Purification and characterization of protein H, the major porin of Pasteurella multocida

Abstract

Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude from this set of structural and functional data that protein H of P. multocida is a pore-forming protein related to the superfamily of the nonspecific bacterial porins.

Virulence gene profiles of Pasteurella multocida strains isolated from cattle and buffalo

“…ompH and oma87 genes were identified in all samples in other studies (DaVIeS et al, 2004;eWerS et al, 2006;Bethe et al, 2009;FUrIaN et al, 2013) and were the most frequently detected genes in isolates in D. Gharibi et al: Virulence gene profile of Pasteurella multocida from cattle and buffalo other studies (taNG et al, 2009;FerreIra et al, 2012;khaMeSIPOUr et al, 2014). Omph is a major outer membrane porin that forms a homotrimeric channel, and has shown some potential as a protective antigen (CheVaLIer et al, 1993). Oma87 is an 87-kDa outer membrane protein of all P. multocida strains that is expressed in vivo (rUFFOLO and aDLer, 1996).…”

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73

“…In native conformation, porin H is a homotrimer, stable in sodium dodecyl sulfate (SDS) at room temperature, and is dissociated into monomers upon boiling. The molecular masses of denatured monomers range between 34 and 42 kDa depending on the serotype and the electrophoretic system used for analysis (8,19,20). The N-terminal amino acid sequence of porin H has been determined for serotype D2 (8).…”

“…The molecular masses of denatured monomers range between 34 and 42 kDa depending on the serotype and the electrophoretic system used for analysis (8,19,20). The N-terminal amino acid sequence of porin H has been determined for serotype D2 (8). This N-terminal sequence is almost identical to that of so-called cross-protection factors OMP 179 and OMP 153 from strain P-1059 (serotype 3), which are in the high-molecular-mass range (42)(43)(44).…”

“…Protein H, or porin H, is the major outer membrane protein in the envelope of P. multocida (20,21). This protein has been purified and characterized as a porin because it is structurally and functionally related to the superfamily of porins of gramnegative bacteria (8,20). In native conformation, porin H is a homotrimer, stable in sodium dodecyl sulfate (SDS) at room temperature, and is dissociated into monomers upon boiling.…”

“…We have designated this gene ompH and the encoded major outer membrane protein OmpH. method described by Chevalier et al (8). Briefly, the bacteria were cultured in brain heart infusion broth at 37°C overnight.…”