The Major Outer Membrane Protein of Chlamydia psittaci Functions as a Porin-Like Ion Channel

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 β-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 (P_Cl/P_K ∼ 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.Members of the order Chlamydiales are distinguishable from other bacteria by their obligate intracellular mode of growth and their distinctive biphasic life cycle in which the small spore-like extracellular and infectious form, the elementary body (EB), alternates with the intracellular vegetative form, the reticulate body (RB). The four main species currently recognized, Chlamydia trachomatis, C. psittaci, C. pneumoniae, and C. pecorum, are diverse pathogens that cause a range of disease in both humans and animals. A common component of all these species is the 40-kDa major outer membrane protein (MOMP), present in both the EB and RB forms. The MOMP is a multifunctional protein which is thought to have a role both in the infectious process (3, 34–36) and in the maintenance of structural rigidity via disulfide bond cross-linking within the EB outer membrane (13, 15, 26).The antigenic properties of MOMP have been studied in detail since the landmark discovery that MOMP purified from sodium dodecyl sulfate (SDS)-gels was capable of raising antibodies which could neutralize the infectivity of C. trachomatis in vitro (6). Protein sequence comparisons of MOMPs both within (33) and between (18) species, combined with epitope mapping studies (8, 43), have shown that the epitopes responsible for neutralization lie within four variable segments. Vaccine preparations based on chlamydial outer membrane complexes, which are highly enriched for the MOMP in its native form, have been shown to be protective against chlamydial disease in sheep (37), guinea pigs (2), and mice (10, 28). However, experimental vaccines based on denatured or nonnative recombinant MOMP preparations have yielded, at best, only partial protection (28). Most recently, protection was demonstrated in mice administered a DNA vaccine comprising only the MOMP gene (42).These results clearly make MOMP the primary candidate for a subunit vaccine against chlamydial infection, but despite many years of intensive study, the paucity of structural information leaves unanswered many questions as to how MOMP fulfills its diverse functions. Structural studies are hampered first by the difficulty of growing chlamydiae in bulk and subsequently by problems with purifying and solubilizing a protein which both is highly cross-linked and normally resides in a hydrophobic environment. These factors have precluded attempts to crystallize the protein and have made it necessary to rely on analysis techniques that require relatively small quantities of protein.A recent report showed that MOMP solubilized with octyl glucoside (OG) in the presence of dithiothreitol (DTT) was oligomeric, with electrophoretic and sedimentation properties consistent with a trimeric structure (21). These oligomers resisted denaturation with SDS in a way similar to that for classical gram-negative porin molecules, which are also trimers (27). The result was consistent with an early observation by Bavoil et al. (3), who used liposome swelling to demonstrate that the chlamydial outer membrane contained pores and, due to its predominance in the outer membrane, that MOMP was the likely pore-forming protein. In this paper, we report direct evidence for porin function obtained by using native, oligomeric MOMP incorporated into planar lipid bilayers. Moreover, due to the traditional view that chlamydiae are required to scavenge ATP from the host cell, we have investigated the transport of ATP through the MOMP channel.