Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts.

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.

Plants and animals share functionally common bacterial virulence factors.

By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins.

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.

Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model.

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans. Using systematic mutagenesis of PA14 to identify mutants that fail to kill C. elegans and a C. elegans mutant that lacks P-glycoproteins, we identified phenazines, secreted P. aeruginosa pigments, as one of the mediators of killing. Analysis of C. elegans mutants with altered responses to oxidative stress suggests that phenazines exert their toxic effects on C. elegans through the generation of reactive oxygen species. Finally, we show that phenazines and other P. aeruginosa factors required for C. elegans killing are also required for pathogenesis in plants and mice, illustrating that this model tackles the dual challenges of identifying bacterial virulence factors as well as host responses to them.

Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis.

We show that a single clinical isolate of the human opportunistic pathogen Pseudomonas aeruginosa (strain PA14), which previously was shown to be pathogenic in mice and plants, also kills Caenorhabditis elegans. The rate of PA14-mediated killing of C. elegans depends on the composition of the agar medium on which PA14 is grown. When PA14 is grown on minimal medium, killing occurs over the course of several days and is referred to as "slow" killing. When PA14 is grown on high-osmolarity medium, killing occurs over the course of several hours and is referred to as "fast" killing. Several lines of evidence, including the fact that heat-killed bacteria are still capable of fast but not slow killing of C. elegans, indicate that fast and slow killing occur by distinct mechanisms. Slow killing involves an infection-like process and correlates with the accumulation of PA14 within worm intestines. Among 10 PA14 virulence-related mutants that had been shown previously to affect pathogenicity in plants and mice, 6 were less effective in killing C. elegans under both fast- and slow-killing conditions, indicating a high degree of commonalty among the P. aeruginosa factors required for pathogenicity in disparate eukaryotic hosts. Thus, we show that a C. elegans pathogenicity model that is genetically tractable from the perspectives of both host and pathogen can be used to model mammalian bacterial pathogenesis.

Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail.

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by quail depends on the level of quail protein overexpression, as well as residual levels of fascin function. In nurse cells that contain excess quail but no fascin, the cytoplasmic actin network initially appears wild type but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of quail overexpression to compensate for the absence of fascin demonstrates that fascin is partially redundant with quail in the Drosophila germline. Quail appears to function as a bundle initiator while fascin provides bundle organization.

The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis.

Mutations in the Drosophila quail gene result in female sterility due to the disruption of cytoplasmic transport from the nurse cells into the oocyte late in oogenesis. Nurse cells from quail mutant egg chambers fail to assemble cytoplasmic actin filament bundles correctly. We have cloned the quail gene and found that it encodes a protein with homology to the vertebrate actin-regulating protein villin. Unlike vertebrate villin, which is restricted to specialized absorptive epithelial cells, the villin-like protein encoded by quail is germline specific in adult flies. Antibodies directed against the quail protein show a striking colocalization with filamentous actin in the nurse cells and the oocyte. Our results demonstrate that the villin-like product of quail is required for the formation of cytoplasmic actin filament bundles in nurse cells, possibly by regulating both the polymerization and organization of actin filaments as demonstrated for vertebrate villin in vitro.