Down-regulation of porin M35 in Moraxella catarrhalis by aminopenicillins and environmental factors and its potential contribution to the mechanism of resistance to aminopenicillins

The outer membrane protein M35 of Moraxella catarrhalis is an antigenically conserved porin. Knocking out M35 significantly increases the MICs of aminopenicillins. The aim of this study was to determine the biological mechanism of this potentially new antimicrobial resistance mechanism of M. catarrhalis and the behaviour of M35 in general stress situations. PCR using m35-specific primers was used to detect the m35 gene in clinical isolates. The m35 mRNA expression of strains 300, O35E and 415 after exposure to amoxicillin and different stress conditions was measured by real-time PCR and normalized in relation to their 16S rRNA expression. The expression of M35 protein was analysed by SDS-PAGE and western blotting. Screening of 52 middle ear isolates resulted in positive PCR products for all tested strains. The analysis of m35 mRNA expression after amoxicillin treatment showed 24%-85% down-regulation compared with the respective amoxicillin-free controls in all three strains tested. Also, analysis of protein concentrations revealed lower M35 expression after growth with amoxicillin. Investigation of M35 during general stress responses showed down-regulation of the porin with growth at 26 degrees C and 42 degrees C, under hyperosmolar stress and under iron restriction. The reduced expression of M35 after aminopenicillin exposure indicates a novel resistance mechanism against aminopenicillins in M. catarrhalis, which may be relevant in vivo. The differences in expression after different stress treatments demonstrate that M35 is involved in general stress responses.