Description of Complex Forms of a Porin in Bacteroides fragilis and Possible Implication of this Protein in Antibiotic Resistance

Abstract Periodic surveys of antibiotic susceptibility patterns among anaerobes have emphasized that new mechanisms of resistance have emerged, especially in the Bacteroides fragilis group. Resistance to the combination of amoxicillin and clavulanic acid among some imipenem-susceptible Bacteroides fragilis strains has been associated with modifications in outer membrane protein electrophoretic patterns with the loss of some porin-like proteins. Porins are outer membrane proteins that play a major part in membrane permeability; if they are under-expressed, they can be responsible for antibiotic resistance. In a previous work, we isolated one outer membrane protein of 45 kDa from Bacteroides fragilis and showed its porin activity. In the present study, we aim to isolate the different complex forms of this protein and to underline their possible role in antibiotic resistance. We therefore compared the electrophoretic patterns of the outer membrane proteins of several strains of Bacteroides fragilis . Although these patterns are similar to each other, some proteins, especially those of high molecular weight, are less visible in the samples heated before electrophoresis. We targeted these high molecular weight proteins (which appeared sensitive to heat) and isolated them by electro-elution. We thus identified two high molecular weight proteins (210 and 130/135 kDa) which seemed to be components of a complex including the 45 kDa outer membrane protein formerly identified by us as a porin protein. Their porin activities were tested by the swelling assay of proteoliposomes which showed that the 210 kDa protein behaved like the 45 kDa protein whereas the 130/135 kDa protein had less porin activity. Furthermore, swelling assays with antibiotic solutions made it possible to compute the role of this protein complex in antibiotic resistance.