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Infection and Immunity, September 2006, p. 5169-5176, Vol. 74, No. 9
0019-9567/06/$08.00+0     doi:10.1128/IAI.00692-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction of a Mutant and Characterization of the Role of the Vaccine Antigen P6 in Outer Membrane Integrity of Nontypeable Haemophilus influenzae

Timothy F. Murphy,1,2,4* Charmaine Kirkham,1 and Alan J. Lesse1,2,3,4

Division of Infectious Diseases, Department of Medicine,1 Department of Microbiology,2 Department of Pharmacology and Toxicology, University at Buffalo, State University of New York,3 Veterans Affairs Western New York Healthcare System, Buffalo, New York4

Received 30 April 2006/ Returned for modification 19 May 2006/ Accepted 2 June 2006

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.

* Corresponding author. Mailing address: VA Western New York Healthcare System, Medical Research 151, 3495 Bailey Avenue, Buffalo, NY 14215. Phone: (716) 862-7874. Fax: (716) 862-6526. E-mail: murphyt{at}buffalo.edu.

Editor: D. L. Burns

Infection and Immunity, September 2006, p. 5169-5176, Vol. 74, No. 9
0019-9567/06/$08.00+0     doi:10.1128/IAI.00692-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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