Functional Study of desKR: a Lineage-Specific Two-Component System Positively Regulating Staphylococcus aureus Biofilm Formation

Abstract

Purpose: Biofilms significantly contribute to the persistence and antibiotic resistance of Staphylococcus aureus infections. However, the regulatory mechanisms governing biofilm formation of S. aureus remain not fully elucidated. This study aimed to investigate the function of the S. aureus lineage-specific two-component system, desKR, in biofilm regulation and pathogenicity.

Methods: Bioinformatic analysis was conducted to assess the prevalence of desKR across various S. aureus lineages and to examine its structural features. The impact of desKR on S. aureus pathogenicity was evaluated using in vivo mouse models, including skin abscess, bloodstream infection, and nasal colonization models. Crystal violet staining and confocal laser scanning microscopy were utilized to examine the impact of desKR on S. aureus biofilm formation. Mechanistic insights into desKR-mediated biofilm regulation were investigated by quantifying polysaccharide intercellular adhesin (PIA) production, extracellular DNA (eDNA) release, autolysis assays, and RT-qPCR.

Results: The prevalence of desKR varied among different S. aureus lineages, with notably low carriage rates in ST398 and ST59 lineages. Deletion of desKR in NCTC8325 strain resulted in decreased susceptibility to β-lactam and glycopeptide antibiotics. Although desKR did not significantly affect acute pathogenicity, the ΔdesKR mutant exhibited significantly reduced nasal colonization and biofilm-forming ability. Overexpression of desKR in naturally desKR-lacking strains (ST398 and ST59) enhanced biofilm formation, suggesting a lineage-independent effect. Phenotypic assays further revealed that the ΔdesKR mutant showed reduced PIA production, decreased eDNA release, and lower autolysis rates. RT-qPCR indicated significant downregulation of icaA, icaD, icaB, and icaC genes, along with upregulation of icaR, whereas autolysis-related genes remained unchanged.

Conclusion: The desKR two-component system positively regulates S. aureus biofilm formation in a lineage-independent manner, primarily by modulating PIA synthesis via the ica operon. These findings provide new insights into the molecular mechanisms of biofilm formation in S. aureus and highlight desKR as a potential target for therapeutic strategies aimed at combating biofilm-associated infections.

Keywords: Staphylococcus aureus; biofilm; desKR; two-component system.

Figure 1

Prevalence and genomic context of the two-component regulatory system desKR. (A) Heatmap showing the prevalence of desKR in different sequence types (STs). (B) Genomic context of desKR. The reference strain NCTC 8325 (ST8), containing the desKR genes, was selected, and strains GCF_000237125 (ST59) and GCF_000009585 (ST398) from NCBI were included for comparison. Black arrows indicate the positions of desK and desR in NCTC 8325.

Figure 2

Simple Modular Architecture Research Tool (SMART) and Swiss-MODEL Analysis of DesKR. (A) Predicted domain of DesK based on SMART (http://smart.embl-heidelberg.de/). (B) 3D protein simulation of DesK using the SWISS-MODEL web server. (C) Predicted domain of DesR based on SMART. (D) 3D protein simulation of DesR using the SWISS-MODEL web server.

Figure 3

Impacts of desKR deletion on S. aureus virulence, adhesion, colonization, and growth. (A) Mouse bloodstream infection model. Kaplan-Meier estimates of survival in mice infected with S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. Sterile PBS was used as a control to exclude the influence of solvents and manipulation. (B) Mouse skin abscess model. Skin abscess area in mice (n=10 per group) two days after infection with S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. (C) Bacterial burden in mouse abscess homogenates determined by serial dilution and culturing on blood agar plates. (D) Mouse nasal colonization model. Bacterial burden in nasal tissues of mice (n=10 per group) 48 hours after infection with S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. (E) 24-hour growth curves of S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. (F) Colony-forming units of S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C cultures following 24 hours of incubation. ****P < 0.0001.

Figure 4

Impacts of desKR on S. aureus biofilm formation ability. (A) Detection of biofilm formation ability of NCTC8325, ΔdesKR, and ΔdesKR-C using crystal violet staining assay. (B) Confocal laser scanning microscopy images of biofilms formed by NCTC8325, ΔdesKR, and ΔdesKR-C. (C) Impacts of desKR overexpression on the biofilm formation ability of clinical strain SA27 (ST59). (D) Impacts of desKR overexpression on the biofilm formation ability of clinical strain SA42 (ST398). ****P < 0.0001.

Figure 5

Impacts of desKR deletion on S. aureus biofilm matrix components. (A) PIA production of S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. (B) Quantification of eDNA in S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. (C) Autolysis ability of S. aureus NCTC8325, ΔdesKR, and ΔdesKR-C. **P < 0.01; ****P < 0.0001.

Figure 6

RT-qPCR detection of the impacts of desKR deletion on the expression of biofilm-related genes. The relative expression level of the wild-type strain NCTC8325 was set to 1. ****P < 0.0001.

Figure 7

Schematic overview of the S. aurues DesKR two-component system and its regulatory effects on biofilm formation and antibiotic susceptibility.