Novel antimicrobial targets from combined pathogen and host genetics.

T identification of drug targets for a given human disease, whether it is mainly environmental or genetic in origin, rests on an understanding of the molecular chain of events that unfold in the disease process. Anatomic pathology, biochemistry, cellular physiology, and pharmacology constitute the main traditional approaches towards identifying potential therapeutic targets. Genetic approaches, by determining the phenotypic consequences of mutations in genes and ordering these genes into functional pathways, are uniquely powerful in identifying novel gene products involved in a disease process. By characterizing mutations that block or reverse the disease phenotype, genetics can provide a direct route to target identification. The wild-type versions of these ‘‘suppressor’’ gene products are potential therapeutic targets, because chemical compounds that phenocopy suppressor gene mutations should similarly block the disease phenotype and thus constitute candidate therapeutic drug leads (1). Such genetic approaches to target identification are most feasible in wellstudied model organisms with short generation times that are easily maintained in the laboratory, principally the yeast Saccharomyces cerevisiae, the soil nematode Caenorhabditis elegans, and the fruit f ly Drosophila melanogaster. Using model organisms to study homologues of genes causally mutated in human disease is relatively well established. This approach has not often been applied to human infectious diseases, however, as most human pathogens have a highly restricted host range. An exception is the Gram-negative bacterium Pseudomonas aeruginosa, strains of which are pathogenic not only to humans but also to C. elegans, Drosophila, and the genetically tractable model plant, Arabidopsis thaliana (2, 3). In the December 21 issue of PNAS, Darby et al. (4) describe a genetic approach toward the identification of potential therapeutic targets for P. aeruginosa infection. P. aeruginosa is a common bacterium in soil and water worldwide and an opportunistic pathogen in humans, causing acute and chronic infections in patients with compromised immunity, severe burns, and cystic fibrosis. A variety of virulence factors, including secreted enzymes as well as toxic chemicals, contribute to the pathogenesis of P. aeruginosa infections (for review see ref. 5). The production and secretion of most known virulence factors is increased at high versus low bacterial cell density, through a cell-to-cell signaling mechanism known as ‘‘quorum sensing’’ that links cell density to gene expression (for review see refs. 6 and 7). In P. aeruginosa two quorum-sensing systems have been described in some detail, las and rhl. Each is composed of two components: an inducer locus, lasI or rhlI, that controls the synthesis of a diffusible, cell-permeant, pheromone and a responder locus, lasR or rhlR, that encodes a transcription factor that binds to and is activated by the pheromone. Activation of LasR enhances the expression of genes encoding secreted proteases, phospholipases, ADP-ribosylating enzymes, and genes that control secretion of virulence factors as well as LasI (thus providing positive feedback to LasR activation) and RhlR. Activation of RhlR controls some genes in parallel to LasR and, in addition, activates the expression of loci responsible for the production of toxic chemicals, including hydrogen cyanide, rhamnolipids, and phenazines. LasR and RhlR are further subject to higher-level positive and negative regulators (8, 9). Two recent PNAS papers extend and expand our knowledge of quorum sensing. First, Whiteley et al. (10) report the isolation of 47 mutants in 39 different genes that are up-regulated by treatment with P. aeruginosa pheromones. The authors note that their screen is far from saturated and speculate that 1–3% of the total 5000– 6000 genes of P. aeruginosa are controlled by quorum sensing. Second, Pesci et al. (11) describe a third cell-to-cell signaling molecule produced by P. aeruginosa, 2-heptyl-3-hydroxy-4-quinolone (a chemical unrelated to the las and rhl pheromones, which are acyl homoserine lactones), that regulates the expression of virulence factors. Synthesis of this new signaling molecule requires both LasR and RhlR. These studies highlight the potential value of targeting the quorumsensing regulatory hierarchy for new antiinfective therapies (7, 12). The complexity and long life cycles of mammalian models have limited the understanding of host factors involved in microbial pathogenesis. To overcome these limitations, Rahme, Ausubel, and colleagues pioneered the use of model organisms to identify ‘‘universal’’ virulence factors for P. aeruginosa pathogenicity. Infection of Arabidopsis thaliana with P. aeruginosa strain PA14, a pathogen derived from a human infection, caused soft-rot, chlorosis, and eventual leaf collapse, and the same strain caused lethal infections in a mouse full-thickness skin burn model (2, 13). Three recent papers from this same laboratory describe PA14 pathogenesis in C. elegans (3, 14, 15). In the nematode, two types of toxicity were observed. Exposure of L4 stage larvae, but not adults, to PA14 grown on nutrientrich media at high osmotic strength killed the nematodes in 4 hr (‘‘fast killing’’), whereas exposure of either L4 or adults to PA14 grown on less rich or minimal media killed after 1 to 2 days (‘‘slow killing’’). Screening for Pseudomonas mutants with reduced pathogenicity in Arabidopsis or in slow or fast killing of C. elegans identified mutations in 23 genes, 19 of which also reduced virulence in the mouse burn model (refs. 13–15; for review see ref. 16). Because most of these genes were identified by only a single mutation, this screen also appears to be far from saturated. Now, Darby et al. (4) describe a third model of Pseudomonas pathogenesis in C. elegans. Exposure of adult C. elegans to P. aeruginosa strain PAO1 (a standard laboratory strain isolated some 50 years ago from a human infection) inhibited feeding of the nematodes within seconds to minutes, more gradually slowed and disorganized locomotory behavior and caused