Construction of a mutant and characterization of the role of the vaccine antigen P6 in outer membrane integrity of nontypeable Haemophilus influenzae

Abstract

Outer membrane protein P6 is the subject of investigation as a vaccine antigen to prevent infections caused by nontypeable Haemophilus influenzae, which causes otitis media in children and respiratory tract infections in adults with chronic lung disease. P6 induces protective immune responses in animal models and is the target of potentially protective immune responses in humans. P6 is a 16-kDa lipoprotein that shares homology with the peptidoglycan-associated lipoproteins of gram-negative bacteria and is highly conserved among strains of H. influenzae. To characterize the function of P6, an isogenic mutant was constructed by replacing the P6 gene with a chloramphenicol resistance cassette. The P6 mutant showed altered colony morphology and slower growth in vitro than that of the parent strain. By electron microscopy, the P6 mutant cells demonstrated increased size, variability in size, vesicle formation, and fragility compared to the parent cells. The P6 mutant showed hypersensitivity to selected antibiotics with different mechanisms of action, indicating increased accessibility of the agents to their targets. The P6 mutant was more sensitive to complement-mediated killing by normal human serum. Complementation of the mutation in trans completely or partially restored the phenotypes. We concluded that P6 plays a structural role in maintaining the integrity of the outer membrane by anchoring the outer membrane to the cell wall. The observation that the absence of expression of P6 is detrimental to the cell is a highly desirable feature for a vaccine antigen, supporting further investigation of P6 as a vaccine candidate for H. influenzae.

FIG. 1.

Coomassie blue-stained SDS gel (left panel) and immunoblot assays of whole bacterial cell lysates (middle and right panels). Lanes a, parent strain; lanes b, P6 mutant; lanes c, complemented P6 mutant. The center panel was probed with the monoclonal antibody 7F3, which recognizes an epitope on outer membrane protein P6. The right panel was probed with the monoclonal antibody 2C7, which recognizes an epitope on outer membrane protein P5. The arrow denotes the location of P6. Molecular mass markers are noted on the left, in kilodaltons.

FIG. 2.

Southern blot assay. DNAs from the parent strain (lanes a), the P6 mutant (lanes b), and the complemented P6 mutant (lanes c) were digested with EcoRI. Panels were probed with ∼200-bp biotinylated PCR fragments of the chloramphenicol resistance cassette (left) and the P6 gene (right). Molecular size markers are noted in kilobases on the left.

FIG. 3.

Results of RT-PCR with the P6 mutant and its parent strain, 49P5H1, as noted at the bottom of the gel. Primers used in reactions corresponding to the P6 gene, the upstream tolB gene, and the downstream hypothetical tRNA/rRNA methyltransferase gene are noted at the top of the gel. Lanes a, purified RNA amplified with reverse transcriptase; lanes b, purified RNA amplified with TaqI polymerase to exclude DNA contamination; lanes c, purified DNA amplified with TaqI polymerase; lanes −, distilled water with reverse transcriptase as a negative control. Molecular size markers are noted in kilobases on the left.

FIG. 4.

Growth curves for H. influenzae strains.

FIG. 5.

Electron micrographs of parent strain 49P5H1, P6 mutant, and complemented mutant. Bar, 1 μm. The graphs indicate the smallest diameters of 100 cells of each strain. The boxes denote 25 and 75% quartiles around the means, and the bars denote 10 and 90% values. Cell diameters lying outside the 10 and 90% values are noted by circles. The y axis shows the cell diameters measured on prints at a magnification of 20,000, in cm.

FIG. 6.

Results of serum bactericidal assays with normal human serum used against H. influenzae strains, as noted. The x axis shows final concentrations of serum in bactericidal reaction mixtures, and the y axis shows percent survival after 30 min.