The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance

Abstract

Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.

FIG. 1.

Comparison of the phenotypes of the wild-type M. catarrhalis strain O35E and the uspA2 mutant O35EΔ2. (A) Western blot analysis of whole-cell lysates of O35E (lane 1) and O35EΔ2 (lane 2) using MAb 17C7 as the primary antibody. This MAb binds both UspA2 (indicated by bracket) and UspA1 (indicated by arrow). (B) Western blot analysis of these whole-cell lysates using the UspA1-specific MAb 24B5 as the primary antibody. (C) Proteins present in outer membrane vesicles from these two strains were resolved by SDS-PAGE and stained with Coomassie blue. The absence of UspA2 in the O35EΔ2 mutant is indicated by the arrow. Whole-cell lysates digested with proteinase K were resolved by SDS-PAGE, and LOS was visualized either by staining with silver (D) or by Western blot analysis (E) using the LOS-reactive MAb 8E7 as the primary antibody. Protein molecular mass markers (in kilodaltons) are present on the left sides of panels A, B, and C.

FIG. 2.

Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35EΔ2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS⁺⁺ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl₂, 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of IgG in killing of the uspA2 mutant O35EΔ2. Wild-type strain O35E (1) and the uspA2 mutant O35EΔ2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.

FIG. 3.

Comparison of UspA2 proteins from M. catarrhalis strains O35E and MC317. (A) Alignment of the deduced amino acid sequences. Identical amino acids are shaded in dark gray, while conserved amino acids are shaded with light gray. This figure was generated by using the ClustalW Alignment program in MacVector (version 6.5). (B) Relative amounts of UspA2 exposed on the surface of the following strains as measured by the indirect antibody accessibility assay: the M. catarrhalis uspA1 uspA2 mutant O35E.12 (1), the uspA1 mutant O35E.1 (2), and the uspA1 mutant MC317.1 (3). The counts per minute of radioiodinated goat anti-mouse IgG bound to MAb 17C7 on the bacterial cell surface (a) are plotted on the y axis. MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of H. ducreyi (28), was used as the negative control (b). These data represent the means of results from two independent experiments plus standard deviations.

FIG. 4.

Effect of uspA2 gene exchange on killing of M. catarrhalis by NHS. (A) Western blot analysis using MAb 17C7 to probe whole-cell lysates of the following strains: O35E-Sm^r, lane 1; the uspA2 mutant O35E.2ZEO, lane 2; transformant strain O35E/317U2 (i.e., O35E expressing the MC317 UspA2 protein), lane 3; MC317-Sm^r, lane 4; the uspA2 mutant MC317.2, lane 5; and transformant strain MC317/35U2 (i.e., MC317 expressing the O35E UspA2 protein), lane 6. The whole-cell lysates used in this Western blot experiment were diluted to be equivalent to 2 × 10⁶ CFU in order to visualize possible changes in the level of expression of UspA2. The bracket indicates the region containing UspA2 and its degradation products. Molecular mass markers are shown to the left in kilodaltons. The MAb 17C7-reactive antigen that migrated near the 105-kDa marker is UspA1. (B) Resistance of these six strains (in the same order as in panel A) to killing by either 10% (vol/vol) heat-inactivated NHS (a) or 10% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors.

FIG. 5.

Effect of expression of the M. catarrhalis UspA2 protein on serum resistance of H. influenzae DB117. (A) Western blot analysis using MAb 17C7 to probe whole-cell lysates of H. influenzae DB117 carrying the following plasmids: pAA-kp (negative control), lane 1; pAA-35U2-kp, lane 2; and pAA-317U2-kp, lane 3. Molecular mass markers (in kilodaltons) are shown to the left. (B) Resistance of these three strains (in the same order as above) to killing by either 5% (vol/vol) heat-inactivated NHS (a) or 5% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors. The asterisk indicates that the difference between column 2b and column 3b is statistically significant (P value, 0.002) as determined by the use of a paired t test.

FIG. 6.

Comparison and analysis of MC317-O35E hybrid UspA2 proteins. (A) Schematic representation of the six hybrids. Open segments and upper numbers represent the amino acid sequence from MC317; filled segments and lower numbers represent the sequence from O35E. Numbering begins with the first residue of the complete protein (containing the signal peptide). All constructs are drawn to scale. (B) Western blot analysis using MAb 17C7 to probe whole-cell lysates of the following strains: MC317-Sm^r, lane 1; hybrid 1, lane 2; hybrid 2, lane 3; hybrid 3, lane 4; hybrid 4, lane 5; hybrid 5, lane 6; hybrid 6, lane 7; MC317/35U2, lane 8; and O35E-Sm^r, lane 9. The whole-cell lysates used in this Western blot experiment were diluted to be equivalent to 2 × 10⁶ CFU in order to visualize any change in the level of expression of UspA2. The bracket indicates the region containing UspA2 and its degradation products. The MAb 17C7-reactive antigen that migrated near the 105-kDa marker is UspA1. Molecular mass markers are shown to the left (in kilodaltons). (C) Resistance of these nine strains (in the same order as in panel B) to killing by either 10% (vol/vol) heat-inactivated NHS (a) or 10% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors.