High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus.

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• Staphylococcus aureus colonization relies on interactions with other microbes.
• The type VII secretion system subtype b (T7SSb) is crucial for S. aureus antagonism and virulence.
• Assessing T7SSb activity is challenging due to low secretion and lack of sensitive assays.

Purpose of the Study:

• To develop a sensitive assay for monitoring T7SSb-dependent protein secretion in S. aureus.
• To investigate variations in T7SSb activity across different S. aureus strains and growth conditions.
• To identify accessory proteins involved in the secretion of TspA, a surface-associated toxin.

Main Methods:

• Utilized NanoLuc binary technology to create a bioluminescence-based secretion assay.
• Fused NanoLuc fragment to the substrate EsxA for extracellular detection.
• Miniaturized the assay to 384-well format for high-throughput analysis and performed co-purification experiments.

Main Results:

• Demonstrated strain- and temperature-dependent differences in T7SSb protein secretion.
• Successfully monitored the secretion of the toxin substrate TspA using the developed assay.
• Identified three conserved accessory proteins essential for TspA secretion, forming a complex with TspA.

Conclusions:

• The NanoLuc-based assay provides a sensitive method to study T7SSb activity in S. aureus.
• T7SSb-dependent secretion is variable and influenced by strain and environmental factors.
• Discovered novel accessory proteins crucial for the secretion pathway of the S. aureus toxin TspA.