Genomic Dissection of Bacterial Pathogens

To better elucidate mechanisms of pathogenicity, the Ausubel laboratory is developing powerful, high-throughput models to study the infectious process and combining them with the power of bacterial genetics and genomics. Our lab has studied a number of microbial pathogens over the past several years, including species of Salmonella and Enterococcus, Staphylococcus aureus, Cryptococcus neoformans, and Fusarium oxysporum. However, the first human pathogen studied in the lab, and the best characterized, is the opportunistic pathogen, Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium isolated from soil, water, and plants, is an opportunistic pathogen that infects cystic fibrosis patients, patients with thermal burns and patients who are immunodeficient or otherwise compromised. A number of years ago, the Ausubel lab discovered that a clinical isolate, called PA14, could infect both plants and animals, and that the ability to cause disease in plants required virulence factors known to be important for pathogenicity in mammalian hosts, arguing for a conservation of mechanism in this process. Using PA14, we have developed a multi-host pathogenesis system in which the bacterium can infect nematodes and insects in addition to traditional cell cultures and murine hosts. These simpler model hosts are genetically tractable, reproduce more rapidly, and are smaller and cheaper to raise than their mammalian counterparts, allowing for large-scale, high-throughput assays of virulence that are impractical or unethical to perform using mice.

The Ausubel lab is currently developing PA14 tools to better characterize its behavior during infection. The most common strain of P. aeruginosa is PAO1, which was sequenced in 2000. However, this strain is generally less virulent than PA14 in a number of hosts. We have therefore sequenced the genome of PA14 to begin to understand the strain differences that account for its enhanced pathogenicity. In conjunction with the sequencing of PA14, we have created a genome-wide collection of defined, non-redundant mutations (http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi). Thirty thousand independent transposon insertion mutations were generated and each isolate was sequenced to determine the site of the lesion. From this master set, we then selected 5474 mutants, each affecting a distinct gene. This unigene library is being made available to the Pseudomonas research community and will allow for rapid, near-saturated screens of PA14 in a number of assays, examining virulence or any other aspect of Pseudomonas biology. Our ultimate goal is to combine the power of this bacterial mutant library with the ability to do genetics in our model hosts to understand the interactions that occur between host and pathogen during the infectious process.