In a previous study, we identified a set of PIB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H) that confer resistance to penicillin and tetracycline (28
). These mutations were hypothesized to reside in loop 3, the extracellular loop that folds into the barrel and forms the constriction zone. Because mutations in loop 3 of other porins are extremely effective in altering pore size and ion and antibiotic permeation properties (23
), we expected that the PIB mutations studied here would have similar effects. Although our data demonstrated that each of the PIB variants had substantially lower predominant conducting states and increased channel noise compared to wild-type PIB, the variants in fact showed no significant differences in pore size, ion permeation, and antibiotic permeation rates. Moreover, our results confirmed and extended earlier studies showing that overexpression of the MtrC-MtrD-MtrE efflux pump in N. gonorrhoeae
is necessary to observe increased resistance to penicillin and tetracycline conferred by penB
In concordance with other porin studies (31
), the structural and functional properties of recombinant wild-type PIB porin, which was overexpressed in inclusion bodies in E. coli
and refolded in vitro, were indistinguishable from native PIB purified from gonococci. Furthermore, wild-type PIB, either native or recombinant, exhibited ion conductance and ion selectivity values in planar lipid bilayers that were very similar to those previously reported for Neisseria
). The wild-type channels were primarily in an open state, but occasionally they were closed in distinct levels reflecting the trimeric porin structure. We infrequently observed a small closed-channel current as described by Mauro et al. (20
). Gonococcal porins have been reported to exhibit voltage-dependent closings at ±80 mV in planar lipid bilayers; however, our experimental design did not address the voltage-dependent gating behaviors of the PIB channels at these potentials (19
The largest differences between the wild-type and variant PIBs were in their ion-conducting properties, including lower conductance states and increased channel noise. The conductance properties of the PIB variants suggest that either the internal regions of the pore are altered or that the individual subunits of the trimers are open less often and for much shorter times than those of the wild type. Even though the ion conductances of the porin variants were markedly altered, liposome-swelling assays showed that mutations in PIB do not alter the size of the pore or the permeation rates of neutral sugars and β-lactam antibiotics. Because the pore sizes were unchanged in the PIB variants, our data are more consistent with changes in the gating properties of the PIB variants and not with alterations within the pore itself.
The increased channel noise and increased time spent in subconductance states observed in the variant PIBs are reminiscent of the changes in the channel properties of the OmpC porin with mutations designed to disrupt the interaction of loop 3 with the outer barrel (17
). The electrophysiological properties of these mutant porins were examined by patch clamp of liposome blisters containing multiple porin channels. Some of the mutant porins displayed a dramatic increase in the closing rate and decrease in open probability relative to wild-type OmpC (17
). These changes were interpreted to be the result of changes in the flexibility of loop 3 within the barrel, caused by the loss of hydrogen bonds and/or salt bridges between loop 3 and the outer barrel. It is tempting to speculate that mutations in loop 3 of PIB also may increase the flexibility of loop 3. Unfortunately, the effects of the OmpC mutations on antibiotic permeation are unknown, so a correlation of changes in electrophysiological properties to changes in permeation of larger compounds cannot be made. The data presented here with gonococcal porins suggest that changes in the electrophysiological properties of PIB do not correlate with changes in solute and antibiotic permeation rates.
Gill et al. (12
) speculated that aspartic acid residues at positions 120 and 121 in loop 3 of PIB decrease antibiotic diffusion by anion repulsion. However, this mechanism is not supported by our data. No differences in the permeation rates of monoanionic β-lactam antibiotics were observed through wild-type and variant PIBs, and both the wild-type and PIB variants showed similar preferences for anions, even with the variants containing either an acidic (Asp) or basic (Lys and Arg) amino acid at position 120. The lack of an effect on ion selectivity by charged amino acid substitutions seems to argue against the prediction that residues 120 and 121 line the channel of the pore. These residues may instead face towards the outer wall or may even be located outside of the pore. A definitive description of the changes induced by these mutations must await the three-dimensional structure of PIB.
Recently, an antibiotic-resistant strain of Enterobacter aerogenes
was identified containing a mutation in the major porin that conferred resistance to a variety of cephalosporin antibiotics. The porin contained a mutation (G112D) in loop 3, in a position similar to that of the PIB mutations, and was shown to have a significantly reduced single-channel ion conductance and reduced pore size (5
). The authors concluded that the smaller pore size of the mutant mediated increased antibiotic resistance, but liposome-swelling assays with β-lactam antibiotics were not performed to determine whether antibiotic fluxes through the mutant porin were also altered. The loop 3 mutation identified in the De et al. study, G112D, was similar to the G120D/A121D variant described in this study (5
). However, the effects of the G112D mutation in the E. aerogenes
porin were very different than the G120D/A121D mutations in PIB, as we saw no significant differences in the pore sizes of wild-type and mutant PIBs.
It is unclear at the present time how the penB
mutations increase antibiotic resistance, especially given that the mutations had no effect on the permeation rates of sugars and antibiotics and the absolute requirement for the presence of the mtrR
determinant to increase antibiotic resistance (and decrease permeation) in strains containing the PIB variants. Some reports have shown that expression of nonspecific porins can be negatively regulated by modulators of the multidrug resistance (Mdr) system, which is homologous to the Mtr system, resulting in decreased permeability of the outer membrane to antibiotics (4
). However, we showed previously that porin expression is not decreased in strains with the PIB variants (28
So why do the PIB mutants increase antibiotic resistance only when the MtrC-MtrD-MtrE efflux pump is overexpressed? One possibility is that only a slight decrease in antibiotic permeation through porins is required to confer resistance when expression of the efflux pump is deregulated. This scenario would be consistent with the small (but not significant) decreases observed in the antibiotic permeation rates through PIB-G120K compared to wild-type PIB. An alternative hypothesis is that the PIB variants may interact directly with the Mtr efflux pump and work cooperatively to decrease the periplasmic concentrations of antibiotic. In this scenario, mutant PIB proteins may form a complex with the MtrC-MtrD-MtrE efflux pump, and the close proximity of the efflux pump may allow for small differences in antibiotic permeation to be magnified.