Isolation and characterization of a conserved porin protein from Helicobacter pylori

Abstract

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50-and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…Porins from the organisms in the ε-proteobacteria, which forms a very deep branch almost reaching the bottom of the whole Proteobacteria group, are indeed quite different in sequence, and I have not been able to align porins from Helicobacter pylori (60,178,196) or Campylobacter jejuni (78,79,362,782) with the porin sequences shown in Fig. 1.…”

“…These proteins (48 to 67 kDa), which are called HopA, HopB, HopC, and HopD, are related to each other, and all produced ion channels in planar lipid bilayers. In a separate study, a 30-kDa protein was suspected of being a porin (681); indeed, this protein, called HopE, was shown to produce 1.5-nS channels in 1 M KCl (178). It also appears to exist as oligomers (most probably as trimers).…”

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

“…Indeed, single-channel conductance measurements have suggested smaller pore sizes for the H. pylori porins HopA, HopB, HopC, HopD (14), HopE (29), and HopV (30) compared with E. coli OmpF with a size exclusion limit of 600 Da. In contrast, Helicobacter HopX might have a larger pore, but this porin is apparently not expressed in detectable quantities (30).…”

Functional Expression in Escherichia coli and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in Helicobacter pylori

“…One of the most striking features of these genomes is a large (32-member) family of sequence-related outer membrane proteins. This family of proteins was discovered in the pregenomic era as a series of five outer membrane proteins named HopA to HopE, which had similar N-terminal sequences, and all reconstituted channels in planar bilayer membranes (8,9). HopE (8) was the smallest of these proteins (31,000 Da) but formed the largest channels with a single channel conductance of 1.5 nS in 1 M KCl.…”

“…This family of proteins was discovered in the pregenomic era as a series of five outer membrane proteins named HopA to HopE, which had similar N-terminal sequences, and all reconstituted channels in planar bilayer membranes (8,9). HopE (8) was the smallest of these proteins (31,000 Da) but formed the largest channels with a single channel conductance of 1.5 nS in 1 M KCl. For this reason it was proposed to be the major nonspecific porin of the H. pylori outer membrane, although it had a considerably lower abundance in the outer membrane than, for example, the major porin of Escherichia coli (OmpF).…”