S. aureus 25923 (B), S. aureus 6538 (C), S. aureus 43300 (D). ∼1.66×106/mL biotinylated S. aureus were added to 5 mL LB medium and cultured at 37 °C. At each time points, 1 mL medium was taken out for detecting the OD₆₀₀, then mixed back with the original ones. N=3 independent biological replicates. (E&F) Less colonies were shown in biotinylated S. aureus 25923. N=6 independent biological replicates. Data are presented as mean ± SEM. Two-sided P values were determined by unpaired student t-test. Data are presented as mean ± SEM. Two-sided P values were determined by a two-way ANOVA with Tukey’s multiple comparisons test.

Fig 3. AgrC biotinylation by Agr-ID inhibited the virulence proteins of S. aureus and promoted phagocytes clearance of S. aureus in vivo. (A) Schematic model for testing the virulence factors of biotinylated S. aureus. (B-E) Virulence proteins from biotinylated S. aureus 25923 led to less cell death. Less virulence protein in supernatant (B&C) and cell lysates (D&E) after incubating with HEK293T cells. The percentage of apoptotic HEK293T cells was detected and measured by Annexin V/DAPI staining and FACS. N=3 independent biological replicates. (F&G) Impaired colonization of biotinylated S. aureus 25923. The percentage of cell death₊ was counted by co-localization of TAMRA and CellTracker under fluorescent microscope. N=5 independent biological replicates. (H&I) Biotinylated S. aureus 25923 had impaired capacity in infecting mice. The enhanced percentage of TAMRA₊ macrophages showed more efficiency in clearance of biotinylated S. aureus 25923 by macrophages. N=5 independent biological replicates. Data are presented as mean ± SEM. Two-sided P values were determined by unpaired student t-test. Data are presented as mean ± SEM. Two-sided P values were determined by a two-way ANOVA with Tukey’s multiple comparisons test.