Variation in the composition and pore function of major outer membrane pore protein P2 of Haemophilus influenzae from cystic fibrosis patients

Abstract

We investigated the relationship between susceptibility to beta-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulated Haemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinct H. influenzae strains from two patients extensively treated with beta-lactam antibiotics. The variants differed in their susceptibilities to at least two beta-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to beta-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences of ompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.

FIG. 1

Analysis of porins from variants strains H. influenzae 70b and 70f. A Coomassie brilliant blue-stained SDS-polyacrylamide gel is shown. The left contains a low-molecular-weight marker (in thousands, as indicated); lane 70b, 2 μg of protein of strain 70b; lane 70f, 1.5 μg of protein of strain 70f.

FIG. 2

Comparison of channel conductance as measured in planar bilayers for porins from H. influenzae OmpP2 strain variants 70b (A) and 70f (B). Conductance steps were recorded at a transmembrane potential of 10 mV and with 1 M KCl as the electrolyte. The bilayer was formed with monoolein dissolved in n-decane. The two porins were diluted with 50 mM Tris-HCl (pH 8.0) to 15 ng/μl. Approximately 1 μl of the diluted sample was added to the Teflon chamber. The total number of conductance steps analyzed was 257 for strain variant 70b and 349 for strain variant 70f.

FIG. 3

Alignment of the deduced amino acid composition of the variable regions of OmpP2 corresponding to surface-exposed loops according to the topology model of OmpP2 of nonencapsulated H. influenzae (3). The OmpP2 sequences of five strains and eight variants of these strains isolated from four CF patients are shown. The amino acid sequences of strain variants 67d, 70a, 77b, and 82e are used as the consensus sequences. The amino acid positions of the loops of strain 67d are indicated above the sequence. Dots indicate identical amino acids, and dashes indicate deletions. Amino acids are given as one-letter codes. Abbreviations: P, patient; S, strain numbers for OmpP2 variants; R, antibiotic-resistant variant; S, antibiotic-susceptible variant.

FIG. 4

Autoradiogram of the binding of [³H]penicillin to the penicillin-binding proteins of strains Rd, 70, 77, and 82, including OmpP2 variants (indicated by the lowercase letters), after the bacteria in the exponential (A) and stationary (B) phases of growth were harvested. The proteins were analyzed by SDS–11% PAGE before autoradiography.