Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique

Technical Summary: Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique

Problem Statement
Staphylococcus aureus secretes bi-component pore-forming toxins (PFTs), known as leukocidins, which target specific host cell receptors (primarily G protein-coupled receptors like chemokine and complement receptors) to form cytotoxic hetero-octameric pores. While the structural architecture of these pores is known, the molecular mechanisms of their formation remain incompletely elucidated. Existing methods for studying these toxins, such as measuring membrane permeability via nucleic acid dyes (e.g., propidium iodide) after long incubation periods (30+ minutes), fail to distinguish between pore-mediated permeability and general cell death. Furthermore, traditional electrophysiological approaches using artificial lipid bilayers require the purification and reconstitution of specific receptors, a technically demanding process that limits the throughput and diversity of receptors that can be studied.

Methodology
The authors developed a high-throughput, real-time electrophysiological method to study receptor-dependent PFTs. The core of this approach involves:

1. Heterologous Expression: Human chemokine and complement receptors (CCR2, CXCR4, ACKR1, C5aR1, CCR5) were expressed on the surface of Xenopus laevis oocytes via mRNA micro-injection.
2. Automated Electrophysiology: Whole-cell currents were recorded in real-time using a two-electrode voltage-clamp (TEVC) robot (HiClamp, Multichannel Systems) operating in a 96-well plate format. This setup requires minimal toxin volumes (~200 µL).
3. Experimental Design: Soluble leukocidin subunits (S and F components) were applied to oocytes either simultaneously or sequentially (with washing steps) to observe pore formation kinetics.
4. Complementary Techniques: To validate binding mechanisms, the authors utilized Plasmon Waveguide Resonance (PWR) on proteoliposomes containing immobilized CCR5 receptors to measure mass changes and anisotropy upon subunit binding.

Key Results

• Real-time Detection and Specificity: The TEVC method successfully detected pore formation within seconds to minutes. Application of cognate leukocidin pairs (e.g., HlgAB on CCR2, HlgCB on C5aR1, LukED on CCR5) generated significant current amplitudes, whereas non-cognate controls (e.g., HlgAB on CXCR4) showed negligible currents.
• Non-cognate Pairing: The method detected functional pore formation by non-cognate subunit pairs, specifically LukE and HlgB, on CCR5-expressing oocytes, confirming the technique's sensitivity to alternative toxin-receptor interactions.
• Concentration Dependence: Pore formation kinetics were highly concentration-dependent. At high concentrations (1 µM), pore formation occurred rapidly (40 s delay). At sub-nanomolar concentrations (0.1 nM), the delay increased significantly (1100 s), and a biphasic current profile was observed at higher concentrations, suggesting a transition from pore formation to massive membrane leakage (cell death).
• Receptor Density as a Limiting Factor: Increasing the expression time of the CCR2 receptor (from 1 to 3 days post-injection) increased the maximum current amplitude by 2.7-fold without altering the kinetics of the initial pore formation. This indicates that the surface density of receptors is a limiting factor for the total number of pores formed under saturating toxin conditions.
• Sequential Application and Subunit Binding:
  • Sequential application of S then F subunits (with washing) resulted in pore formation, though with lower amplitude than co-application, suggesting a role for soluble subunits in maximizing pore efficiency.
  • Crucially, sequential application of the F subunit (HlgB) followed by the S subunit (HlgA) also resulted in pore formation. This demonstrates that F subunits can bind to the cell surface independently of the S subunit and remain available to oligomerize upon the arrival of the second subunit.
  • PWR data confirmed that HlgB binds directly to CCR5-containing membranes, supporting the observation of F-subunit surface retention.

Significance and Claims
The paper claims that this TEVC-based approach offers a simple, sensitive, and high-throughput alternative to traditional methods for characterizing receptor-dependent PFTs. Its primary significance lies in:

1. Eliminating Purification Steps: It allows for the functional study of PFTs on a wide diversity of receptors without the need for receptor purification and reconstitution into artificial bilayers.
2. Real-time Kinetics: It enables the observation of pore formation stages preceding cell lysis, providing kinetic data (lag times, saturation) that indirect dye-based assays cannot capture.
3. Mechanistic Insight: The results challenge the strict "S-first" recruitment model by demonstrating that F subunits can bind to the membrane independently. The authors propose a "hybrid model" of pore formation where oligomerization can occur via a combination of soluble subunit recruitment and pre-bound subunits on the membrane, depending on subunit concentration.
4. Screening Potential: The method is presented as a viable tool for screening therapeutic agents that block cytotoxicity, identifying new target receptors, and characterizing the pore-forming properties of various PFT variants across different bacterial lineages.

The authors acknowledge limitations, including the requirement for sufficient receptor expression in oocytes, the use of non-endogenous cells, and the inability to resolve single-pore unitary conductance (as TEVC measures macroscopic currents). However, they assert that the method's ability to maintain a natural lipid asymmetry and provide real-time data makes it a valuable addition to the toolkit for studying staphylococcal leukocidins.