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 β-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulatedHaemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinctH. influenzae strains from two patients extensively treated with β-lactam antibiotics. The variants differed in their susceptibilities to at least two β-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 β-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 ofompP2 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.

Protected environments allow parallel evolution of a bacterial pathogen in a patient subjected to long‐term antibiotic therapy

“…2B). Such changes are common among genes for surface structures that may be subject to strong selective pressure by the host immune response (Smith et al ., 1995; Regelink et al ., 1999). The mutations, primarily transitions C → T on the coding strand (Table 1), caused a reduction in the GC content of the gene by 2.3% relative to K‐12, which was atypical of other genes analysed in these isolates.…”

Mechanisms of Bacterial Resistance to Antibiotics in Infections of COPD Patients

“…These proteins can frequently be involved in antimicobial resistance (reviewed in [79]). Porin alterations that are associated with a decrease in outer membrane permeability for H. influenzae have been shown to only partially contribute to resistance to ampicillin, penicillin, cephalothin and chloramphenicol [81,82] and this has not been considered a major mechanism of antimicrobial resistance in H. influenzae, despite P2 proteins exhibiting significant sequence variations between strains [80]. Porin alterations that are associated with a decrease in outer membrane permeability for H. influenzae have been shown to only partially contribute to resistance to ampicillin, penicillin, cephalothin and chloramphenicol [81,82] and this has not been considered a major mechanism of antimicrobial resistance in H. influenzae, despite P2 proteins exhibiting significant sequence variations between strains [80].…”

Multiple facets of bacterial porins

“…A divergence between these strains in the OmpC amino acid sequence, consisting of 15 amino acid substitutions and two gaps (a five‐residue deletion and a four‐residue insertion) was reported [38]. Variation in the composition and pore function of major outer membrane pore protein P2 of Haemophilus influenzae , involved in cystic fibrosis has been reported [39]. DNA sequencing of ompP2 gene from clinical isolates revealed very few non‐synonymous base differences, affecting the amino acid sequence of surface‐exposed loops 4, 5, 6 and 8.…”

“…DNA sequencing of ompP2 gene from clinical isolates revealed very few non‐synonymous base differences, affecting the amino acid sequence of surface‐exposed loops 4, 5, 6 and 8. These changes were suggested to affect the permeability of the porin channel and thus their susceptibility to β‐lactam antibiotics [39].…”