The major outer membrane protein of Chlamydia psittaci functions as a porin-like ion channel

Abstract

The major outer membrane protein (MOMP) of Chlamydia species shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified from Chlamydia psittaci has a predominantly beta-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the "porin channel" hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (PCl/PK approximately 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.

FIG. 1

SDS-PAGE and Western blot analyses of OG-DTT-solubilized MOMP. (A) SDS-PAGE analysis of 2% (wt/vol) OG–10 mM DTT-solubilized MOMP-enriched preparations by silver staining. (B) The OG-DTT-solubilized preparation was subjected to SDS-PAGE on 12.5% gels, immunoblotted, and probed with MAb 4/11 (sample loaded after boiling; lane a) and MAb A11 (sample loaded without boiling; lane b). Molecular masses are indicated.

FIG. 2

SDS-PAGE analysis of the hydroxyapatite-purified MOMP-enriched preparation. Fractions eluted from the hydroxyapatite column (5 by 1 cm) loaded with the 2% (wt/vol) SDS-solubilized MOMP-enriched preparation (see Materials and Methods) were subjected to SDS-PAGE on a 12.5% gel, under reducing conditions (not boiled), and silver stained. Molecular masses are indicated.

FIG. 3

CD spectra of hydroxyapatite-purified MOMP and bovine heart mitochondrial porin in the presence of 0.1% (wt/vol) OG.

FIG. 4

Ion channel recordings from OG-DTT-solubilized MOMP in a bilayer bathed in 50 mM KCl–10 mM Tris-HCl (pH 7.4). (A) Holding potential switched from 0 to +80 mV. (B) Holding potential switched from 0 to −80 mV. Recordings were low-pass filtered at 100 Hz and show decaying capacitative transients representing the discharge of the typically large bilayer capacitance (∼300 pF).

FIG. 5

Effects of MAb A11 on the MOMP channel. The bilayer was bathed in 50 mM KCl–10 mM Tris-HCl (pH 7.4); MAb A11 was added to both the cis and trans chambers to a final dilution of 1/1,000 in the presence of BSA (1 mg/ml). (A) Traces obtained after switching the holding potential from 0 to −80 mV before (left) and 30 min after (right) addition of MAb A11. In each recording, there is a typical bilayer capacitative current transient superimposed on channel openings, but the openings are of much lower amplitude after addition of MAb A11. (B) Detailed appearance of an individual unit current before (left) and 30 min after (right) addition of MAb A11. Note the difference in amplitude and the reduction in the number of long-lived channel closures. The dotted line represents the baseline level, corresponding to the closure of the unit conductance. Currents were filtered at 100 Hz.

FIG. 6

Analysis of the minimal conducting unit of the MOMP channel. (A) Contiguous 40-s recording of MOMP activity in a bilayer bathed in 50 mM KCl–10 mM Tris-HCl (pH 7.4) (holding potential −70 mV, filtered at 200 Hz). The closed level of the channel (0) and three unit open levels (1, 2, and 3) are indicated. (B) Maximum number of unit open levels counted in 25 individual reconstitution experiments.

FIG. 7

Current-voltage relationship of MOMP channels in symmetrical concentrations of KCl (each buffered with 10 mM Tris-HCl [pH 7.4]). KCl concentrations are 300 mM (▴), 150 mM (○), and 50 mM (•). Error bars represent ± standard error of the mean for four independent experiments.