The lipopolysaccharide (LPS) of Pseudomonas aeruginosa is an immunodominant antigen and a major virulence factor of this opportunisti pathogenic bacterium. We are investigating the genetic basis of LPS O antigen production in P. aeruginosa. There is an obvious interest in understanding the mechanisms underlying this pathogen's virulence, and O antigen production and variation are clearly at the forefront of this process. While there are twenty recognized serogroups of P. aeruginosa that differ from one another in monosaccharide composition and structure of the O antigen repeating units, ten of these serogroups are responsibl for the vast majority of infections. Development of effective vaccination strategies to prevent or treat P. aeruginosa infections are needed as the limits of antibiotic and current therapies are reached. O-antigen-specific antibodies are protective against P. aeruginosa infections, however the protection observed is often serogroup specific. One of our long-term objectives, already partially realized, will be to elicit P. aeruginosa O antigen production by enteric organisms that can be used as oral vaccines. We have cloned the genes required for expression of O antigen (the rfb gene cluster) from P. aeruginosa strain PA103, a serogroup 11 strain and, as such, representative of the most commonly found serogroup among environment strains, and one which is often responsible for infections. We have expressed the P. aeruginosa serogroup 11 O antigen on Escherichia coli and Salmonella, and, after oral delivery of these recombinant organisms to mice, have elicited protection against subsequent challenge with serogroup 11 P.aeruginosa. These results indicate the feasibility of such an approach to create an effective vaccine, and represent the basis for the specific aims of the current proposal: to compare the cloned genes encoding two different rf gene clusters of P. aeruginosa (from serogroup 11 and serogroup 5) in terms of genetic organization, number of proteins required for O antigen expression, and the functions of the genes that are common and unique to these serogroups. This information will advance our basic understanding of the biology of P. aeruginosa LPS production and allow us to develop the tools necessary to realize our long-term goals. These include determining the role that the O antigen of P. aeruginosa plays in virulence, both in acute infections and in chronic lung infections of patients with cystic fibrosis. We will be able to create isogenic strains differing only in O antigen structure, and examine these strains in animal models and in vitro, investigating the interaction of LPS with host immune system effectors such as complement and phagocyte cells. We also hope to develop a recombinant oral vaccine against P. aeruginosa based on our ability to clone the O antigen gene cluster of any serogrou of this organism. By expressing many different P. aeruginosa serogroups in Salmonella strains, we will prepare a recombinant oral 'cocktail' protective against all serogroups important in clinical infections. Unlike other P. aeruginosa vaccines, these recombinant oral vaccines wil have the advantage of being genetically defined, easily administered and well tolerated, and should be capable of eliciting immune responses at various mucosal sites. These studies will also provide new insights int the expression and genetic regulation of LPS O antigen by P. aeruginosa.
