1.6.2S
ARABIDOPSIS THALIANA AND PSEUDOMONAS AERUGINOSA: A HOST-PATHOGEN SYSTEM TO DEFINE MOLECULAR INTERACTIONS REQUIRED FOR BACTERIAL PATHOGENICITY

LG RAHME1, L LE1, J PLOTNIKOVA 2, E DRENKARD2, H CAO2, RG TOMPKINS1 and FM AUSUBEL2

1Department of Medicine, Harvard Medical School and Department of Surgery and Shriners Burns Institute and 2Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA

Background and objectives
Our goal is to gain insight into the molecular interactions that underlie the pathogenicity of Pseudomonas aeruginosa, a Gram-negative human bacterial pathogen that infects burned, cystic fibrosis, immunodeficient or otherwise compromised individuals. Identification of some clinical strains of P. aeruginosa that cause disease in plants, and evidence of similarities in virulence mechanisms of plant and animal pathogens, prompted us to search for a strain of P. aeruginosa capable of eliciting disease in both plants and animals. The advantage of using a plant system as an adjunct to an animal model is that thousands of bacterial clones from a mutagenized P. aeruginosa library can be individually screened for avirulent mutants on plants, therefore overcoming the inherent limitations that an animal model presents. After establishing that a clinical isolate of P. aeruginosa strain UCBPP-PA14 is infectious in a mouse burn model as well as in the cruciferous plant model Arabidopsis thaliana [1], we conducted a large-scale screening in plants for non-virulent P. aeruginosa mutants. P. aeruginosa strain UCBPP-PA14 genome was mutagenized using the transposon TnphoA. To date nine TnphoA-mutant derivatives of P. aeruginosa that were identified in a plant leaf assay as less pathogenic mutants also exhibited significantly reduced virulence in a mouse burn model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts [2].

Results and conclusions
Seven out of the nine TnphoA mutants identified correspond to previously unknown genes. All of the mutants elicited either weak or moderate symptoms on Arabidopsis thaliana ecotype Ll-O, except mutant 33C7, which caused no symptoms as compared to the wild-type strain. In a mouse burn model, all mutants caused reduced mortality and showed statistically significant differences from the wild type. Complete DNA sequence analysis of the region corresponding to pho34B12, 33A9 and 33C7 mutants showed no homology to other known virulence-related genes. The pho34B12 mutation abolishes the production of pyocyanin, partially affects elastase activity and leads to production of higher levels of auto-inducer(s). The type of auto-inducer(s) produced by pho34B12 and its role in the quorum-sensing cascade will be discussed. In addition, results concerning the ability of mutants 33A9 and 33C7 to invade both mammalian and plant cells will be presented. Furthermore, studies of the structure and distribution of P. aeruginosa strain UCBPP-PA 14 in Arabidopsis leaves, using Nomarski technique and confocal microscopy, showed that P. aeruginosa penetrated the plant mesophyll cells within 24 h and propagated quickly at the infection locus. Bacteria were spread along the Arabidopsis leaf veins and were observed both intercellularly and intracellularly. Scanning electron microscopy of freeze-fractured leaves showed that UCBPP-PA 14 is situated vertically on the cell wall and enters the cell volume by perforating the plant primary walls at the point of contact. In summary, our results establish that that there are a variety of common virulence factors for bacterial pathogenicity in plants and animals. In addition, it is demonstrated that a high-throughput screen can be performed using plants as an alternative non-vertebrate host of P. aeruginosa to identify novel bacterial factors involved in mammalian pathogenesis. The microscopy studies of Arabidopsis-P. aeruginosa interactions will provide a further understanding of P. aeruginosa infectious process.

References
1. Rahme LG, Stevens EJ, Wolfort SF et al., Science 268, 1899-1902.
2. Rahme LG, Tan MW, Le L et al., 1997. Proceedings of the National Academy of Sciences, USA 94, 13245-13250.