Staphylococcus aureus(S. aureus) is a common pathogen that is responsible for numerous aspects of acute infections. A majority of the virulence factors are controlled by a cell to cell interaction pathway called quorum sensing(QS). The quorum sensing system of S. aureus is considered as an autocatalytic sensory transduction system which uses accessory gene regulator(agr). The agr operon encodes four protein(AgrA-AgrD) to complete this process. In the beginning, AgrB, an integral membrane endopeptidase modified AgrD, resulting that the N-terminal of AgrD cleave and the mature AIP signal outside the cell liberate. As the sufficient concentration of AIP has accumulated, AIP binds to AgrC, a transmembrane receptor histidine kinase（Vasquez et al., 2019）. Then, AgrC transautophosphorylates and then phosphorylates AgrA, an intracellular transcription factor. After that, AgrA binds to P2 promoter in order to upregulates agr operon, therefore amplifying the QS signal. Thus, a typical autoinduction cycle of QS systems is established.
The experiment of batrachotoxin-modified sodium channels incorporated in planar lipid bilayers can illustrate this. The conductance of the channel has increased as the negative charges on the channel protein is near the channel entrances.The negative charges are provided by excess amino acids and the negatively charged carbonhydrate chains attached to the protein. The experiment uses synaptosomes, incorporated sodium channel in the bilayer and a standard two-electrode voltage-clamp, which is used for electrical measurements. It results that BTX-modified Sodium Channels Disappear Spontaneously without any observable(Green et al., 1987). One possible explanation for channel disappearances is the dissociation of BTX from the channels, in which case the disappearance rate is an upper estimate for the BTX dissociation rate constant.
We apply proximity labeling technology to fulfill this process. TurboID is an ideal enzyme for the application, which has faster labeling kinetics using the same non-toxic labeling conditions. TurboID enables probing of dynamic biological processes with much higher temporal resolution, because it only requires labeling times as short as 10 min or less in cell culture. Moreover, TurboID retain catalytic activity at lower temperatures. Two TurboID fragments (Tb(N), an N-terminal fragment, and Tb(C), a C-terminal fragment) called Split-TurboID can be brought together by a protein–protein interaction or membrane–membrane apposition to reconstitute an active enzyme(Cho et al., 2020).TurboID uses biotin and ATP as substrates , generating biotin–adenosine monophosphate (biotin–5ʹ-AMP), a reactive intermediate that can covalently label proximal endogenous proteins.
References:
(1) Joseph K. Vasquez et al.(2019), Simplified Autoinducing Peptide Mimetics with Single-Nanomola Activity Against the Staphylococcus aureus AgrC Quorum Sensing Receptor, ACS Infectious Diseases, vol.5, pp.484−492
(2) Kelvin F. Cho et al.(2020), Proximity Labeling in Mammalian Cells with
TurboID and Split-TurboID, Natural Protocols,vol.15, pp.3971–3999
(3)W. N. Green et al.(1987), Batrachotoxin-modified Sodium Channels in Planar Lipid Bilayers, The Journal of General Physiology, vol.89, pp.841-872