Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.


The secondary structures of PorB in liposomes and its soluble form are different
We compared the secondary structure of PorB in solution and in detergent solution with the corresponding structure of the protein in liposomes using circular dichroism (CD) spectroscopy (Fig. S7A,B). Spectra were analyzed with the CDpro package [1][2][3] using three different algorithms to fit the spectra and in addition with a neural network approach [4]. As expected [5] PorB reveals a significantly higher β-sheet content as compared to α-helical structures. Spectra of PorB in solution and in the presence of decylmaltoside (DM) showed similar shapes (Fig. S7A). However, the shape of the PorB CD spectrum in liposomes was significantly different from the one of the soluble form (Fig. S7B). Analysis of this spectrum showed a considerable increase of β-sheet structures and an almost complete loss of α-helical structures after incorporation of the protein into liposomes. Both CD spectra and calculations of the spectra clearly demonstrate the large structural changes of PorB during membrane insertion.

Calculation of membrane potential dissipation
The capacity of a biological membrane is the product of the specific capacity (for a typical biological membrane this can be estimated with c M = 1.0 µF · cm -2 ) and the surface.
Considering a mitochondrion as a prolate with an axial ratio of a = c = 1/5 b and a length of b = 5 µm the membrane capacity can be estimated as follows: The surface of the mitochondrion is: Where A M is the surface and a, b and c are the axes of the spheroid.
Thus the capacity (C) of a mitochondrion is:

Assessment of proton pump rates and leak currents I) Proton pumping capacity of a mitochondrion
Measurements with rat heart mitochondria (1) and model calculations (2)

Mammalian mitochondria experiments
Mitochondrial isolation and import of proteins were performed essentially as described previously [9]. Following the import, mitochondria were either treated with 50 µg/ml of proteinase K in the case of VDAC, or subjected to carbonate extraction. To this end, mitochondria (50 µg of protein) were solubilized in 100 µl of 100 mM Na 2 CO 3 , pH 11.5 (or pH 10.8 where indicated), incubated for 30 min on ice, and centrifuged for 30 min at 100 000 g. The pellet was then solubilized in Laemmli buffer and subjected to SDS-PAGE. Where supernatant was analyzed, proteins were first precipitated with trichloroacetic acid (TCA).
Radiolabelled proteins were visualized using Fuji FLA3000 imaging system and the intensity of the bands was quantified using AIDA Image Analyzer software.

Yeast mitochondria experiments
Mitochondria of the temperature-sensitive strain sam50-1 were preincubated for 15 min at 37°C to induce the mutant phenotype; the corresponding wildtype mitochondria were treated in the same way [10]. The radiolabelled proteins were synthesized in rabbit reticulocyte lysate

Circular dichroism-spectroscopy
Purified proteins were analyzed under three different conditions. PorB in solution: the protein was dialyzed over night at 4°C against a buffer containing 20 mM K 2 HPO 4 /KH 2 PO 4 , 10 mM KCl, pH 7.0. PorB solubilized in detergent solution: the protein was dialyzed against the same P i -buffer in addition with 8 mM N-Decyl-β-D-Maltopyranosid. To insert PorB into membrane vesicles, small azolectin-liposomes were formed as described [11]. Liposomes were incubated with purified protein for 2.5 h at room temperature and subsequently dialyzed over night at 4°C against 20 mM K 2 HPO 4 /KH 2 PO 4 , 10 mM KCl, pH 7.0. All three samples were adjusted to the protein concentration of 550 µg / ml. CD spectra were recorded using a Jasco J-810 spectrapolarimeter. All measurements were carried out in a quartz cuvette with an optical path length of 0.01 cm at room temperature. The scans (n = 16) were averaged to improve the signal / noise ratio. Blank buffer spectra were collected and subtracted from the sample spectra. To compare the