Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore

Abstract

Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor-acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA-HlgB or HlgB-HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA-HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB-HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

Figure 1. Schematic diagram of the FRET planning

The three-dimensional model of the possible organization of HlgA and HlgB components inside the pore and localization of the single cysteine residue introduced near the protomer–protomer interfaces. HlgA protomers are shown in dark grey and HlgB protomers in light grey. Highlighted residues are Ser²² (HlgA_la), Gln²⁰² (HlgA_lb) and Ser¹⁴⁸ (HlgA_r) of HlgA, and Ser²⁷ (HlgB_l) and Arg¹⁵⁵ (HlgB_r) of HlgB. The corresponding names of the mutants are reported in parentheses. The mutated residues correspond to Tyr²⁸, Ser²²¹ and Ser¹⁵⁹ of α-toxin, which were used to generate this image.

Figure 2. Fluorescence of labelled HlgA–HlgB couples in the presence of lipid vesicles

(A) Emission spectra of HlgA S148C (HlgA_r) and HlgB S27C (HlgB_l) labelled with donor (D) and acceptor (A) ALEXA fluorophores respectively. The excitation wavelength was 490 nm for each experiment (excitation and emission slits were 1 nm). The labelled couple was premixed in a cuvette at a molar concentration of 300 nM for each component. Thereafter, LUVs were added at a final lipid concentration of 4 μM. The solid line is the spectrum taken 105 min after LUV addition. The dashed line is the corresponding result when only the HlgA component (HlgA_r) is labelled and used together with unlabelled HlgB_l. The dotted line is the corresponding result when only the HlgB component (HlgB_l) is labelled and mixed with unlabelled HlgA_r. (B) Fluorescence changes that ensue during the development of the interaction of the γ-haemolysins with the added LUV. Each spectrum is the difference between the spectrum taken at time t and time 0 (i.e. immediately after the addition of the vesicles). (C) Fluorescence changes observed as difference from controls. In this case, each spectrum was obtained by subtracting the two control spectra from the spectrum in which both components are labelled. All the spectra were taken at the same time t. In the two control experiments only one component was labelled. In (B and C), the time in minutes is indicated next to each trace.

Figure 3. Fluorescence changes during the interaction of HlgA_l and different partners with LUVs

HlgA Q202C (HlgA_lb) was coupled with one among HlgB R155C (HlgB_r, A), HlgB wt and HlgA S148C (HlgA_r, B) and HlgB S27C (HlgB_l, C). Differential spectra were obtained as described in Figure 2(B). HlgA_l was labelled as acceptor in (A and B) and as donor in (C); the partner mutant carried the complementary label. All other experimental conditions are as in Figure 2.

Figure 4. Time course of fluorescence changes and LUV permeabilization

An estimate of the energy transfer is obtained by taking the difference between the acceptor fluorescence at 570 nm and that of the donor at 516 nm from spectra such as those in Figures 2(B) or 3 (Δ₁=F_t−F₀). The time course of this difference is compared with the fluorescence increase due to the marker release, as shown in a separate experiment run with calcein-loaded LUV under the same experimental conditions (insets). The couple HlgA S148C (HlgA_r)–HlgB S27C (HlgB_l) was used in (A) and the couple HlgA Q202C (HlgA_lb)–HlgA S148C (HlgA_r)+HlgB wt was used in (B). The left position of each mutant was labelled with the acceptor and the right with the donor. All other experimental conditions are as described in Figure 2.

Figure 5. Fluorescence changes during the interaction of HlgB_l and HlgB_r with HlgA wt and LUVs

(A) Differential spectra were obtained as described in Figure 2(C). Donor-labelled HlgB R155C (HlgB_r^D) and acceptor-labelled HlgB S27C (HlgB_l^A) were mixed with wt HlgA before adding the LUVs. (B) Time course of the energy transfer obtained by taking the difference between the acceptor and the donor fluorescence difference between the acceptor fluorescence at 570 nm and that of the donor at 516 nm from the spectra reported in (A) (Δ₂=F_t−F_t(a+d)). This elaboration of the data was chosen in order to emphasize the peculiar initial change after the addition of the vesicles, which could not be seen with the elaboration of Figure 4. The time course of LUV permeabilization (obtained by calcein-release assay in a separate experiment) is compared in the inset under the same experimental conditions.

Figure 6. Topology and assembly pathway of the HlgA–HlgB pore

As summarized in Table 2, a strong FRET is observed only with the heterologous couples HlgA_r–HlgB_l or HlgA_{la or b}–HlgB_r. At the steady state, neither couple HlgA_lb–HlgA_r nor HlgB_l–HlgB_r gave an intense transfer when mixed with the competent wt component. Our results suggest that the heptameric complexes are much less probable events. Hexamers or octamers, containing only one type of heterologous interface, are equally possible. The pathway towards the assembly of the pore proceeds through the formation of a transient unstable homologous HlgB_l–HlgB_r interface.