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

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.

Antimicrobial susceptibility of Haemophilus influenzae in the respiratory tracts of patients with cystic fibrosis.

We analyzed the antimicrobial susceptibilities of Haemophilus influenzae isolates from 157 sputum specimens prospectively collected from 39 cystic fibrosis (CF) patients during a 2-year study. These isolates were characterized by random amplified polymorphic DNA analysis and major outer membrane protein (MOMP) analysis to identify H. influenzae strains and MOMP variants and to assess their persistence in the respiratory tract. Among the 247 H. influenzae isolates, 16 (6.5%) produced beta-lactamase. The 231 beta-lactamase-negative isolates represented 85 H. influenzae strains, 61 MOMP variants derived from 27 of these strains, and 85 persistent isolates identical to strains or MOMP variants. All beta-lactamase-negative isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, cefuroxime, cefotaxime, cefaclor, imipenem, tetracycline, and trimethoprim-sulfamethoxazole by disk diffusion testing. Eleven (13%) H. influenzae strains, 18 (30%) MOMP variants, and 30 (35%) persistent isolates were resistant to one or more of the antibiotics tested. Antimicrobial susceptibility was decreased among MOMP variants and persistent isolates compared to nonpersistent H. influenzae strains, and changes in susceptibility occurred irrespective of MOMP variation. We conclude that the decreased antimicrobial susceptibility of H. influenzae during persistence contributes to the poor eradication of H. influenzae from the respiratory tracts of CF patients.

Multiple Haemophilus influenzae strains and strain variants coexist in the respiratory tract of patients with cystic fibrosis.

To investigate the epidemiology of nontypeable Haemophilus influenzae in the respiratory tract of cystic fibrosis (CF) patients, H. influenzae isolates from sputum specimens of 40 CF patients were analyzed longitudinally for 2 years. The isolates were characterized by analysis of the major outer membrane protein (MOMP) patterns. MOMP variant H. influenzae strains were discriminated from distinct strains by randomly amplified polymorphic DNA analysis of genomic DNA. Multiple H. influenzae strains and MOMP variant strains were isolated from single sputum specimens of 29 patients. In 22 patients, a distinct H. influenzae strain persisted over time (median persistence, 8 months; range 2-24). In general, the appearance of MOMP variant strains did not coincide with the occurrence of exacerbations.

The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.