We have developed a novel model system to identify previously unknown virulence-related genes in the human opportunistic pathogen Pseudomonas aeruginosa and to identify host genes that are involved in the defense response against pathogenic microorganisms. We have identified a P. aeruginosa clinical isolate, strain PA14, that is not only infectious in several mouse models, but also causes disease in the model plant Arabidopsis thaliana and in the insects Drosophila melanogaster and Galleria mellonella (wax moth caterpillar). Moreover, PA14 also kills the nematode Caenorhabditis elegans. From the perspective of the pathogen, the advantage of using model genetic hosts is that thousands of bacterial clones from a mutagenized P. aeruginosa library can be individually screened for avirulent mutants in separate plants, in separate insects or on separate petri plates seeded with C. elegans. This approach is not feasible or ethical using a vertebrate host because of large numbers of host organisms involved. The advantage of studying pathogenesis in model genetic hosts is that host genetic analysis can be used to identify host genes involved in pathogen defense by screening for host mutants that are more susceptible or more resistant to pathogen attack.
Surprisingly, many previously known bacterial genes that are required for pathogenesis, including toxA, gacA, dsbA, lasR and degP are required for pathogenesis in all of our model hosts. We therefore screened for non-pathogenic P. aeruginosa PA14 transposon-induced mutants in the Arabidopsis and C. elegans models in an attempt to identify previously unknown bacterial virulence-related genes. To date we have identified a total of 22 P. aeruginosa PA14 mutants that are less pathogenic in the Arabidopsis and/or C. elegans models and most of these mutants are less pathogenic in the insect and mouse models as well. Remarkably, 8 out of 10 P. aeruginosa mutants that were identified in the Arabidopsis screen correspond to previously unknown genes.
From the host perspective, we have isolated a set of Arabidopsis and C. elegans mutants that exhibit enhanced susceptibility or enhanced resistance to Pseudomonas. We have also studied a set of existing C. elegans mutants for susceptibility to PA14 killing and using these mutants we have demonstrated that a small molecule (pyocyanin) excreted by P. aeruginosa plays a key role in C. elegans killing, most likely by exposing C. elegans to high levels of oxidative stress.
Selected Publications:
Mahajan-Miklos, S., M.-W. Tan, L.G. Rahme and F.M. Ausubel. (1999)
Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas
aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96:47-56.
Tan, M.-W., S. Mahajan-Miklos, and F.M. Ausubel. (1999) Killing of C. elegans by P. aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl. Acad. Sci. USA 96:715-720.
Tan, M.-W., L.G. Rahme, J. Sternberg, R.G. Tompkins and F.M. Ausubel.
(1999) Pseudomonas aeruginosa killing of Caenorhabditis elegans used to
identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA
96:2408-2413.