Modeling In Vivo Phosphate Depletion in the C elegansP aeruginosa System Discovery of Red Death

In order to verify that acute Pi depletion alone was sufficient to shift the lethality of P. aeruginosa in vivo, we modeled Pi depletion in the P. aeruginosa-C. elegans system (Fig. 9.2) (Zaborin et al. 2009). C. elegans normally feed on lawns of E. coli OP50 that grow on nematode growth media (NGM), a high [Pi] media (25 mM). We used P. aeruginosa PAO1, known to be non-lethal to C. elegans on NGM media, and depleted the media of Pi to determine if low [Pi] would shift P. aeruginosa to express lethality. To accomplish this, C. elegans feeding on E. coli OP50 were transferred onto lawns of P. aeruginosa PAO1 growing on NGM media at high [Pi] (25 mM) or low [Pi] (~0.1 mM). Although initially we did not observe any mortality under low [Pi], C. elegans progeny formation, an important marker of viability, was significantly attenuated. In order to deplete the worm digestive tube of any residual Pi upon transfer to the lawn of P. aeruginosa, we subjected worms to a period of short-term starvation (18 h) and performed reiterative studies. Results demonstrated that following this approach, C. elegans survival dramatically decreased on low [Pi] lawns compared to those feeding on high [Pi] lawns (30% versus 100% at 48 h - Fig. 9.2). To determine if these results were due to the stress of starvation versus nutrient deprivation itself, we exposed non-starved worms to mild heat stress (35°C 2 h), performed reiterative studies, and obtained similar results (data not shown) (Note: worms normally thrive and feed at 20°C). Thus analogous to our mouse model, host stress is required for intestinal P. aeruginosa to express a lethal phenotype against its host. Importantly, the Pi concentration at which P. aeruginosa produced mortality in C. elegans (~0.1 mM) was similar to that which increased the virulence output of P. aeruginosa in vitro and to its concentration in the intestinal tract of mice following surgical injury.

Fig. 9.2 C. elegans growing on lawns of P. aeruginosa on Nematode growth media (NGM) at high and low phosphate [Pi]. Worms are transferred onto lawns of E. coli or P. aeruginosa at high or low [Pi]. Only P. aeruginosa growing on low [Pi] NGM causes mortality with red material accumulating in the digestive tube (arrows). Red material was not observed in surviving worms

Fig. 9.2 C. elegans growing on lawns of P. aeruginosa on Nematode growth media (NGM) at high and low phosphate [Pi]. Worms are transferred onto lawns of E. coli or P. aeruginosa at high or low [Pi]. Only P. aeruginosa growing on low [Pi] NGM causes mortality with red material accumulating in the digestive tube (arrows). Red material was not observed in surviving worms

We unexpectedly observed red pigmented material in the digestive tube of worms and discovered that this was due to the release of a toxic PQS iron complex in response to low [Pi] which we termed red death (Zaborin et al. 2009). We also determined that genes encoding the byosynthesis of rhamnolipids, surfactants known to solubilize PQS, were also overexpressed during low [Pi] and that the triple mixture (PQS, Fe, rhamnolipids) induced severe epithelial apoptosis in the cecum of mice (Fig. 9.3) (Zaborin et al. 2009). Taken together, these data provide compelling evidence that low [Pi] is sufficient to shift P. aeruginosa to express a lethal phenotype from within the digestive tube of C. elegans and mice.

Lastly, we tested the synergistic effect of both low [Pi] and exposure to the various host compounds (i.e., opioids) and found that low [Pi] enhances the responsiveness of P. aeruginosa to host compounds released during stress (unpublished observations). Thus, it is likely that multiple aspects of the local microenvironment within which pathogens like P. aeruginosa colonize and feed are important for the activation of virulence and lethality. As these aspects are uncovered and pathways identified that are conserved across a variety of pathogens, compounds can be developed that might molecularly silence P. aeruginosa and other pathogens from expressing virulence as they course through the intestinal tract of critically ill and immunocompromised patients.

Tissue Injury/Inflammation/Hypoxia

Tissue Injury/Inflammation/Hypoxia

Interferon-Y Opioids (Dynorphin) Adenosine

Interferon-Y Opioids (Dynorphin) Adenosine

Depletes Phosphates

Fig. 9.3 Multi-pronged attack of P aeruginosa against the intestinal epithelium in response to host stress tissue compounds and local environmental "cues" present in the gut during surgical injury. Host death is hypothesized to result from various toxins and barrier disrupting compounds released by P aeruginosa in response to host stress compounds and low phosphate conditions

Fig. 9.3 Multi-pronged attack of P aeruginosa against the intestinal epithelium in response to host stress tissue compounds and local environmental "cues" present in the gut during surgical injury. Host death is hypothesized to result from various toxins and barrier disrupting compounds released by P aeruginosa in response to host stress compounds and low phosphate conditions

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