Characterization of the Moraxella catarrhalis opa-like protein, OlpA, reveals a phylogenetically conserved family of outer membrane proteins

Abstract

Moraxella catarrhalis is a human-restricted pathogen that can cause respiratory tract infections. In this study, we identified a previously uncharacterized 24-kDa outer membrane protein with a high degree of similarity to Neisseria Opa protein adhesins, with a predicted beta-barrel structure consisting of eight antiparallel beta-sheets with four surface-exposed loops. In striking contrast to the antigenically variable Opa proteins, the M. catarrhalis Opa-like protein (OlpA) is highly conserved and constitutively expressed, with 25 of 27 strains corresponding to a single variant. Protease treatment of intact bacteria and isolation of outer membrane vesicles confirm that the protein is surface exposed yet does not bind host cellular receptors recognized by neisserial Opa proteins. Genome-based analyses indicate that OlpA and Opa derive from a conserved family of proteins shared by a broad array of gram-negative bacteria.

FIG. 1.

Moraxella catarrhalis OlpA. A. Alignment of OlpA proteins from M. catarrhalis strains ATTC4 3617, 035E, 046E, 4223, ATTC 25238, 7169, and TTA37. Red indicates high (>90%) conservation; blue indicates >50% conservation. !, I or V; $, indicates L or M; %, F or Y; #, any one of N, D, Q, or E. Gene sequence accession numbers are listed in Table 2. B. Predicted 2D structure of OlpA. Residues depicted in filled circles with white lettering are identical in OlpA and Neisseria NspA; those in black are unique to OlpA. The two-residue insertion in the second extracellular loops of M. catarrhalis strains O35E and O46E are encircled in black and green. C. Predicted OlpA 3D structure. The structure was derived by threading over the crystal structure of the neisserial NspA.

FIG. 2.

Detection of OlpA protein by immunoblotting. Western blots of bacterial lysates were probed with the OlpA-specific antiserum. A. M. catarrhalis cell lysates. B. Cell lysates from recombinant E. coli strains expressing either OlpA cloned from the indicated M. catarrhalis strains or a kanamycin cassette (Kan) as a negative control. C. Immunoblot analysis to detect reactivity with M. catarrhalis strain O35E-specific OlpA antiserum with lysates prepared from the indicated strains. Molecular mass position markers (in kilodaltons) are shown on the left.

FIG. 3.

A. Detection of OlpA in EDTA-induced outer membrane vesicles of M. catarrhalis. OMVs prepared from either strain O35E or the USPA1-deficient 035E mutant (O35EΔU) were probed with specific antibodies to detect OlpA, USPA1, and CopB. B. Surface exposure of OlpA. Intact E. coli expressing either OlpA or the periplasmic MBP were subjected to increasing doses of trypsin and proteinase K. Lysates were resolved and probed for either OlpA or MBP, as indicated. C. Heat-modifiability of OlpA was detected by using SDS-PAGE and immunoblot analysis following incubation of bacterial cell lysates for 20 min at either 100°C or 23°C. Molecular mass position markers (in kilodaltons) are shown on the left.

FIG. 4.

Relationship between diverse Opa-like proteins. A. The neighbor-joining tree diagram depicted is based on ClustalW protein alignments using Phylodraw, as outlined in Materials and Methods. Listed are bacterial species, previously used names and functions of OlpA-related proteins, and sequence accession numbers. ETEC, enterotoxigenic E. coli. B. Sequence alignment of diverse Opa-like proteins representing the indicated bacterial species illustrated in the tree diagram (A). Predicted surface loops are indicated as L1 to L4, while transmembrane regions are indicated by TM1 to TM8. Amino acid sequence identities are indicated by red (>90%) or blue (>50%).