Identification of a novel two-partner secretion locus in Moraxella catarrhalis

Abstract

Although Moraxella catarrhalis continues to be a significant cause of disease in both children and adults, the steps involved in pathogenesis remain poorly understood. We have identified three open reading frames in the M. catarrhalis genome that encode homologues of the two-partner secretion system (TPS). The sequenced M. catarrhalis hemagglutinin-like locus of strain 7169 has a unique gene organization composed in the order of mchA1, mchB, and mchA2, where mchA1 is divergent. MchA1 and MchA2 are 74% identical at the amino acid level and diverge only in the C-terminal regions. The TPS motif identified in the common N-terminal regions of MchA1 and MchA2 was found to be homologous to the filamentous hemagglutinin of Bordetella pertussis, and MchB has homology to other TpsB transporters. The presence of MchA1 and MchA2 in outer membrane protein preparations and concentrated culture supernatants (CCSs) of strain 7169 was confirmed by immunoblotting using specific antisera. Nanoscale liquid chromatography-tandem mass spectrometry peptide sequencing of the antibody-reactive bands from the CCSs was performed and demonstrated that 13 different peptides mapped to identical regions of MchA1 and MchA2. Quantitative adherence assays revealed a decrease of binding to primary normal human bronchial epithelial cells by the mch mutants 7169mchB and 7169mchA1A2B compared to that by the wild-type strain. These studies show that MchA1, MchA2, and MchB are components of a novel TPS identified in M. catarrhalis and suggest that these proteins may be involved in colonization.

FIG. 1.

Genetic organization and orientation of the M. catarrhalis 7169 hemagglutinin-like locus. The solid areas in the maps of mchA1 and mchA2 define the homologous regions of the two genes corresponding to the N termini of the products (100% identical), while the hatched areas represent the divergent regions corresponding to the C termini (24% identical). The 2-kb region common to the portion of mchA1 and mchA2 corresponding to the N termini that is used to express rMchA-His is also shown.

FIG. 2.

Comparative analysis of M. catarrhalis 7169 and mutant WCLs (A), OMPs (B), and CCSs (C) by immunoblotting using the rMchA-His polyclonal antisera. Lane 1, 7169; lane 2, 7169mchA1A2B; lane 3, 7169mchB; lane 4, 7169mchB-R. Molecular size standards are shown in kilodaltons.

FIG. 3.

Conservation of MchA1 and MchA2 in the OMPs from a panel of M. catarrhalis clinical isolates. Lane 1, 7169; lane 2, HF-084; lane 3, HF-165; lane 4, 3P8B1; lane 5, 5P26B1; lane 6, 7P94B1. Molecular size standards are shown in kilodaltons.

FIG. 4.

Adherence to NHBE cells. The values represent the means of results from separate experiments plus the standard errors. ***, P < 0.001.