Moraxella catarrhalis strain O35E expresses two filamentous hemagglutinin-like proteins that mediate adherence to human epithelial cells

Abstract

Two-partner secretion (TPS) systems are a family of proteins being rapidly identified and characterized in a growing number of gram-negative bacteria. TPS systems mediate the secretion of proteins, many involved in virulence traits such as hemolysis, adherence to epithelial cells, inhibition of bacterial growth, and immunomodulation of the host. A TPS system typically consists of a transporter located in the bacterial outer membrane (OM) which is responsible for the recognition and secretion of at least one large exoprotein. Two of the better-characterized TPS systems specify the Bordetella pertussis FHA and Haemophilus influenzae HMW1/HMW2 proteins. We identified three gene products of Moraxella catarrhalis strain O35E that resemble TPS proteins and designated them MhaC (transporter), MhaB1 (exoprotein), and MhaB2 (exoprotein). Western blot analysis using anti-MhaC, or antibodies reacting to both MhaB1 and MhaB2 (MhaB-reactive), revealed that these antigens are expressed in the OM of 63% of isolates tested. Mutations in the mhaC gene specifying the putative transporter of the M. catarrhalis wild-type strains O35E, O12E, and McGHS1 resulted in the absence of MhaB1/MhaB2 in the OM of mutants. These results are therefore consistent with the Mha proteins functioning as a TPS system. Furthermore, we discovered that these mhaC mutants exhibit markedly decreased binding to human epithelial cells relevant to pathogenesis by M. catarrhalis (Chang, HEp2, A549, and/or 16HBE14o(-)). Expression of O12E MhaC and MhaB1 in a nonadherent strain of Escherichia coli was found to increase the adherence of recombinant bacteria to HEp2 monolayers by sevenfold, thereby demonstrating that this M. catarrhalis TPS system directly mediates binding to human epithelial cells. The construction of isogenic mutants in the mhaB1 and mhaB2 genes of strain O35E also suggests that the MhaB proteins play distinct roles in M. catarrhalis adherence.

FIG. 1.

Schematic representation of the M. catarrhalis mhaB1 mhaC mhaB2 locus. The open bars represent the regions of the mhaB1 and mhaB2 gene products that are virtually identical, whereas the patterned arrowheads illustrate the regions of divergence.

FIG. 2.

Western blot analysis of selected OM proteins expressed by M. catarrhalis strain O35E and isogenic mha mutants. OM vesicles were obtained from the WT isolate O35E (lane 1) and from its mhaC (lane 2), mhaB1 (lane 3), mhaB2 (lane 4), and mhaB1 mhaB2 (lane 5) mutant strains and analyzed by immunoblotting with the indicated antibodies (Ab). Molecular mass markers are shown to the left in kilodaltons.

FIG. 3.

Adherence of M. catarrhalis strain O35E (black bars) and mha mutants (open bars) to human epithelial cells in vitro. Adherence is expressed as the percentage (±standard error) of inoculated bacteria bound to epithelial cells following a 5-min incubation (shown on y axes). The Mann-Whitney test was used to determine whether decreases in adherence were statistically significant (*, P < 0.05).

FIG. 4.

Western blot analysis of selected OM proteins expressed by M. catarrhalis isolates of various origins. OM vesicles were resolved on 7.5% polyacrylamide gels, transferred to PVDF membranes, and probed with murine sera against MhaC and MhaB. McaP antibodies (Ab) were also included in these experiments as loading indicators. Molecular mass markers are shown to the left in kilodaltons. This figure is a composite of separate Western blots, and therefore, O35E (lanes 1 and 10) was included as a reference strain.

FIG. 5.

Western blot analysis of selected OM proteins expressed by M. catarrhalis O35E, O12E, and McGHS1 and strains derived from these isolates. OM preparations from the WT, mhaC mutants, and repaired mhaC strains were electrophoresed, transferred to PVDF membranes, and analyzed by immunoblotting with the indicated antibodies (Ab). Molecular mass markers are shown to the left in kilodaltons.

FIG. 6.

Adherence of M. catarrhalis WT (black bars) and mhaC mutant (open bars) strains to human epithelial cells in vitro. Adherence is expressed as the percentage (±standard error) of inoculated bacteria bound to epithelial cells following a 5-min incubation (shown on y axes). The Mann-Whitney test was used to determine whether decreases in adherence were statistically significant (*, P < 0.05).

FIG. 7.

Western blot analysis and adherence assays of recombinant E. coli bacteria expressing O12E MhaC and MhaB1. (A) Sarkosyl-insoluble OM proteins were extracted from E. coli strains carrying the plasmids pCC1.3 (lane 1) and pRB.O12E.B1C (lane 2) and were then analyzed by immunoblotting with murine sera against MhaB and MhaC; molecular mass markers are shown in kilodaltons. (B) Duplicate adherence assays were performed on at least four separate occasions. The adherence is expressed as the percentage (±standard error) of inoculum bound to epithelial cells following a 3-h incubation. The Mann-Whitney test was used to determine whether the increase in adherence was statistically significant (*, P < 0.05). Ab, antibody.